新型含苯并咪唑基噻唑烷二酮类化合物的合成及其抗糖尿病脑功能障碍的研究
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摘要
背景：
糖尿病脑病（Diabetes encephalopathy, DE）是糖尿病（Diabetes mellitus, DM）严重并发症之一，损害患者的认知和记忆。传统治疗采用噻唑烷二酮（Thiazolidinedione, TZD）类过氧化物酶体增殖物激活受体（Perioxisomeproliferator-activated receptor-γ, PPAR-γ）激动剂控制血糖，辅以改善脑细胞功能的药物，目前尚无特效治疗药物。TZD类药控制血糖作用良好，但因脂溶性低，难以通过血脑屏障（Blood brain barrier, BBB），防治神经损伤作用有限。由此，研发降糖效果好、能顺利通过血脑屏障的药物将填补这一空白，具有非常重要的临床意义。
目的：
烷基取代的苯并咪唑基团亲水性低脂溶性高，生物相容性好。为此，我们将烷基取代的苯并咪唑基团与TZD基团连接，合成含苯并咪唑基的TZD类PPAR-γ激动剂系列化合物H2、H4和H6；采用体外高糖损伤神经元模型，结合细胞免疫荧光、蛋白免疫印迹和流式细胞术，筛选出神经保护作用药效最好的化合物H2；初步药代动力学结果提示，化合物H2透过BBB；建立2型糖尿病（Type2Diabetes Mellitus，T2DM）大鼠模型，进一步考察化合物H2对糖尿病脑病的治疗作用。结果显示H2显著提高大鼠生存率、降低血糖，同时，H2明显改善大鼠的学习、记忆行为；生化指标提示，化合物H2神经保护作用可能是通过降低血中炎性因子白细胞介素1β（Interleukin-1β, IL-1β）、肿瘤坏死因子-（Tumor necrosis factor-, TNF-）和脑内丙二醛（Malondialdehyde, MDA）水平，以及提高血中抗炎因子IL-10和脑内抗氧化物质超氧化物歧化酶（Superoxide dismutase, SOD）、过氧化氢酶（Catalase, CAT）和谷胱甘肽过氧化物酶（Glutathione peroxidase, GSH-Px）水平发挥作用。本研究为发掘新型糖尿病脑病治疗药物初步奠定了基础。
方法：
一、新型含苯并咪唑基的TZD类化合物H2、H4和H6的合成
1．酸性条件下，取代的邻氨基苯与1-氯直链羧酸缩合得到取代苯并咪唑缩合产物；
2.4-羟基苯甲醛与2,4-噻唑烷二酮反应生成5-(4-羟基苯亚甲基)噻唑烷-2,4-二酮（H7），再经硼氢化钠还原得到中间体5-(4-羟基苯甲基)噻唑烷-2,4-二酮（H8）；
3.合成苯并咪唑中间体2-氯甲基-1H-苯并咪唑（H1）、2-(氯甲基)-5-甲基-1H-苯并咪唑（H3）、2-(氯甲基)-1-甲基-1H-苯并咪唑（H5）；
4.苯并咪唑中间体H1、H3、H5与噻唑烷二酮中间体H8缩合得到目标化合物H2、H4、H6；
二、新型含苯并咪唑基的TZD类化合物H2、H4和H6的体外神经保护作用研究
1.蛋白免疫印迹实验
培养的神经元高糖（30mM）刺激24h后，给予不同浓度（0.1、1.0、10μM）化合物H2、H4、H6继续作用24h。Western blot方法检测与糖尿病病理进展、神经毒性相关的蛋白醛糖还原酶（Aldose reductase，AR）、缺氧诱导因子-1（Hypoxiainducible factor-1，HIF-1）、Bax，Pro-caspase-3等在神经元表达的变化，同时设立等渗透压的甘露醇（Mannitol）及母核药物罗格列酮（Rosiglitazone, RGZ）作为对照。
2.细胞免疫荧光实验
培养的神经元高糖（30mM）刺激24h后，给予不同浓度（0.1、1.0、10μM）化合物H2继续作用24h，同时设立等渗透压的甘露醇及母核药物RGZ作为对照。细胞免疫荧光染色神经元骨架蛋白MAP2（Microtubule-associated protein2），观察合成化合物对神经元形态学的影响。
3.流式细胞实验
培养的神经元高糖（30mM）刺激24h后，给予不同浓度（0.1、1.0、10μM）化合物H2作用24h，同时设立等渗透压的甘露醇及母核药物RGZ作为对照。流式细胞术检测合成化合物H2对神经元凋亡的影响。
三、化合物H2药代动力学研究
1．用反相高效液相色谱法，流动相为0.01mol/L NH4Ac：CH3OH（35:65，V/V），流速1mL/min，检测波长247nm，RGZ为内标。大鼠腹腔注射25mg/kg化合物H2，于给药后0、0.083、0.25、0.5、1、1.5、2、3、4h眼眶采血，高效液相色谱（High performanceliquid chromatography，HPLC）检测血中H2浓度，确定H2在SD大鼠血浆中的药代动力学参数。
2.根据预实验结果，大鼠腹腔注射25mg/kg化合物H2后0.25h，取脑组织，制作脑组织匀浆，HPLC法检测脑中H2的浓度。
四、化合物H2对糖尿病脑病的治疗作用
1．建立T2DM模型和急性焦虑模型
T2DM模型：高糖高脂饲料喂养SD大鼠4周，诱导发生胰岛素抵抗，再腹腔注射链脲佐菌素（Streptozotocin，STZ）30mg/kg维持高糖高脂饮食8周，即T2DM模型。
急性焦虑模型：SD大鼠束缚2h/d，连续2次，建立急性焦虑模型。
2．Morris水迷宫实验
将造模成功的T2DM大鼠分为T2DM组，低、中、高剂量H2治疗组（1mg/kg，3mg/kg，10mg/kg）和RGZ治疗组（3mg/kg），动物给予药物治疗4周，正常组给予相同体积生理盐水。Morris水迷宫实验以逃避潜伏期和穿越平台次数考察H2对T2DM大鼠学习记忆的保护作用。
3．高架十字迷宫实验
SD大鼠分为正常组、H2（3mg/kg）组和焦虑组。将大鼠面朝开臂放在中央区域，迷宫上方采用视频记录并分析5min内大鼠进入开臂或闭臂（四爪全部入臂）的次数与时间。以进入开臂的次数和滞留时间作为衡量焦虑的指标，总入臂次数则用于自主活动的检测。
4．生化指标检测
水迷宫实验结束后，SD大鼠心脏取血并取脑组织制作脑组织匀浆，用南京建成试剂盒测定血浆IL-1β、TNF-和IL-10及脑皮质匀浆SOD、CAT、GSH-Px和MDA的水平。
结果：
一、H2、H4和H6的合成
合成得到3个目标化合物H2、H4、H6，并通过1H NMR和MS鉴定其结构。另外，合成过程中还得到中间体H5及副产物H55的晶体，并用X-射线表征其结构。
二、新型含苯并咪唑基的TZD类化合物H2、H4和H6的体外神经保护作用研究
1.化合物H2、H4和H6对高糖培养神经元细胞的影响
Western blot结果显示高糖可升高AR、HIF-1、Bax，降低Pro-caspase-3在神经元的表达；与正常对照组比较，高糖组神经元AR、HIF-1、Bax和Pro-caspase-3表达有显著差异；为排除渗透压对培养体系的影响，同时设置等渗透压即30mM甘露醇作为对照，结果显示，甘露醇对神经元中AR、HIF-1、Bax和Pro-caspase-3的表达没有影响，与正常对照组相当，排除渗透压对神经元细胞生长状态的影响。化合物H2、H4、H6可以逆转高糖培养神经元AR、HIF-1、Bax和Pro-caspase-3的表达水平，且H2效果最佳。
2.化合物H2对神经元细胞形态的影响
高糖处理神经元24h，可造成明显的细胞骨架形态改变，表现为骨架蛋白MAP2染色神经元突起不连续，成断线状改变。给予化合物H2处理24h则神经元突起形态基本恢复正常。
3.化合物H2对神经元细胞凋亡的影响
高糖损伤24h未引起显著的神经细胞凋亡，各治疗组亦未见细胞凋亡；可能高糖条件不能诱导细胞凋亡，流式细胞术检测高糖引起的神经元凋亡不是敏感指标，无法评价H2的神经保护作用。
三、化合物H2药代动力学研究
本研究建立一种简单、安全、经济的方法检测血和脑组织中H2的HPLC-UV方法，分析结果准确、灵敏。大鼠腹腔注射给药25mg/kg时，AUC(0-)为（14.58±1.45）mg/L*h，Tmax为（0.17±0）h，CLz/F为（1.73±0.17）L/h/kg，t1/2为（0.73±0.075）h，Cmax为（26.39±1.02）μg/mL。H2能透过血脑屏障，给药后0.0833h脑中浓度为1.57ng/g。
四、化合物H2对糖尿病脑功能异常的治疗作用
1．一般指标观察
T2DM大鼠造模成功后体重持续下降，且出现多饮、多尿、多食等症状，空腹血糖>7.8mmol/L。正常组大鼠体重持续增长，毛皮及精神状态良好。
给予药物治疗4周后，与T2DM组比较，低、中、高剂量H2组和RGZ组大鼠空腹血糖显著下降（p <0.01）；T2DM组和RGZ组大鼠生存率分别为67.5%和75%，低、中、高剂量H2组大鼠全部存活，明显提高了T2DM大鼠生存率。
2．Morris水迷宫结果
药物治疗4周后，与正常组大鼠比较，T2DM组大鼠逃避潜伏期显著升高（p <0.01），提示T2DM大鼠出现认知功能障碍。药物治疗组中，与T2DM组比较，低、中、高剂量H2组及RGZ组逃避潜伏期显著减小（p <0.05、p <0.01、p <0.01和p <0.05）。高剂量H2组逃避潜伏期显著小于RGZ组（p＜0.05）。
在d6撤去平台后，与正常组比较，T2DM模型组大鼠跨越平台次数显著减少（p<0.01）。药物治疗组中，中、高剂量H2组比RGZ组跨越平台次数显著增加（p <0.05，p <0.01）。
3．H2对T2DM大鼠白内障的预防
T2DM大鼠治疗过程中，观察到各组T2DM大鼠不同程度出现肉眼可见的晶状体浑浊。根据本校第二附属医院眼科晶状体分级系统，对大鼠晶状体的浑浊程度进行分级。与正常对照组比较，T2DM组晶状体浑浊显著（p <0.01）；与T2DM组比较，低、中、高剂量H2治疗组晶状体浑浊程度显著减轻（p <0.01）；与RGZ组比较，中、高剂量H2治疗组晶状体浑浊程度显著减轻（p <0.01）。
4．高架十字实验
焦虑模型组大鼠在开臂内滞留时间与空白对照组比较具有显著性差异（p <0.05），说明急性焦虑模型成功。但H2治疗组与焦虑模型组在进入开臂的次数和滞留时间上未见显著性差异。
5．生化指标检测
大鼠血浆炎症因子IL-1β、TNF-和抗炎因子IL-10水平：与正常组比较，T2DM组IL-1β和TNF-显著升高（p <0.05，p <0.01），IL-10显著降低（p <0.01）；与T2DM组大鼠比较，低、中、高剂量H2组IL-1β水平有显著降低（p <0.05，p <0.01，p <0.01）；与T2DM组大鼠比较，中、高剂量H2组TNF-水平有显著降低（p <0.05，p <0.01），其中高剂量H2组与RGZ比较有显著降低（p <0.05）；与T2DM组大鼠比较，低、中、高剂量H2组IL-10水平均有显著升高（p <0.05，p <0.05，p <0.01），其中高剂量H2组与RGZ组比较显著升高（p <0.05）。
大鼠脑匀浆SOD、CAT、GSH-Px和MDA水平：与正常组比较，T2DM组SOD、CAT和GSH-Px水平显著降低（p <0.01），MDA水平显著升高（p <0.01）；与T2DM组大鼠比较，低、中、高剂量H2组SOD水平显著升高（p <0.05，p <0.01，p <0.05），且与RGZ组比较有显著升高（p <0.05，p <0.01，p <0.05）；与T2DM组大鼠比较，中、高剂量H2组CAT水平显著升高（p <0.05），且与RGZ组比较有显著升高（p <0.05）；与T2DM组大鼠比较，低、中、高剂量H2组GSH-Px水平显著升高（p <0.05），且高剂量H2组与RGZ组比较有显著升高（p <0.05）；与T2DM组大鼠比较，低、中、高剂量H2组MDA水平均有显著降低（p <0.01），且中、高剂量H2组与RGZ组比较有显著降低（p <0.05，p <0.01）。
结论：
1．体外神经保护作用
Western blot结果表明，高糖损伤24h后，糖代谢相关蛋白AR、HIF-1表达水平增高；凋亡相关蛋白Bax的表达水平增高，Pro-Caspase-3蛋白的表达水平降低，表明高糖造成神经元糖代谢异常，引起神经元的早期损伤。合成化合物H2、H4和H6降低AR、HIF-1和Bax蛋白的表达，从而抑制了神经元的损伤。这些结果表明，化合物H2、H4和H6能抑制高糖引起的糖代谢异常，对高糖损伤的神经元有一定的保护作用，其中H2药效强于H4和H6。
高糖损伤24h可造成神经元骨架形态的改变，表现为骨架蛋白MAP2染色神经元突起不连续，成断线状。合成药H2治疗后神经元突起形态基本恢复正常，发挥神经保护作用。
高糖处理神经元24h，细胞凋亡不显著，这可能由于糖尿病引起的神经损伤是一长期过程，体外高糖作用时间较短，因而流式细胞术检测高糖引起的神经元凋亡不是敏感指标。
2．化合物H2药代动力学研究
SD大鼠腹腔给药后15min脑中H2含量为1.57μg/g，此时间点血浆中浓度为26.4μg/mL，说明H2能够通过血脑屏障。有文献报道，脑脊液中母核药物RGZ浓度仅为血中的0.0045%[1]。我们的检测体系未能检测到脑组织中的RGZ，可能是由于脑内RGZ浓度低于检测基线。具体原因还有待进一步考证。相同方法下，脑组织中能检测到H2而不能检测到RGZ，提示H2通过血脑屏障的能力要强于RGZ。
3．化合物H2对糖尿病脑功能障碍的治疗作用
大量研究结果显示，T2DM患者出现学习记忆、认知功能障碍等中枢神经系统（Central nervous system, CNS）损害的临床症状。Morris水迷宫实验是研究啮齿类动物学习、记忆能力的公认模型，能客观、准确地评价动物的认知功能。通过考察动物的逃避潜伏期、穿越平台次数等主要指标来衡量动物的学习、记忆功能。本实验两项指标的结果提示，H2能改善T2DM大鼠的认知功能障碍，且效果优于阳性对照药RGZ。通过检测T2DM大鼠血浆和脑匀浆相关生化指标发现，H2抑制炎症相关因子IL-1β和TNF-的表达，提高抗炎性细胞因子IL-10的表达，从而减轻糖尿病时自身免疫性和慢性炎症反应，发挥治疗作用。另外，H2治疗后脑中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）活性提高以及脂肪氧化终产物丙二醛（MDA）水平降低，且中、高剂量药效要优于RGZ。该结果提示H2能改善脑内酶抗氧化系统的活性，提高机体的抗氧化能力，清除各种活性氧对机体的损伤，减少脂肪氧化产生毒性物质（MDA），发挥神经保护作用。
另有研究表明，焦虑情绪的缓解对糖尿病患者的治疗有积极意义[2]。我们考察H2对焦虑是否具有潜在的治疗意义，结果提示H2对急性焦虑没有明显作用，对于慢性焦虑的作用还有待研究。
4．对白内障的预防作用
糖尿病患者晚期往往眼部病变。实验中，我们观察到T2DM大鼠先后出现白内障， H2治疗组白内障的发生率及严重程度均显著下降。此结果提示，化合物H2在T2DM大鼠模型白内障形成及预防方面的潜在治疗价值。
综上所述，离体实验中化合物H2与罗格列酮对高糖介导的神经元损伤具有相似的保护作用；T2DM大鼠的治疗中，合成化合物H2的药效要优于RGZ。
Backgroud:
Diabetic encephalopathy (DE), one of the serious complications of Diabetes Mellitus(DM), impaired cognition and memory of DM patients. Traditional treatment for DE is thecombination of thiazolidinedione (TZD), peroxisome proliferator-activated receptor(PPAR-γ) agonists to control blood glucose, with drugs to improve the function of braincells; however, there is still no specific medicine for DE therapy. Although the TZD drugsis effective to control blood glucose, the roles in prevention and treatment of neural injuryof DM patients is very limited, because TZD is difficult to pass the blood-brain barrier(BBB) due to its low liposolubility. Therefore, it has a very important clinical significanceto fill this gap to develop a therapeutic medicine for DE treatment, and this medicineshould have hypoglycemic effect as well as high liposolubility to pass the blood-brainbarrier.
Aims：
Alkyl-substituted benzimidazole group has low hydrophilic, high liposolubility and good biocompatibility. To this end, we synthesized benzimidazole derivative named ascompounds H2, H4and H6, by conjuncting the alkyl-substituted benzimidazole group toTZD group. The evaluation of these compounds on neural protection were carried outusing a neuron-injury model induced by high glucose stimulation in vitro, combined withimmunofluorescence staining, Western blot and flow cytometry methods. Among them,compound H2was identified to have the best neuroprotective effects. The preliminarypharmacokinetic results suggested that compound H2could pass the BBB of rats. Inaddition, the therapeutic effects of compound H2for diabetic encephalopathy wereinvestigated using a type2diabetes (T2DM) rat model. The results showed that H2significantly promoted the survival rate, decreased the level of blood glucose, andimproved learning and memory behaviors of T2DM rats. The underlying mechanisms ofH2neuroprotection were determined by biochemical markers related to inflammation andantioxidant system. We found that the levels of inflammatory cytokines IL-1β and TNF-decreased, while anti-inflammatory cytokine IL-10increased in the blood in H2-treatedT2DM rats. Furthermore, H2decreased the level of malondialdehyde (MDA), improvedthe antioxidant substances superoxide dismutase (SOD), catalase (CAT) and glutathioneperoxidase (GSH-Px) in the T2DM rats brain. This study provides preliminary basis toexplore a new candidate for the diabetes encephalopathy treatment.
Methods:
1. Synthesis of new benzimidazole derivative componds H2, H4and H6
1) Under acidic conditions, benzimidazoles were obtained by the condensationreaction using substituted anthranilic benzene and1-chloro-straight-chain carboxylic acid;
2)5-methylenebis (4-hydroxyphenyl) thiophene-2,4-dione (H7) was harvestedthrough the reaction of4-hydroxybenzaldehyde with2,4-thiazolidine dione, then theintermediate5-(4-hydroxybenzyl) thiazolidine-2,4-dione (H8) was obtained by sodiumborohydride reduction;
3) Synthesis of benzimidazole intermediates2-chloro-methyl-1H-benzimidazole (H1),2-(chloromethyl)-5-methyl-1H-benzimidazole (H3),2-(chloro-methyl yl)-1-methyl-1H-benzimidazole (H5);
4) Condensation reatcion of benzimidazole intermediates H1, H3and H5withthiazolidinedione intermediates H8to form the target compounds H2, H4, H6;
2. The evaluation of the neuroprotective effects of new TZD derivative, compondsH2, H4and H6, in vitro
1) Western blot analysis
The neuroprotective effects of compounds H2, H4and H6were investigated in thecultured cortical neurons. The neurons were stimulated with high glucose (30mM) for24h, followed by treatment with compounds H2, H4and H6at different concentrations(0.1,1.0,10μM) for another24h. Western blot analysis were used to detect the changes ofexpressions levels of aldose reductase (AR), hypoxia-inducible factor1-alpha (HIF-1),Bax and pro-caspase-3, which are related to the pathological progress of diabetes andneurotoxicity. Mannitol was used as the osmotic pressure control and prosome structurerosiglitazone (RGZ) was used as positive control.
2) Immunofluorescence experiments
The cultured mature neurons were stimulated with high glucose (30mM) for24h,followed by treatment with compound H2at different concentrations (0.1,1.0,10μM) foranother24h. The effects of H2on neuron morphology after treatment were checked byimmunofluorescence staining of neuronal cytoskeletal proteins microtubule-associatedprotein-2(MAP2). Mannitol and RGZ weres used as control.
3) Flow cytometry experiments
The effects of H2on neuron apoptosis were determined by flow cytometry afterneurons were stimulated with30mM glucose for24h followed by H2treatment at0.1,1.0,10μM for another24h. Mannitol and RGZ were both used as control.
3. The pharmacokinetic study of compound H2
1) Reversed-phase liquid chromatography was used to detect the concentration of H2in samples, and the working system was used as following: mobile phase0.01mol/LNH4Ac: CH3OH (35:65, V/V), flow rate1mL/min, detection wavelength247nm. RGZwas used as the internal standard. Compound H2was adimistrated to normal rats byintraperitoneal injection at25mg/kg. Orbital blood was collected at0,0.083,0.25,0.5,1.0, 1.5,2.0,3.0and4.0h after H2administration, and the H2concentrations in blood weredetermined by HPLC assay, therefore, the pharmacokinetic parameters of H2in SD rats’plasma were obtained.
2) Brain tissues were harvested after intraperitoneal injection of H2(25mg/kg) for0.25h and homogenized according to the preliminary experiments, HPLC assay was usedto detect H2concentrations in the brain tissue.
4. The therapeutic effects of compound H2for diabetic encephalopathy treatment
1) Establishment of T2DM and acute anxiety models
To establish type2diabetes (T2DM) and rat insulin resistance model, SD rats werefed continously with high sugar and fat diet for4weeks, then streptozotocin (STZ) wasadministrated to rats by intraperitoneal injection at30mg/kg, and the same diet wasmaintained for another8weeks to get T2DM model.
To induced acute anxiety model, SD rats were constrainted in well-ventilated Perspexrestraining tubes for2h for two consecutive days.
2) Morris water maze (MWM) test
To check the therapeutic effects of H2, T2DM rats were divided into DM controlgroup, H2-treated group (1mg/kg,3mg/kg,10mg/kg) and RGZ-treated group (3mg/kg)besides the normal control group, the animals were administrated with H2or RGZintragastricly for4weeks. Same volume saline was used as control. The effects of H2onspatial learning and memory were evaluated by the Morris water maze test, and the escapelatency and the number of crossing the platform were collected for analysis.
3) Elevated plus maze (EPM) experiment
The elevated plus maze (EPM) was conducted to evaluated the potential side effectsof compound H2. SD rats were randomly divided into control group, H2-treated group (3mg/kg) and acute anxiety group. Individual animals were placed in the centre square,facing an open arm, and allowed to move freely for5min. Rats were videotaped using acamera fixed above the maze and analyzed with a video-tracking system. Open and closedarm entries (all four paws in an arm) and times were recorded. The number of entriesand time spent in open arms were used to measure anxiety degree, and the number of total entries in four arms was used for the detection of locomotor activity.
4) Detection of the biochemical markers
After the MWM test was finished, blood and brain tissue were harvested immediately.The concentrations of IL-1β, TNF-and IL-10in plasma, the activities of SOD, CAT,GSH-Px and MDA levels in brain homogenates were determined by kits purchased fromNanjing Jiancheng company.
Results:
1. Synthesis of componds H2, H4and H6
Three target compounds of H2, H4, H6were synthesized, and their structures wereidentified by1H NMR and MS. In addition, single crystal of the intermediate H5andbyproduct H55were obtained during the synthesis process, and their structures werecharacterized by X-ray.
2. The neuroprotective effects of new TZD derivative, componds H2, H4and H6, invitro
1) Effects of compounds H2, H4and H6on neurons stimulated with high glucose
Western blot results showed that the expression levels of AR, HIF-1and Bax incultured neurons increased markedly upon high glucose stimulation, while the level ofpro-caspase-3reduced, compared with normal controls. Treatment with compounds H2,H4and H6revesed the expression levels of AR, HIF-1, Bax and Pro-caspase-3to somedegree.30mM mannitol was set as a osmotic control to exclude the influence of osmoticpressure in culture system at the same time. The results showed that mannitol had noeffects on AR, HIF-1, Bax and Pro-caspase-3expression, similar to the results from thenormal control group. Therefore, the impact of osmotic pressure on the growth state of theneurons was excluded.
2) Effects of compound H2on neuron morphology
The morphological changes of the cytoskeleton in cultured neurons were observedafter30mM glucose stimulation for24h, which was characterized as discontinuous andbreak-like neurite by cytoskeletal protein MAP2staining. Treatment with compound H2for24h, the neurite morphology was rescued to normal.
3) Effects of compound H2on neuron apoptosisThere was no significant apoptosis in neurons upon high glucose stimulation for24h aswell as in other treated groups. It is possibile that early and acute apoptosis can not beinduced under high glucose condition in short time. Therefore, flow cytometry methodwas not sensitive to evaluate the neuroprotective effects of synthetic compounds in theearly phase.
3. The pharmacokinetic study of compound H2
A simple, safe and economical way was established in this study to detect theconcentrations of H2in the plasma and brain tissue using HPLC-UV method. Accurateand sensitive data were obtained for pharmacokinetic analysis. Administration of H2at25mg/kg(i.p), the parameters were obtained as following: the AUC (0-)14.581.45mg/L*h, Tmax0.17±0h, CLz/F1.73±0.17L/h/kg, t1/20.73±0.075h, Cmax26.39±1.02g/mL.The concentration of H2in brain after administration for0.0833h was1.57ng/g, and thisresult implicated that H2passed through the blood-brain barrier.
4. The therapeutic effects of compound H2on the diabetic brain functions
1) General observation
Type2diabetic rats showed a continued decline in weight, accompanied withpolydipsia, polyuria and polyphagia which was observed in diabetes patients, and thefasting blood glucose was more than7.8mmol/L. The weight of normal rats continued togrow and they survived healthily with smooth and glossy fur and in good mental status.The blood glucose decreased significantly in each treatment of H2and RGZ, comparedwith the T2DM group (p <0.01).
2) Treatment of H2rescued deficits of learning and memory in T2DM rats
The T2DM rats had impaired learning using the available visuospatial cues to locatethe submerged escape platform, as indicated by prolonged escape latency acrossconsecutive trials compared with the normal group (p <0.01), suggesting that T2DM ratsdevelops cognitive deterioration with increasing age. After medication of H2or RGZ for4weeks, the reduced escape latency across the trials of rats in H2groups (low, medium andhigh dose) and RGZ group were observed compared with the control T2DM group (p < 0.05, p <0.01, p <0.01and p <0.05). Furthermore, we confirmed the escape latency ofrats in high-dose H2group was significantly reduced compared with the RGZ-treated rats(p <0.05).
The platform in the water maze was removed at day6, the numbers across theplatform in T2DM reduced markedly compared with the normal rats (p <0.01). Thenumbers across the platform increased significantly in H2-treated groups (medium andhigh dose), compared with RGZ-treated group (p <0.05, p <0.01).
3) H2treatment prevented cataract development in T2DM rats
There was visible lens opacification observed in T2DM rats from each group in thelate phase of the DM models. According to the lens grading system, the lens opacificationof the rats were graded. The worst grade of lens opacification happened in T2DM ratscompared with normal control and H2-treated rats (p <0.01). H2treatment (low, mediumand high dose) ameliorated significantly the severity of opacification compared withT2DM rats (p <0.05), and this effect was not observed in RGZ-treated group, implicatingthat H2treatment effectively prevent cataract development in diabetic rats.
4) Elevated plus maze experiment
The acute anxiety rat model was established by restraint rats for two consecutive days,and the time spent in open arms increased compared with control rats (p <0.05). While,there was no significant difference of the number of entries and time spent in open armbetween H2-treated group and anxiety model group.
5) Biochemical markers
We first checked the levels of inflammatory cytokines IL-1β and TNF-, andanti-inflammatory cytokine IL-10in rat plasma at the end of experiments. The levels ofIL-1β and TNF-in T2DM group were increased significantly compared with normalgroup (p <0.05, p <0.01), while levels of IL-10were decreased significantly (p <0.01).Compared with T2DM rats, levels of IL-1β decreased significantly in each of H2-treatedgroup (p <0.05). The levels of TNF-were significantly decreased in H2-treated group atboth middle and high dose compared with the T2DM group (p <0.05, p <0.01). Levels ofTNF-decreased markedly in high-dose H2group compared with RGZ-treated group (p < 0.05). The levels of IL-10in each H2-treated group were significantly increased comparedwith T2DM rats (p <0.05, p <0.05, p <0.01). Furthermore, the levels of IL-10were evenhigher in high-dose H2group than in RGZ-treated group (p <0.05).
We next checked the activities of SOD, CAT and GSH-Px and the levels of MDA inbrain homogenate. The activities of SOD, CAT and GSH-Px decreased significantly inT2DM group compared with normal group (p <0.01). SOD acitvities increasedsignificantly in each H2-treated group compared with both T2DM group and RGZ-treatedgroup (p <0.05, p <0.01, p <0.05). The levels of CAT were significantly increased inmedium and high dose H2group compared with both T2DM and RGZ-treated group (p <0.05). Levels of GSH-Px activity in each H2-treated group were significantly highercompared with T2DM rats (p <0.05), and was significantly higher in high-dose H2groupcompared with RGZ group (p <0.05). Levels of MDA reduced significantly in eachH2-treated group compared with T2DM rats (p <0.01), and even lower significantly inmiddle and high dose H2group compared with the RGZ-treated group (p <0.05, p <0.01).
Conclusion:
1. Neuroprotective effects of compound H2in vitro
Western blot results showed that the expression level of AR and HIF-1, which arerelated to glucose metabolism, increased markedly in neurons upon the stimulation withhigh glucose for24h, as well as apoptosis-related protein Bax. While, compared withcontrol, the levels of pro-caspase-3were decreased, indicating that high glucose led toabnormal glucose metabolism of neurons followed by early neuronal injury. The syntheticcompounds H2, H4and H6decreased the expression of AR, HIF-1and Bax, therebyinhibiting the neuronal injury induced by high glucose. These results suggested thatsynthetic H2, H4and H6rescued abnormal glucose metabolism mediated by high glucose.Among these compounds, H2was the most effective one for neuroprotection.
There were some morphological changes in neurons characterized as discontinuousand break-like neurite by cytoskeletal protein MAP2staining after treatment with highglucose. Treatment with compound H2for24h after high glucose injury, the neurite morphology was rescued to normal, implicating that synthetic H2may play aneuroprotective role.
There was no significant apoptosis in neurons upon high glucose stimulation for24has well as in other different treatment groups. It is possible that neuronal injury is along-term process in diabetic patients, therefore, flow cytometry method was not sensitiveto evaluate the neuroprotective effects of these compounds in such a short-time treatment.
2. The pharmacokinetics research of compound H2
In the present study, H2content in the rat brain was1.57μg/g after administration for15min, and H2in plasma was26.4mg/mL at this corresponding time point, implicatingthat H2passed through the blood-brain barrier. It has been reported by other group that theconcentration of rosiglitazone, the positive control drug, in the cerebrospinal fluid wasonly0.0045%of that in blood. We failed to detect rosiglitazone in brain tissue other thanin blood using the same method in the present study, maybe due to rosiglitazoneconcentrations was below the detection line. Detailed reasons needs to be furtherinvestigated. Taken together, we identified the ability of the compond H2passing throughblood-brain barrier into the brain tissue, which was more effective than rosiglitazone.
3. The mechanisms of compound H2in the treatment of diabetes brain dysfunction
Evidence showed that T2DM develops cognitive deterioration due to the centralnervous system (CNS) injury under high blood glucose. Morris water maze is a widelyused in the assessment of rodent learning and memory, objectively and accurately. Theescape latency and number of crossing the platform were used to evaluate the cognitivefunction of the animals. In this study, H2treatment improved the cognitive dysfunction ofT2DM rats, which was better than the positive control RGZ. The preliminary mechanismsof H2protection were further explored by detection of inflammatory andanti-inflammatory cytokines in blood, antioxidant system in brain. The results showed thatH2treatment inhibited IL-1β and TNF-expression, and promoted the anti-inflammatorycytokine IL-10expression, thereby reducing autoimmune and chronic inflammationreaction in diabetes. In addition, the activities of SOD, CAT and GSH-Px increased andMDA decreased significantly in H2-treated brains. Furthermore the improvement of H2at the medium and high dose was much better than RGZ-treated rats. All these datasuggested that H2could improve brain enzyme activity of antioxidant system to clear avariety of reactive oxygen species which is harmful to the health, as well as to reduceMDA, a fat oxidation toxic substance, to play a neuroprotective effect.
It is reported by another group that the relief of anxiety is positive to the therapy ofdiabetic patients. Then we observed whether H2had potential therapeutic effects onanxiety. The results of EPM showed that there was no significant acute anxiolytic effect ofH2, and chronic anxiolytic effects need to be further elucidated.
4. Prevention against cataract by H2
Aged diabetic patients are often complicated by Ophthalmopathy. This phenomenonwas also confirmed in this study. We found visible lens opacification in T2DM rats ofeach group. H2treatment decreased the incidence rate and ameliorated significantlyseverity of opacification compared with T2DM rats. This data indicated that compoundH2might have potential therapeutic effects on cataract development.
In summary, compound H2showed similar neuroprotective effects in vitro as RGZ.However, H2had greater efficacy of potential therapeutic effects on diabeticencephalopathy in T2DM rats as compared to RGZ.
糖尿病脑病（Diabetes encephalopathy, DE）是糖尿病（Diabetes mellitus, DM）严重并发症之一，损害患者的认知和记忆。传统治疗采用噻唑烷二酮（Thiazolidinedione, TZD）类过氧化物酶体增殖物激活受体（Perioxisomeproliferator-activated receptor-γ, PPAR-γ）激动剂控制血糖，辅以改善脑细胞功能的药物，目前尚无特效治疗药物。TZD类药控制血糖作用良好，但因脂溶性低，难以通过血脑屏障（Blood brain barrier, BBB），防治神经损伤作用有限。由此，研发降糖效果好、能顺利通过血脑屏障的药物将填补这一空白，具有非常重要的临床意义。
目的：
烷基取代的苯并咪唑基团亲水性低脂溶性高，生物相容性好。为此，我们将烷基取代的苯并咪唑基团与TZD基团连接，合成含苯并咪唑基的TZD类PPAR-γ激动剂系列化合物H2、H4和H6；采用体外高糖损伤神经元模型，结合细胞免疫荧光、蛋白免疫印迹和流式细胞术，筛选出神经保护作用药效最好的化合物H2；初步药代动力学结果提示，化合物H2透过BBB；建立2型糖尿病（Type2Diabetes Mellitus，T2DM）大鼠模型，进一步考察化合物H2对糖尿病脑病的治疗作用。结果显示H2显著提高大鼠生存率、降低血糖，同时，H2明显改善大鼠的学习、记忆行为；生化指标提示，化合物H2神经保护作用可能是通过降低血中炎性因子白细胞介素1β（Interleukin-1β, IL-1β）、肿瘤坏死因子-（Tumor necrosis factor-, TNF-）和脑内丙二醛（Malondialdehyde, MDA）水平，以及提高血中抗炎因子IL-10和脑内抗氧化物质超氧化物歧化酶（Superoxide dismutase, SOD）、过氧化氢酶（Catalase, CAT）和谷胱甘肽过氧化物酶（Glutathione peroxidase, GSH-Px）水平发挥作用。本研究为发掘新型糖尿病脑病治疗药物初步奠定了基础。
方法：
一、新型含苯并咪唑基的TZD类化合物H2、H4和H6的合成
1．酸性条件下，取代的邻氨基苯与1-氯直链羧酸缩合得到取代苯并咪唑缩合产物；
2.4-羟基苯甲醛与2,4-噻唑烷二酮反应生成5-(4-羟基苯亚甲基)噻唑烷-2,4-二酮（H7），再经硼氢化钠还原得到中间体5-(4-羟基苯甲基)噻唑烷-2,4-二酮（H8）；
3.合成苯并咪唑中间体2-氯甲基-1H-苯并咪唑（H1）、2-(氯甲基)-5-甲基-1H-苯并咪唑（H3）、2-(氯甲基)-1-甲基-1H-苯并咪唑（H5）；
4.苯并咪唑中间体H1、H3、H5与噻唑烷二酮中间体H8缩合得到目标化合物H2、H4、H6；
二、新型含苯并咪唑基的TZD类化合物H2、H4和H6的体外神经保护作用研究
1.蛋白免疫印迹实验
培养的神经元高糖（30mM）刺激24h后，给予不同浓度（0.1、1.0、10μM）化合物H2、H4、H6继续作用24h。Western blot方法检测与糖尿病病理进展、神经毒性相关的蛋白醛糖还原酶（Aldose reductase，AR）、缺氧诱导因子-1（Hypoxiainducible factor-1，HIF-1）、Bax，Pro-caspase-3等在神经元表达的变化，同时设立等渗透压的甘露醇（Mannitol）及母核药物罗格列酮（Rosiglitazone, RGZ）作为对照。
2.细胞免疫荧光实验
培养的神经元高糖（30mM）刺激24h后，给予不同浓度（0.1、1.0、10μM）化合物H2继续作用24h，同时设立等渗透压的甘露醇及母核药物RGZ作为对照。细胞免疫荧光染色神经元骨架蛋白MAP2（Microtubule-associated protein2），观察合成化合物对神经元形态学的影响。
3.流式细胞实验
培养的神经元高糖（30mM）刺激24h后，给予不同浓度（0.1、1.0、10μM）化合物H2作用24h，同时设立等渗透压的甘露醇及母核药物RGZ作为对照。流式细胞术检测合成化合物H2对神经元凋亡的影响。
三、化合物H2药代动力学研究
1．用反相高效液相色谱法，流动相为0.01mol/L NH4Ac：CH3OH（35:65，V/V），流速1mL/min，检测波长247nm，RGZ为内标。大鼠腹腔注射25mg/kg化合物H2，于给药后0、0.083、0.25、0.5、1、1.5、2、3、4h眼眶采血，高效液相色谱（High performanceliquid chromatography，HPLC）检测血中H2浓度，确定H2在SD大鼠血浆中的药代动力学参数。
2.根据预实验结果，大鼠腹腔注射25mg/kg化合物H2后0.25h，取脑组织，制作脑组织匀浆，HPLC法检测脑中H2的浓度。
四、化合物H2对糖尿病脑病的治疗作用
1．建立T2DM模型和急性焦虑模型
T2DM模型：高糖高脂饲料喂养SD大鼠4周，诱导发生胰岛素抵抗，再腹腔注射链脲佐菌素（Streptozotocin，STZ）30mg/kg维持高糖高脂饮食8周，即T2DM模型。
急性焦虑模型：SD大鼠束缚2h/d，连续2次，建立急性焦虑模型。
2．Morris水迷宫实验
将造模成功的T2DM大鼠分为T2DM组，低、中、高剂量H2治疗组（1mg/kg，3mg/kg，10mg/kg）和RGZ治疗组（3mg/kg），动物给予药物治疗4周，正常组给予相同体积生理盐水。Morris水迷宫实验以逃避潜伏期和穿越平台次数考察H2对T2DM大鼠学习记忆的保护作用。
3．高架十字迷宫实验
SD大鼠分为正常组、H2（3mg/kg）组和焦虑组。将大鼠面朝开臂放在中央区域，迷宫上方采用视频记录并分析5min内大鼠进入开臂或闭臂（四爪全部入臂）的次数与时间。以进入开臂的次数和滞留时间作为衡量焦虑的指标，总入臂次数则用于自主活动的检测。
4．生化指标检测
水迷宫实验结束后，SD大鼠心脏取血并取脑组织制作脑组织匀浆，用南京建成试剂盒测定血浆IL-1β、TNF-和IL-10及脑皮质匀浆SOD、CAT、GSH-Px和MDA的水平。
结果：
一、H2、H4和H6的合成
合成得到3个目标化合物H2、H4、H6，并通过1H NMR和MS鉴定其结构。另外，合成过程中还得到中间体H5及副产物H55的晶体，并用X-射线表征其结构。
二、新型含苯并咪唑基的TZD类化合物H2、H4和H6的体外神经保护作用研究
1.化合物H2、H4和H6对高糖培养神经元细胞的影响
Western blot结果显示高糖可升高AR、HIF-1、Bax，降低Pro-caspase-3在神经元的表达；与正常对照组比较，高糖组神经元AR、HIF-1、Bax和Pro-caspase-3表达有显著差异；为排除渗透压对培养体系的影响，同时设置等渗透压即30mM甘露醇作为对照，结果显示，甘露醇对神经元中AR、HIF-1、Bax和Pro-caspase-3的表达没有影响，与正常对照组相当，排除渗透压对神经元细胞生长状态的影响。化合物H2、H4、H6可以逆转高糖培养神经元AR、HIF-1、Bax和Pro-caspase-3的表达水平，且H2效果最佳。
2.化合物H2对神经元细胞形态的影响
高糖处理神经元24h，可造成明显的细胞骨架形态改变，表现为骨架蛋白MAP2染色神经元突起不连续，成断线状改变。给予化合物H2处理24h则神经元突起形态基本恢复正常。
3.化合物H2对神经元细胞凋亡的影响
高糖损伤24h未引起显著的神经细胞凋亡，各治疗组亦未见细胞凋亡；可能高糖条件不能诱导细胞凋亡，流式细胞术检测高糖引起的神经元凋亡不是敏感指标，无法评价H2的神经保护作用。
三、化合物H2药代动力学研究
本研究建立一种简单、安全、经济的方法检测血和脑组织中H2的HPLC-UV方法，分析结果准确、灵敏。大鼠腹腔注射给药25mg/kg时，AUC(0-)为（14.58±1.45）mg/L*h，Tmax为（0.17±0）h，CLz/F为（1.73±0.17）L/h/kg，t1/2为（0.73±0.075）h，Cmax为（26.39±1.02）μg/mL。H2能透过血脑屏障，给药后0.0833h脑中浓度为1.57ng/g。
四、化合物H2对糖尿病脑功能异常的治疗作用
1．一般指标观察
T2DM大鼠造模成功后体重持续下降，且出现多饮、多尿、多食等症状，空腹血糖>7.8mmol/L。正常组大鼠体重持续增长，毛皮及精神状态良好。
给予药物治疗4周后，与T2DM组比较，低、中、高剂量H2组和RGZ组大鼠空腹血糖显著下降（p <0.01）；T2DM组和RGZ组大鼠生存率分别为67.5%和75%，低、中、高剂量H2组大鼠全部存活，明显提高了T2DM大鼠生存率。
2．Morris水迷宫结果
药物治疗4周后，与正常组大鼠比较，T2DM组大鼠逃避潜伏期显著升高（p <0.01），提示T2DM大鼠出现认知功能障碍。药物治疗组中，与T2DM组比较，低、中、高剂量H2组及RGZ组逃避潜伏期显著减小（p <0.05、p <0.01、p <0.01和p <0.05）。高剂量H2组逃避潜伏期显著小于RGZ组（p＜0.05）。
在d6撤去平台后，与正常组比较，T2DM模型组大鼠跨越平台次数显著减少（p<0.01）。药物治疗组中，中、高剂量H2组比RGZ组跨越平台次数显著增加（p <0.05，p <0.01）。
3．H2对T2DM大鼠白内障的预防
T2DM大鼠治疗过程中，观察到各组T2DM大鼠不同程度出现肉眼可见的晶状体浑浊。根据本校第二附属医院眼科晶状体分级系统，对大鼠晶状体的浑浊程度进行分级。与正常对照组比较，T2DM组晶状体浑浊显著（p <0.01）；与T2DM组比较，低、中、高剂量H2治疗组晶状体浑浊程度显著减轻（p <0.01）；与RGZ组比较，中、高剂量H2治疗组晶状体浑浊程度显著减轻（p <0.01）。
4．高架十字实验
焦虑模型组大鼠在开臂内滞留时间与空白对照组比较具有显著性差异（p <0.05），说明急性焦虑模型成功。但H2治疗组与焦虑模型组在进入开臂的次数和滞留时间上未见显著性差异。
5．生化指标检测
大鼠血浆炎症因子IL-1β、TNF-和抗炎因子IL-10水平：与正常组比较，T2DM组IL-1β和TNF-显著升高（p <0.05，p <0.01），IL-10显著降低（p <0.01）；与T2DM组大鼠比较，低、中、高剂量H2组IL-1β水平有显著降低（p <0.05，p <0.01，p <0.01）；与T2DM组大鼠比较，中、高剂量H2组TNF-水平有显著降低（p <0.05，p <0.01），其中高剂量H2组与RGZ比较有显著降低（p <0.05）；与T2DM组大鼠比较，低、中、高剂量H2组IL-10水平均有显著升高（p <0.05，p <0.05，p <0.01），其中高剂量H2组与RGZ组比较显著升高（p <0.05）。
大鼠脑匀浆SOD、CAT、GSH-Px和MDA水平：与正常组比较，T2DM组SOD、CAT和GSH-Px水平显著降低（p <0.01），MDA水平显著升高（p <0.01）；与T2DM组大鼠比较，低、中、高剂量H2组SOD水平显著升高（p <0.05，p <0.01，p <0.05），且与RGZ组比较有显著升高（p <0.05，p <0.01，p <0.05）；与T2DM组大鼠比较，中、高剂量H2组CAT水平显著升高（p <0.05），且与RGZ组比较有显著升高（p <0.05）；与T2DM组大鼠比较，低、中、高剂量H2组GSH-Px水平显著升高（p <0.05），且高剂量H2组与RGZ组比较有显著升高（p <0.05）；与T2DM组大鼠比较，低、中、高剂量H2组MDA水平均有显著降低（p <0.01），且中、高剂量H2组与RGZ组比较有显著降低（p <0.05，p <0.01）。
结论：
1．体外神经保护作用
Western blot结果表明，高糖损伤24h后，糖代谢相关蛋白AR、HIF-1表达水平增高；凋亡相关蛋白Bax的表达水平增高，Pro-Caspase-3蛋白的表达水平降低，表明高糖造成神经元糖代谢异常，引起神经元的早期损伤。合成化合物H2、H4和H6降低AR、HIF-1和Bax蛋白的表达，从而抑制了神经元的损伤。这些结果表明，化合物H2、H4和H6能抑制高糖引起的糖代谢异常，对高糖损伤的神经元有一定的保护作用，其中H2药效强于H4和H6。
高糖损伤24h可造成神经元骨架形态的改变，表现为骨架蛋白MAP2染色神经元突起不连续，成断线状。合成药H2治疗后神经元突起形态基本恢复正常，发挥神经保护作用。
高糖处理神经元24h，细胞凋亡不显著，这可能由于糖尿病引起的神经损伤是一长期过程，体外高糖作用时间较短，因而流式细胞术检测高糖引起的神经元凋亡不是敏感指标。
2．化合物H2药代动力学研究
SD大鼠腹腔给药后15min脑中H2含量为1.57μg/g，此时间点血浆中浓度为26.4μg/mL，说明H2能够通过血脑屏障。有文献报道，脑脊液中母核药物RGZ浓度仅为血中的0.0045%[1]。我们的检测体系未能检测到脑组织中的RGZ，可能是由于脑内RGZ浓度低于检测基线。具体原因还有待进一步考证。相同方法下，脑组织中能检测到H2而不能检测到RGZ，提示H2通过血脑屏障的能力要强于RGZ。
3．化合物H2对糖尿病脑功能障碍的治疗作用
大量研究结果显示，T2DM患者出现学习记忆、认知功能障碍等中枢神经系统（Central nervous system, CNS）损害的临床症状。Morris水迷宫实验是研究啮齿类动物学习、记忆能力的公认模型，能客观、准确地评价动物的认知功能。通过考察动物的逃避潜伏期、穿越平台次数等主要指标来衡量动物的学习、记忆功能。本实验两项指标的结果提示，H2能改善T2DM大鼠的认知功能障碍，且效果优于阳性对照药RGZ。通过检测T2DM大鼠血浆和脑匀浆相关生化指标发现，H2抑制炎症相关因子IL-1β和TNF-的表达，提高抗炎性细胞因子IL-10的表达，从而减轻糖尿病时自身免疫性和慢性炎症反应，发挥治疗作用。另外，H2治疗后脑中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）活性提高以及脂肪氧化终产物丙二醛（MDA）水平降低，且中、高剂量药效要优于RGZ。该结果提示H2能改善脑内酶抗氧化系统的活性，提高机体的抗氧化能力，清除各种活性氧对机体的损伤，减少脂肪氧化产生毒性物质（MDA），发挥神经保护作用。
另有研究表明，焦虑情绪的缓解对糖尿病患者的治疗有积极意义[2]。我们考察H2对焦虑是否具有潜在的治疗意义，结果提示H2对急性焦虑没有明显作用，对于慢性焦虑的作用还有待研究。
4．对白内障的预防作用
糖尿病患者晚期往往眼部病变。实验中，我们观察到T2DM大鼠先后出现白内障， H2治疗组白内障的发生率及严重程度均显著下降。此结果提示，化合物H2在T2DM大鼠模型白内障形成及预防方面的潜在治疗价值。
综上所述，离体实验中化合物H2与罗格列酮对高糖介导的神经元损伤具有相似的保护作用；T2DM大鼠的治疗中，合成化合物H2的药效要优于RGZ。
Backgroud:
Diabetic encephalopathy (DE), one of the serious complications of Diabetes Mellitus(DM), impaired cognition and memory of DM patients. Traditional treatment for DE is thecombination of thiazolidinedione (TZD), peroxisome proliferator-activated receptor(PPAR-γ) agonists to control blood glucose, with drugs to improve the function of braincells; however, there is still no specific medicine for DE therapy. Although the TZD drugsis effective to control blood glucose, the roles in prevention and treatment of neural injuryof DM patients is very limited, because TZD is difficult to pass the blood-brain barrier(BBB) due to its low liposolubility. Therefore, it has a very important clinical significanceto fill this gap to develop a therapeutic medicine for DE treatment, and this medicineshould have hypoglycemic effect as well as high liposolubility to pass the blood-brainbarrier.
Aims：
Alkyl-substituted benzimidazole group has low hydrophilic, high liposolubility and good biocompatibility. To this end, we synthesized benzimidazole derivative named ascompounds H2, H4and H6, by conjuncting the alkyl-substituted benzimidazole group toTZD group. The evaluation of these compounds on neural protection were carried outusing a neuron-injury model induced by high glucose stimulation in vitro, combined withimmunofluorescence staining, Western blot and flow cytometry methods. Among them,compound H2was identified to have the best neuroprotective effects. The preliminarypharmacokinetic results suggested that compound H2could pass the BBB of rats. Inaddition, the therapeutic effects of compound H2for diabetic encephalopathy wereinvestigated using a type2diabetes (T2DM) rat model. The results showed that H2significantly promoted the survival rate, decreased the level of blood glucose, andimproved learning and memory behaviors of T2DM rats. The underlying mechanisms ofH2neuroprotection were determined by biochemical markers related to inflammation andantioxidant system. We found that the levels of inflammatory cytokines IL-1β and TNF-decreased, while anti-inflammatory cytokine IL-10increased in the blood in H2-treatedT2DM rats. Furthermore, H2decreased the level of malondialdehyde (MDA), improvedthe antioxidant substances superoxide dismutase (SOD), catalase (CAT) and glutathioneperoxidase (GSH-Px) in the T2DM rats brain. This study provides preliminary basis toexplore a new candidate for the diabetes encephalopathy treatment.
Methods:
1. Synthesis of new benzimidazole derivative componds H2, H4and H6
1) Under acidic conditions, benzimidazoles were obtained by the condensationreaction using substituted anthranilic benzene and1-chloro-straight-chain carboxylic acid;
2)5-methylenebis (4-hydroxyphenyl) thiophene-2,4-dione (H7) was harvestedthrough the reaction of4-hydroxybenzaldehyde with2,4-thiazolidine dione, then theintermediate5-(4-hydroxybenzyl) thiazolidine-2,4-dione (H8) was obtained by sodiumborohydride reduction;
3) Synthesis of benzimidazole intermediates2-chloro-methyl-1H-benzimidazole (H1),2-(chloromethyl)-5-methyl-1H-benzimidazole (H3),2-(chloro-methyl yl)-1-methyl-1H-benzimidazole (H5);
4) Condensation reatcion of benzimidazole intermediates H1, H3and H5withthiazolidinedione intermediates H8to form the target compounds H2, H4, H6;
2. The evaluation of the neuroprotective effects of new TZD derivative, compondsH2, H4and H6, in vitro
1) Western blot analysis
The neuroprotective effects of compounds H2, H4and H6were investigated in thecultured cortical neurons. The neurons were stimulated with high glucose (30mM) for24h, followed by treatment with compounds H2, H4and H6at different concentrations(0.1,1.0,10μM) for another24h. Western blot analysis were used to detect the changes ofexpressions levels of aldose reductase (AR), hypoxia-inducible factor1-alpha (HIF-1),Bax and pro-caspase-3, which are related to the pathological progress of diabetes andneurotoxicity. Mannitol was used as the osmotic pressure control and prosome structurerosiglitazone (RGZ) was used as positive control.
2) Immunofluorescence experiments
The cultured mature neurons were stimulated with high glucose (30mM) for24h,followed by treatment with compound H2at different concentrations (0.1,1.0,10μM) foranother24h. The effects of H2on neuron morphology after treatment were checked byimmunofluorescence staining of neuronal cytoskeletal proteins microtubule-associatedprotein-2(MAP2). Mannitol and RGZ weres used as control.
3) Flow cytometry experiments
The effects of H2on neuron apoptosis were determined by flow cytometry afterneurons were stimulated with30mM glucose for24h followed by H2treatment at0.1,1.0,10μM for another24h. Mannitol and RGZ were both used as control.
3. The pharmacokinetic study of compound H2
1) Reversed-phase liquid chromatography was used to detect the concentration of H2in samples, and the working system was used as following: mobile phase0.01mol/LNH4Ac: CH3OH (35:65, V/V), flow rate1mL/min, detection wavelength247nm. RGZwas used as the internal standard. Compound H2was adimistrated to normal rats byintraperitoneal injection at25mg/kg. Orbital blood was collected at0,0.083,0.25,0.5,1.0, 1.5,2.0,3.0and4.0h after H2administration, and the H2concentrations in blood weredetermined by HPLC assay, therefore, the pharmacokinetic parameters of H2in SD rats’plasma were obtained.
2) Brain tissues were harvested after intraperitoneal injection of H2(25mg/kg) for0.25h and homogenized according to the preliminary experiments, HPLC assay was usedto detect H2concentrations in the brain tissue.
4. The therapeutic effects of compound H2for diabetic encephalopathy treatment
1) Establishment of T2DM and acute anxiety models
To establish type2diabetes (T2DM) and rat insulin resistance model, SD rats werefed continously with high sugar and fat diet for4weeks, then streptozotocin (STZ) wasadministrated to rats by intraperitoneal injection at30mg/kg, and the same diet wasmaintained for another8weeks to get T2DM model.
To induced acute anxiety model, SD rats were constrainted in well-ventilated Perspexrestraining tubes for2h for two consecutive days.
2) Morris water maze (MWM) test
To check the therapeutic effects of H2, T2DM rats were divided into DM controlgroup, H2-treated group (1mg/kg,3mg/kg,10mg/kg) and RGZ-treated group (3mg/kg)besides the normal control group, the animals were administrated with H2or RGZintragastricly for4weeks. Same volume saline was used as control. The effects of H2onspatial learning and memory were evaluated by the Morris water maze test, and the escapelatency and the number of crossing the platform were collected for analysis.
3) Elevated plus maze (EPM) experiment
The elevated plus maze (EPM) was conducted to evaluated the potential side effectsof compound H2. SD rats were randomly divided into control group, H2-treated group (3mg/kg) and acute anxiety group. Individual animals were placed in the centre square,facing an open arm, and allowed to move freely for5min. Rats were videotaped using acamera fixed above the maze and analyzed with a video-tracking system. Open and closedarm entries (all four paws in an arm) and times were recorded. The number of entriesand time spent in open arms were used to measure anxiety degree, and the number of total entries in four arms was used for the detection of locomotor activity.
4) Detection of the biochemical markers
After the MWM test was finished, blood and brain tissue were harvested immediately.The concentrations of IL-1β, TNF-and IL-10in plasma, the activities of SOD, CAT,GSH-Px and MDA levels in brain homogenates were determined by kits purchased fromNanjing Jiancheng company.
Results:
1. Synthesis of componds H2, H4and H6
Three target compounds of H2, H4, H6were synthesized, and their structures wereidentified by1H NMR and MS. In addition, single crystal of the intermediate H5andbyproduct H55were obtained during the synthesis process, and their structures werecharacterized by X-ray.
2. The neuroprotective effects of new TZD derivative, componds H2, H4and H6, invitro
1) Effects of compounds H2, H4and H6on neurons stimulated with high glucose
Western blot results showed that the expression levels of AR, HIF-1and Bax incultured neurons increased markedly upon high glucose stimulation, while the level ofpro-caspase-3reduced, compared with normal controls. Treatment with compounds H2,H4and H6revesed the expression levels of AR, HIF-1, Bax and Pro-caspase-3to somedegree.30mM mannitol was set as a osmotic control to exclude the influence of osmoticpressure in culture system at the same time. The results showed that mannitol had noeffects on AR, HIF-1, Bax and Pro-caspase-3expression, similar to the results from thenormal control group. Therefore, the impact of osmotic pressure on the growth state of theneurons was excluded.
2) Effects of compound H2on neuron morphology
The morphological changes of the cytoskeleton in cultured neurons were observedafter30mM glucose stimulation for24h, which was characterized as discontinuous andbreak-like neurite by cytoskeletal protein MAP2staining. Treatment with compound H2for24h, the neurite morphology was rescued to normal.
3) Effects of compound H2on neuron apoptosisThere was no significant apoptosis in neurons upon high glucose stimulation for24h aswell as in other treated groups. It is possibile that early and acute apoptosis can not beinduced under high glucose condition in short time. Therefore, flow cytometry methodwas not sensitive to evaluate the neuroprotective effects of synthetic compounds in theearly phase.
3. The pharmacokinetic study of compound H2
A simple, safe and economical way was established in this study to detect theconcentrations of H2in the plasma and brain tissue using HPLC-UV method. Accurateand sensitive data were obtained for pharmacokinetic analysis. Administration of H2at25mg/kg(i.p), the parameters were obtained as following: the AUC (0-)14.581.45mg/L*h, Tmax0.17±0h, CLz/F1.73±0.17L/h/kg, t1/20.73±0.075h, Cmax26.39±1.02g/mL.The concentration of H2in brain after administration for0.0833h was1.57ng/g, and thisresult implicated that H2passed through the blood-brain barrier.
4. The therapeutic effects of compound H2on the diabetic brain functions
1) General observation
Type2diabetic rats showed a continued decline in weight, accompanied withpolydipsia, polyuria and polyphagia which was observed in diabetes patients, and thefasting blood glucose was more than7.8mmol/L. The weight of normal rats continued togrow and they survived healthily with smooth and glossy fur and in good mental status.The blood glucose decreased significantly in each treatment of H2and RGZ, comparedwith the T2DM group (p <0.01).
2) Treatment of H2rescued deficits of learning and memory in T2DM rats
The T2DM rats had impaired learning using the available visuospatial cues to locatethe submerged escape platform, as indicated by prolonged escape latency acrossconsecutive trials compared with the normal group (p <0.01), suggesting that T2DM ratsdevelops cognitive deterioration with increasing age. After medication of H2or RGZ for4weeks, the reduced escape latency across the trials of rats in H2groups (low, medium andhigh dose) and RGZ group were observed compared with the control T2DM group (p < 0.05, p <0.01, p <0.01and p <0.05). Furthermore, we confirmed the escape latency ofrats in high-dose H2group was significantly reduced compared with the RGZ-treated rats(p <0.05).
The platform in the water maze was removed at day6, the numbers across theplatform in T2DM reduced markedly compared with the normal rats (p <0.01). Thenumbers across the platform increased significantly in H2-treated groups (medium andhigh dose), compared with RGZ-treated group (p <0.05, p <0.01).
3) H2treatment prevented cataract development in T2DM rats
There was visible lens opacification observed in T2DM rats from each group in thelate phase of the DM models. According to the lens grading system, the lens opacificationof the rats were graded. The worst grade of lens opacification happened in T2DM ratscompared with normal control and H2-treated rats (p <0.01). H2treatment (low, mediumand high dose) ameliorated significantly the severity of opacification compared withT2DM rats (p <0.05), and this effect was not observed in RGZ-treated group, implicatingthat H2treatment effectively prevent cataract development in diabetic rats.
4) Elevated plus maze experiment
The acute anxiety rat model was established by restraint rats for two consecutive days,and the time spent in open arms increased compared with control rats (p <0.05). While,there was no significant difference of the number of entries and time spent in open armbetween H2-treated group and anxiety model group.
5) Biochemical markers
We first checked the levels of inflammatory cytokines IL-1β and TNF-, andanti-inflammatory cytokine IL-10in rat plasma at the end of experiments. The levels ofIL-1β and TNF-in T2DM group were increased significantly compared with normalgroup (p <0.05, p <0.01), while levels of IL-10were decreased significantly (p <0.01).Compared with T2DM rats, levels of IL-1β decreased significantly in each of H2-treatedgroup (p <0.05). The levels of TNF-were significantly decreased in H2-treated group atboth middle and high dose compared with the T2DM group (p <0.05, p <0.01). Levels ofTNF-decreased markedly in high-dose H2group compared with RGZ-treated group (p < 0.05). The levels of IL-10in each H2-treated group were significantly increased comparedwith T2DM rats (p <0.05, p <0.05, p <0.01). Furthermore, the levels of IL-10were evenhigher in high-dose H2group than in RGZ-treated group (p <0.05).
We next checked the activities of SOD, CAT and GSH-Px and the levels of MDA inbrain homogenate. The activities of SOD, CAT and GSH-Px decreased significantly inT2DM group compared with normal group (p <0.01). SOD acitvities increasedsignificantly in each H2-treated group compared with both T2DM group and RGZ-treatedgroup (p <0.05, p <0.01, p <0.05). The levels of CAT were significantly increased inmedium and high dose H2group compared with both T2DM and RGZ-treated group (p <0.05). Levels of GSH-Px activity in each H2-treated group were significantly highercompared with T2DM rats (p <0.05), and was significantly higher in high-dose H2groupcompared with RGZ group (p <0.05). Levels of MDA reduced significantly in eachH2-treated group compared with T2DM rats (p <0.01), and even lower significantly inmiddle and high dose H2group compared with the RGZ-treated group (p <0.05, p <0.01).
Conclusion:
1. Neuroprotective effects of compound H2in vitro
Western blot results showed that the expression level of AR and HIF-1, which arerelated to glucose metabolism, increased markedly in neurons upon the stimulation withhigh glucose for24h, as well as apoptosis-related protein Bax. While, compared withcontrol, the levels of pro-caspase-3were decreased, indicating that high glucose led toabnormal glucose metabolism of neurons followed by early neuronal injury. The syntheticcompounds H2, H4and H6decreased the expression of AR, HIF-1and Bax, therebyinhibiting the neuronal injury induced by high glucose. These results suggested thatsynthetic H2, H4and H6rescued abnormal glucose metabolism mediated by high glucose.Among these compounds, H2was the most effective one for neuroprotection.
There were some morphological changes in neurons characterized as discontinuousand break-like neurite by cytoskeletal protein MAP2staining after treatment with highglucose. Treatment with compound H2for24h after high glucose injury, the neurite morphology was rescued to normal, implicating that synthetic H2may play aneuroprotective role.
There was no significant apoptosis in neurons upon high glucose stimulation for24has well as in other different treatment groups. It is possible that neuronal injury is along-term process in diabetic patients, therefore, flow cytometry method was not sensitiveto evaluate the neuroprotective effects of these compounds in such a short-time treatment.
2. The pharmacokinetics research of compound H2
In the present study, H2content in the rat brain was1.57μg/g after administration for15min, and H2in plasma was26.4mg/mL at this corresponding time point, implicatingthat H2passed through the blood-brain barrier. It has been reported by other group that theconcentration of rosiglitazone, the positive control drug, in the cerebrospinal fluid wasonly0.0045%of that in blood. We failed to detect rosiglitazone in brain tissue other thanin blood using the same method in the present study, maybe due to rosiglitazoneconcentrations was below the detection line. Detailed reasons needs to be furtherinvestigated. Taken together, we identified the ability of the compond H2passing throughblood-brain barrier into the brain tissue, which was more effective than rosiglitazone.
3. The mechanisms of compound H2in the treatment of diabetes brain dysfunction
Evidence showed that T2DM develops cognitive deterioration due to the centralnervous system (CNS) injury under high blood glucose. Morris water maze is a widelyused in the assessment of rodent learning and memory, objectively and accurately. Theescape latency and number of crossing the platform were used to evaluate the cognitivefunction of the animals. In this study, H2treatment improved the cognitive dysfunction ofT2DM rats, which was better than the positive control RGZ. The preliminary mechanismsof H2protection were further explored by detection of inflammatory andanti-inflammatory cytokines in blood, antioxidant system in brain. The results showed thatH2treatment inhibited IL-1β and TNF-expression, and promoted the anti-inflammatorycytokine IL-10expression, thereby reducing autoimmune and chronic inflammationreaction in diabetes. In addition, the activities of SOD, CAT and GSH-Px increased andMDA decreased significantly in H2-treated brains. Furthermore the improvement of H2at the medium and high dose was much better than RGZ-treated rats. All these datasuggested that H2could improve brain enzyme activity of antioxidant system to clear avariety of reactive oxygen species which is harmful to the health, as well as to reduceMDA, a fat oxidation toxic substance, to play a neuroprotective effect.
It is reported by another group that the relief of anxiety is positive to the therapy ofdiabetic patients. Then we observed whether H2had potential therapeutic effects onanxiety. The results of EPM showed that there was no significant acute anxiolytic effect ofH2, and chronic anxiolytic effects need to be further elucidated.
4. Prevention against cataract by H2
Aged diabetic patients are often complicated by Ophthalmopathy. This phenomenonwas also confirmed in this study. We found visible lens opacification in T2DM rats ofeach group. H2treatment decreased the incidence rate and ameliorated significantlyseverity of opacification compared with T2DM rats. This data indicated that compoundH2might have potential therapeutic effects on cataract development.
In summary, compound H2showed similar neuroprotective effects in vitro as RGZ.However, H2had greater efficacy of potential therapeutic effects on diabeticencephalopathy in T2DM rats as compared to RGZ.
引文
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