益气解毒化瘀法抗肾小球硬化实验研究
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摘要
肾小球硬化是慢性肾衰的主要病理基础，也是各种肾小球疾病进行性进展的最终结局，临床上表现为慢性肾功能衰竭。其主要病理特征为系膜细胞(MC)增殖和细胞外基质(ECM)的过度积聚。目前西医在肾小球硬化的发病机理方面做了许多深入的研究工作，但是对于在肾小球硬化的治疗，保护肾脏，控制慢性肾功能衰竭的进展方面的研究，报道较少，最近几年，中药的复方、单味草药及中药提取物在治疗肾小球硬化，保护肾脏，延缓肾功能衰竭进展，取得了许多新进展，因此对于中药治疗肾小球硬化，保护肾脏，值得深入研究。
本实验是在前期课题“愈肾颗粒拆方重组对大鼠肾小球系膜细胞的影响研究”的基础上开展的，前期课题研究，按照中医病机和组方原则为依据进行拆方，将原处方按益气化瘀解毒、益气化瘀、益气解毒的组方原则，经试验研究，初步结论复方1号组黄芪，丹参，白花蛇舌草组更具有显著抑制肾小球系膜细胞增殖，降低细胞外基质合成，防治肾小球硬化的功效。本实验即针对该益气解毒化瘀小复方（黄芪，丹参，白花蛇舌草），开展进一步研究，观察益气解毒化瘀小复方及其单味药组干预肾小球硬化的作用及作用机制，并探讨益气解毒化瘀同治对提高防治肾小球硬化的理论意义和实用价值，为在临床方面防治肾小球硬化，提供理论与方法依据，为下一步的深入科学研究中药防治肾小球硬化打下基础。
实验一：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞增殖及凋亡影响的研究
目的：
探讨益气解毒化瘀小复方及其单味药对血管紧张素Ⅱ诱导的大鼠肾小球系膜细胞增殖，凋亡的影响。
材料与方法：
1.主要材料：Wistar大鼠60只，体重（200±20）g；大鼠肾小球系膜细胞；中药材：小复方由黄芪，丹参、白花蛇舌草组成，单味药组分别为黄芪，丹参，白花蛇舌草。西药对照组：替米沙坦；DMEM培养基干粉、标准胎牛血清、血管紧张素Ⅱ、MTT、AnnexinV-FITC、台盼蓝；
2.主要仪器：超净工作台；CO2恒温培养箱；倒置荧光显微镜；Benehmark酶标仪；电子精密天平；超声波细胞粉碎机；流式细胞仪；蛋白核酸分析仪；电泳仪；凝胶图像分析系统；高速冷冻离心机；电热恒温鼓风干燥箱；电动玻璃匀浆机；细胞培养瓶；细胞培养板（96孔、12孔）、离心管、微孔滤器；
3．含药大鼠血清的制备：将各组中药饮片分别放入冷水中浸泡大约30分钟，先用武火将中药煎沸腾，再用文火保持沸腾30分钟，然后将各组药液滤出来，再加水煎煮，煮沸后文火保持大约20分钟，每一剂煎煮三次之后混合一起，煎煮浓缩至所需剂量的浓度。将中药黄芪水煎并浓缩至0.7g/ml，丹参水煎并浓缩0.25g/ml，白花蛇舌草水煎并浓缩至0.3g/ml，复方组水煎并浓缩至1.25g/ml，替米沙坦加入蒸馏水配成浓度为0.6mg/ml。将60只Wistar大鼠按照随机分组的原则分为6组，分为：复方组（丹参、黄芪、白花蛇舌草），正常对照组黄芪组，丹参组，白花蛇舌草组，对照西药替米沙坦组。正常组每日灌服蒸馏水4ml，分早晚两次，中药组分别每日灌服药液4ml，分早晚两次，西药替米沙坦组每日灌服药液4ml，分早晚两次灌药，连续三日，在第三天灌胃后1小时采取腹主动脉采血，采血后离心取得血清（1500rpm离心5分钟），在56℃水浴，时间为30min，灭活补体后，用0.22μm无菌滤器进行过滤，之后放入-20℃冰箱中冻存备用
4.系膜细胞的培养：每天要观察大鼠肾小球生长状态，放到倒置显微镜下观察，细胞生长状态比较好的表现为，细胞呈现为梭型，并且在细胞中间可见到一清晰卵圆形细胞核，细胞紧贴在细胞培养瓶上。一般2-3天换液一次，换液时候弃去培养液的总量的1/2-1/3，然后加入新的培养液，新加入的培养液能够覆盖在培养瓶底就可以，换完培养液后，旋紧培养瓶盖并松半扣，放入5%CO2孵箱中，37℃中进行培养。
5.随机分组系膜细胞：血管紧张素Ⅱ组：加入浓度为10-6mol/L的AngⅡ和浓度为10%胎牛血清的DMEM液；
正常血清组：加入10%浓度未给予药物的正常大鼠血清的DMEM液和10-6mol/L的AngⅡ中药小复方组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的中药小复方组大鼠血清。黄芪组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的黄芪组大鼠血清丹参组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的丹参组大鼠血清白花蛇舌草组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的白花蛇舌草组大鼠血清西药对照替米沙坦组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的替米沙坦中药组大鼠血清
6.系膜细胞增殖与凋亡的检测：采用MTT比色法，用酶联免疫检测仪测吸光度值A(570nm处)，将生长状态良好的大鼠肾小球系膜细胞调整细胞浓度为1×104/ml，转种于96孔板每孔200μl，每组随机设6个复孔，培养24h待细胞贴壁后，换成无血清DMEM培养液培养24h，使多数细胞同步于G0/G1期，然后分别加入含10%正常大鼠血清和10%含药血清的，每孔200μl，继续培养48小时后，弃去培养液，分别于培养结束前6小时加入MTT20μl/孔。培养结束后，移去MTT溶液，然后每孔加入150μl二甲亚砜（DMSO），于37oC微孔振荡器振荡10分钟，待结晶后完全溶解后，用酶联免疫检测仪测吸光度值A(570nm处)。流式细胞仪测定含药血清对肾小球系膜细胞增殖的抑制及凋亡作用。
结果：
与正常对照组比较，AngⅡ组有极显著性差异(P<0.01)，提示血管紧张素Ⅱ组(AngⅡ)能促进肾小球系膜细胞异常增殖，系膜细胞增殖模型建立成功。各药物组明显降低系膜细胞增殖情况，与AngⅡ组对比有显著性差异(P<0.01)，说明各药物干预组对AngⅡ刺激的系膜细胞增殖均有抑制作用；复方组降低系膜细胞增殖最为明显，与各单味药，西药对照组比较，有显著性差异(P<0.01或P<0.05)，提示复方组抑制肾小球系膜细胞增殖作用最强；其余各组之间比较无显著性差异。药物血清在作用24、48小时之间无显著性差异，提示药物抑制大鼠肾小球系膜细胞增殖作用不随时间的延长而又显著性变化。流式细胞仪检测结果显示：与正常对照组，血管紧张素组相比，各治疗组凋亡率显著增加（P<0.05），并且复方组抑制肾小球系膜细胞凋亡作用最强，与单味药及西药组比较有显著差异（P<0.05），其余各药物组之间比较没有显著性差异（P>0.05）。
结论：
益气解毒化瘀小复方可有效地抑制肾小球系膜细胞增殖，有效地促进系膜细胞凋亡，并优于单味药，从而达到抗肾小球硬化，保护肾脏的目的。实验二：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞FN，Col-IV胶原蛋白影响的研究
目的：
探讨益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞细胞外基质FN，Col-IV胶原蛋白的影响。
材料与方法：
主要材料：血清IV型胶原酶联免疫测定试剂盒、血清纤维连接蛋白(FN)酶联免疫测定试剂盒、Col-IV型胶原酶。主要方法：采用双抗体ELISA夹心法对系膜细胞上清进行测定，取培养液100ul，按血清按照血清FN、Col-IV酶联免疫试剂操作说明书进行检测，结果为酶标仪测定的OD值，减去正常对照组的OD值
结果：
血管紧张素组可明显增高FN，Col-IV胶原蛋白水平，与空白对照组比较有极显著性差异（P<0.01），说明血管紧张素能够促进ECM合成增加，加速肾小球硬化形成；与血管紧张素组对比，替米沙坦组，丹参组，黄芪组，白花蛇舌草组，复方组均有显著性差异（P<0.05），说明上述中药能明显降低FN，Col-IV胶原蛋白水平，抑制ECM合成，防止肾小球硬化；各组降低FN，Col-IV胶原蛋白水平比较，复方组最为明显，与替米沙坦组，黄芪组，丹参组，白花蛇舌草组比较有显著性差异（P<0.05），以上结果显示复方组能够下调FN，Col-IV胶原蛋白水平，降低细胞外基质的沉积，延缓肾小球硬化的进展，并且优越于各单味药组。
结论：
益气解毒化瘀小复方可有效减少FN，Col-IV胶原蛋白含量，降低系膜细胞外基质（ECM）合成，并优于单味药，达到防治肾小球硬化的目的。
实验三：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞MMP3，TIMP1影响研究
目的：
探讨益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞MMP3，TIMP1的基因表达影响，初步明确其抗肾小球硬化的作用机理。
材料与方法：
主要材料与仪器：Trizol、异丙醇、氯仿、DNA-MARKER、RT-PCR试剂盒、MMP-3，TIMP-1。超低温冰箱；超纯水机；电动玻璃匀浆机；超声波细胞粉碎机；蛋白核酸分析仪低温高速离心机；低温冰箱；洁净工作台；快速混匀器；PCR仪；电泳仪；电泳槽；微波炉仪固液相分子杂交仪。
方法：RT-PCR法检测各组含药血清对大鼠肾小球系膜细胞MMP3，TIMP1mRNA的表达影响。
结果：
各药物组对大鼠肾小球系膜细胞MMP3mRNA，TIMP1mRNA表达影响的研究：各组肾小球系膜细胞MMP-3mRNA，TIMP1mRNA明显不均一。各治疗组组均能不同程度地促进MMP3mRNA的表达，降低TIMP1mRNA的表达，而以中药复方最为显著，说明中药复方降低ECM合成，抗肾小球硬化作用与促进MMP3mRNA的表达，降低TIMP1mRNA表达有关。
结论：
益气解毒化瘀小复方抑制系膜细胞增殖，降低细胞外基质合成，与降低MMP3mRNA的表达，促进TIMP1mRNA表达有关。
实验四：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞Smad3，Smad7mRNA的影响研究
目的：
探讨益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞信号通路Smad3，Smad7mRNA的基因表达影响，初步明确其抗肾小球硬化的作用机理。
材料与方法：
Smad3，Smad7试剂。
RT-PCR法检测各组含药血清对大鼠肾小球系膜细胞细胞内信号传递Smad3/7检测：分子Smad3/7mRNA的表达影响
结果：各药物组对大鼠肾小球系膜细胞细胞内信号通路Smad3/Smad7mRNA表达影响：采取益气解毒化瘀小复方及个单味药干预后，可以明显抑制Smad3的表达，促进Smad7的表达，并且益气解毒化瘀复方优越于各单味药组。所以益气解毒化瘀小复方中药可以抑制大鼠肾小球系膜细胞TGF-β/smad信号通路的过程，从而延缓肾小球硬化。这一结论从基因水平阐述了益气解毒化瘀小复方抑制肾小球系膜细胞增殖及细胞外基质（ECM）积聚，保护肾脏的作用机理。
结论：
益气解毒化瘀小复方中药可以抑制大鼠肾小球系膜细胞TGF-β/smad信号通路的过程，从而延缓肾小球硬化。从基因和蛋白水平阐述了益气解毒化瘀小复方抑制肾小球系膜细胞增殖及抑制ECM积聚，保护肾脏的作用机理
Glomerular sclerosis is the main pathological basis of chronic renal failureand the final outcome of various glomerular diseases, with chronic renal failureas the clinical performance. And the main pathological features include theproliferation of mesangial cell (MC) and excessive accumulation of extracellularmatrix (ECM), which can lead to the formation of glomerular sclerosis. Anincreasing number of basic and clinical researches regarding traditional Chinesemedicine and western medicine treatments for glomerular sclerosis have obtainedgreat achievements, mainly focusing on the glomerular sclerosis mechanisms.There is little evidence about the treatment. Many scholars are trying to explorevarious measures for preventing glomerular sclerosis, delaying diseasedevelopment, and retarding renal failure. Single Chinese herbs, single herbalextracts and Chinese herbal compound prescription can inhibit MC proliferation,reduce ECM accumulation, antagonize adverse effects caused by cytokines, preventand treat glomerular sclerosis.
Previous studies of our research group have investigated the change of MCsin rats after treated with separated prescriptions of Yushen granules, includingone for benefiting qi, removing stasis and relieving toxicity, one for benefitingqi and removing stasis, and one for benefiting qi and relieving toxicity.Preliminary results showed that, compound No.1that was consisted of Astragalus,Danshen Root and Oldenlandia could significantly inhibit the MC proliferation,decrease the ECM synthesis, and prevent glomerular sclerosis. According to ahypothesis that “stagnant toxin blocks kidney channel" raised by ProfessorZhang Jun, the pathogenesis of various preliminary and secondary glomerulardiseases attributes to the deficiency-and toxin-caused blood stasis. This studyaims to observe the efficacy and mechanisms underlying small compoundprescriptions (Astragalus, Danshen Root and Oldenlandia) for benefiting qi,detoxification and removing stasis on the glomerular sclerosis, in a broader attempt to provide theoretical and practical significance for the preventionand treatment of glomerular sclerosis.
Experiment1: Influence of compound prescription and itssingle herbs on MC proliferation and apoptosis in ratsPurpose:
The aim of this study was to discuss the effect of compound prescriptionand its single herbs on the angiotensin II (Ang II)-induced MC proliferationand apoptosis in rats.
Material and method:
1. Main materials: Sixty Wistar rats, weighing200±20g； rat MCs； Chineseherbal medicine compound consisting of Astragalus, Danshen Root and Oldenlandia；three single herb groups: Astragalus, Danshen Root and Oldenlandia,respectively；western medicine control group: telmisartan；Dulbecco's modifiedEagle's medium (DMEM) powder, fetal bovine serum, Ang II, MTT, AnnexinV-FITC,trypan blue.
2. Main instruments: Superclean bench； CO2thermostatic incubator； invertedfluorescence microscope； Benehmark microplate reader； electronic precisebalance； ultrasonic cell grinder； flow cytometry； protein nucleic acidspectrophotometer； electrophoresis system； EPS-300(Tanon, Shanghai, China)；gelgraph analyzing system； high-speed freezing centrifuge； DHG series heatingand drying oven； glass Dounce homogenizer； cell culture flask； cell cultureplate (96-well,12-well)； centrifuge tube, frozen pipe, microporous filter.
3. Serum pharmacological method: Traditional Chinese medicine preparations:decoction pieces of Chinese herbs were immersed in cold water for30minutesand boiled in strong fire for30minutes, then the herbal liquid was filteredand the herbs were boiling with water for additional20minutes. High-doseprescriptions can be obtained after three times of decoctions. Astragalusdecoction was condensed to0.7g/mL, Danshen Root decoction was condensed to 0.25g/mL, Oldenlandia decoction was condensed to0.3g/mL, compound decoctionwas condensed1.25g/mL, and telmisartan concentration was adjusted into0.6mg/mL.
Drug serum preparation: Sixty Wistar rats were randomly divided into six groups,with ten rats in each group, namely normal group, compound group, Astragalusgroup, Danshen Root group, Oldenlandia group, and western medicine control group(telmisartan). Rats of the normal group, Chinese herb groups, and telmisartangroup were respectively administrated with4mL of distilled water andcorresponding drug liquids, twice per day, for3consecutive days. At1hourafter the last administration, the abdominal aorta blood was sampled andcentrifuged at1500r/min for5minutes, to obtain blood serum. The complementwas deactivated in56℃water bath for30minutes, filtered with0.22-μm sterilefilter, stored at-20℃for further use.
4. MC culture: The rat MC morphology was observed under inverted microscope.Results showed that, MCs were well growing and spindle-shaped, with a clear ovalnucleus in the center, cells adhered to the wall of culture flask and the majorityof cells were fused.1/2-2/3of the culture medium was replenished with DMEMcontaining10%fetal bovine serum, and the cells were incubated at37℃,5%CO2humidity. The culture medium was changed every2-3days.
5. Random grouping:
Ang II group: Culture medium was added with10-6mol/L Ang II and DMEM containing10%fetal bovine serum；
Normal serum group: Culture medium was added with10-6mol/L Ang II and10%normalrat serum with no Chinese herb.
Compound prescription group: Culture medium was added with10-6mol/L Ang IIand10%serum of rats treated with compound prescription.Astragalus group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Astragalus.
Danshen Root group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Danshen Root. Oldenlandia group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Oldenlandia.Western medicine control group: Culture medium was added with10-6mol/L AngII and10%serum of rats treated with telmisartan.6. Detection of MC proliferation and apoptosis: The absorbance value at570nmwas determined with ELISA colorimetric assay, using MTT method. The rat MCs at1×104/mL were seeded onto96-well culture plate, with200μL in each well. Therewere six parallel wells in each group. The adherent cells were cultured withserum-free DMEM for additional24hours, and the majority of cells weresynchronized in G0/G1phase and cultured with10%normal rat serum and10%serumcontaining drugs,200μL in each well, for48hours. Then culture medium wasremoved and each well was supplemented with20μL MTT6hours prior to thecompletion of culture. After the cultivation was completed, the MTT solutionwas removed, and each well was added with150μL dimethyl sulfoxide, shakenat37oC microporous oscillator for10minutes. After all crystallizations werecompletely dissolved, the absorbance value was read with ELISA assay at570nm.Flow cytometry was used to determine the effect of drug-contained serum on theMC proliferation and apoptosis.
Results:
The proliferation and apoptosis of rat MCs showed extremely significantdifferences in Ang II group comparison with normal control group (P<0.01),suggesting that Ang II can promote abnormal proliferation of rat MCs, and MCproliferation model is successfully established. There were significantdifferences between Ang II group and various drug groups (P<0.01), whichindicated that all the drug interventions can inhibit the Ang II-induced MCproliferation. The compound group also showed significant differences incomparison to other drug groups (P<0.01or P<0.05). This is evidence that thecompound prescription has the strong inhibition effect on MC proliferation. Nosignificant difference was found between any of other drug groups. The efficacy of drug-contained serum was similar at24hours and48hours, which clearlydemonstrated that the inhibition effect on the MC proliferation was not dependenton the time. Flow cytometry results showed that, the apoptosis rate was increasedsignificantly compared with normal control group and Ang II group (P<0.05), withno significant difference between drug groups (P>0.05).
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasisis superior to single Chinese herbs on inhibiting the MC proliferation andpromoting the MC apoptosis, thus can prevent and treat glomerular sclerosis,as well as protect the kidney.
Experiment2: Influence of compound prescription and itssingle herbs on fibronectin and collagen type IV proteinlevels in rat MCs
Purpose:
The aim of this study was to observe the effect of compound prescription andits single herbs on fibronectin and collagen type IV protein levels inextracellular matrix of rat MCs.
Material and method:
1. Main materials: Serum collagen type IV ELISA kit (Beijing Bangding TaikeBiotechnology Co., Ltd., Beijing, China), serum fibronectin ELISA kit (BeijingBangding Taike Biotechnology Co., Ltd., Beijing, China), collagenase type IV (Sigma,USA).
2. Main methods: The MC supernatant was determined using double antibody ELISAsandwich assay.100μL culture medium was taken out and the absorbance valuewas detected with a microplate reader according to the instructions of ELISAkit for serum fibronectin and collagen type IV. The absorbance value=(absorbance value of tested well–absorbance value of normal controlled well).
Results:
The serum fibronectin and collagen type IV protein levels had extremelysignificant differences between Ang II group and normal control group (P <0.01),indicating Ang II can lead to a significantly increased level of fibronectinand collagen type IV proteins, promote ECM synthesis, and accelerate glomerularsclerosis formation. There were also significant differences between Ang IIgroup and drug groups (P <0.05), which suggested that Chinese herbal medicineprescriptions can significantly decrease level of fibronectin and collagen typeIV proteins, inhibit the ECM synthesis, and prevent glomerular sclerosis. Thechanges in the compound prescription group were significantly different withthose in telmisartan group and other single herbal groups (P <0.05). The compoundprescription has the strongest effect to down-regulate the serum levels offibronectin and collagen type IV protein, and delay glomerular sclerosis.
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasiscan effectively reduce levels of fibronectin and collagen type IV protein anddecrease ECM synthesis in MCs, and the compound is superior to single herbs onthe prevention of glomerular sclerosis.Experiment3: Influence of compound prescription and itssingle herbs on matrix metalloproteinases (MMPs) and tissueinhibitor of metalloproteinases (TIMPs) in rat MCs
Purpose:
The aim of this study was to investigate the effect of compound prescriptionand its single herbs on MMP3and TIMP1genetic expression in rat MCs, and topreliminarily reveal the underlying mechanism.
Material and method:
1. Main materials and equipments: Trizol, isopropyl alcohol, chloroform, DNA-MARKER, RT-PCR kit, MMP-3, TIMP-1. Ultra-cold freezer； ultra-pure water；electric glass homogenate machine； ultrasonic cell grinder； protein nucleicacid spectrophotometer； deep-freeze refrigerator； superclean bench； swiftmixing； PCR instrument； electrophoresis system； electrophoresis tank；microwave； solid-liquid phase hybridization instrument.
2. Methods: RT-PCR assay was used for the detection of each drug-contained serumon the MMP3and TIMP1mRNA expression in rats.
Results:
The MMP-3and TIMP1mRNA expression greatly varied in different groups. All drugtreatments could significantly promote the MMP3mRNA expression and decreasethe TIMP1mRNA expression, especially the compound prescription group. This isevidence that the compound prescription can reduce ECM synthesis and preventglomerular sclerosis, and the effect is associated with the increased MMP3mRNAexpression and the decreased TIMP1mRNA expression.
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasisinhibits the MC proliferation and reduces the ECM synthesis, which contributesto the decrease of MMP3mRNA expression and the increase of TIMP1mRNA expression.Experiment4: Influence of compound prescription and itssingle herbs on rat MCs
Purpose:
The aim of this study was to investigate the effect of compound prescriptionand its single herbs on MC signaling pathway Smad3/Smad7mRNA genetic expressionin rats, and preliminarily reveal the underlying mechanism.
Material and method:
1. Main materials: Smad3and Smad7reagent.
2Main methods: The signal transmission of Smad3/7in MCs was detected. TheSmad3/7mRNA expression was also determined. Results:
The effect of each drug treatment on the intercellular signaling pathwaySmad3/Smad7mRNA expression in rats was investigated. Results showed that, thecompound prescription and its single herbs can significantly inhibit Smad3expression and promote Smad7expression, and the compound was superior to singleherbs. So compound prescription can suppress the TGF-beta/Smad signaling pathwayin the MCs, thus delay glomerular sclerosis formation. This is the geneticexplanation for the mechanism underlying compound prescription inhibits MCproliferation and ECM accumulation, as well as protects the kidney.
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasissuppresses the TGF-beta/Smad signaling pathway in the MCs, thereby delaying theformation of glomerular sclerosis. We have revealed the underlying mechanismsassociated with compound prescription inhibiting MC proliferation and ECMaccumulation, as well as protecting the kidney in the gene and protein levels.
本实验是在前期课题“愈肾颗粒拆方重组对大鼠肾小球系膜细胞的影响研究”的基础上开展的，前期课题研究，按照中医病机和组方原则为依据进行拆方，将原处方按益气化瘀解毒、益气化瘀、益气解毒的组方原则，经试验研究，初步结论复方1号组黄芪，丹参，白花蛇舌草组更具有显著抑制肾小球系膜细胞增殖，降低细胞外基质合成，防治肾小球硬化的功效。本实验即针对该益气解毒化瘀小复方（黄芪，丹参，白花蛇舌草），开展进一步研究，观察益气解毒化瘀小复方及其单味药组干预肾小球硬化的作用及作用机制，并探讨益气解毒化瘀同治对提高防治肾小球硬化的理论意义和实用价值，为在临床方面防治肾小球硬化，提供理论与方法依据，为下一步的深入科学研究中药防治肾小球硬化打下基础。
实验一：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞增殖及凋亡影响的研究
目的：
探讨益气解毒化瘀小复方及其单味药对血管紧张素Ⅱ诱导的大鼠肾小球系膜细胞增殖，凋亡的影响。
材料与方法：
1.主要材料：Wistar大鼠60只，体重（200±20）g；大鼠肾小球系膜细胞；中药材：小复方由黄芪，丹参、白花蛇舌草组成，单味药组分别为黄芪，丹参，白花蛇舌草。西药对照组：替米沙坦；DMEM培养基干粉、标准胎牛血清、血管紧张素Ⅱ、MTT、AnnexinV-FITC、台盼蓝；
2.主要仪器：超净工作台；CO2恒温培养箱；倒置荧光显微镜；Benehmark酶标仪；电子精密天平；超声波细胞粉碎机；流式细胞仪；蛋白核酸分析仪；电泳仪；凝胶图像分析系统；高速冷冻离心机；电热恒温鼓风干燥箱；电动玻璃匀浆机；细胞培养瓶；细胞培养板（96孔、12孔）、离心管、微孔滤器；
3．含药大鼠血清的制备：将各组中药饮片分别放入冷水中浸泡大约30分钟，先用武火将中药煎沸腾，再用文火保持沸腾30分钟，然后将各组药液滤出来，再加水煎煮，煮沸后文火保持大约20分钟，每一剂煎煮三次之后混合一起，煎煮浓缩至所需剂量的浓度。将中药黄芪水煎并浓缩至0.7g/ml，丹参水煎并浓缩0.25g/ml，白花蛇舌草水煎并浓缩至0.3g/ml，复方组水煎并浓缩至1.25g/ml，替米沙坦加入蒸馏水配成浓度为0.6mg/ml。将60只Wistar大鼠按照随机分组的原则分为6组，分为：复方组（丹参、黄芪、白花蛇舌草），正常对照组黄芪组，丹参组，白花蛇舌草组，对照西药替米沙坦组。正常组每日灌服蒸馏水4ml，分早晚两次，中药组分别每日灌服药液4ml，分早晚两次，西药替米沙坦组每日灌服药液4ml，分早晚两次灌药，连续三日，在第三天灌胃后1小时采取腹主动脉采血，采血后离心取得血清（1500rpm离心5分钟），在56℃水浴，时间为30min，灭活补体后，用0.22μm无菌滤器进行过滤，之后放入-20℃冰箱中冻存备用
4.系膜细胞的培养：每天要观察大鼠肾小球生长状态，放到倒置显微镜下观察，细胞生长状态比较好的表现为，细胞呈现为梭型，并且在细胞中间可见到一清晰卵圆形细胞核，细胞紧贴在细胞培养瓶上。一般2-3天换液一次，换液时候弃去培养液的总量的1/2-1/3，然后加入新的培养液，新加入的培养液能够覆盖在培养瓶底就可以，换完培养液后，旋紧培养瓶盖并松半扣，放入5%CO2孵箱中，37℃中进行培养。
5.随机分组系膜细胞：血管紧张素Ⅱ组：加入浓度为10-6mol/L的AngⅡ和浓度为10%胎牛血清的DMEM液；
正常血清组：加入10%浓度未给予药物的正常大鼠血清的DMEM液和10-6mol/L的AngⅡ中药小复方组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的中药小复方组大鼠血清。黄芪组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的黄芪组大鼠血清丹参组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的丹参组大鼠血清白花蛇舌草组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的白花蛇舌草组大鼠血清西药对照替米沙坦组：加入浓度为10-6mol/L的AngⅡ和浓度为10%的替米沙坦中药组大鼠血清
6.系膜细胞增殖与凋亡的检测：采用MTT比色法，用酶联免疫检测仪测吸光度值A(570nm处)，将生长状态良好的大鼠肾小球系膜细胞调整细胞浓度为1×104/ml，转种于96孔板每孔200μl，每组随机设6个复孔，培养24h待细胞贴壁后，换成无血清DMEM培养液培养24h，使多数细胞同步于G0/G1期，然后分别加入含10%正常大鼠血清和10%含药血清的，每孔200μl，继续培养48小时后，弃去培养液，分别于培养结束前6小时加入MTT20μl/孔。培养结束后，移去MTT溶液，然后每孔加入150μl二甲亚砜（DMSO），于37oC微孔振荡器振荡10分钟，待结晶后完全溶解后，用酶联免疫检测仪测吸光度值A(570nm处)。流式细胞仪测定含药血清对肾小球系膜细胞增殖的抑制及凋亡作用。
结果：
与正常对照组比较，AngⅡ组有极显著性差异(P<0.01)，提示血管紧张素Ⅱ组(AngⅡ)能促进肾小球系膜细胞异常增殖，系膜细胞增殖模型建立成功。各药物组明显降低系膜细胞增殖情况，与AngⅡ组对比有显著性差异(P<0.01)，说明各药物干预组对AngⅡ刺激的系膜细胞增殖均有抑制作用；复方组降低系膜细胞增殖最为明显，与各单味药，西药对照组比较，有显著性差异(P<0.01或P<0.05)，提示复方组抑制肾小球系膜细胞增殖作用最强；其余各组之间比较无显著性差异。药物血清在作用24、48小时之间无显著性差异，提示药物抑制大鼠肾小球系膜细胞增殖作用不随时间的延长而又显著性变化。流式细胞仪检测结果显示：与正常对照组，血管紧张素组相比，各治疗组凋亡率显著增加（P<0.05），并且复方组抑制肾小球系膜细胞凋亡作用最强，与单味药及西药组比较有显著差异（P<0.05），其余各药物组之间比较没有显著性差异（P>0.05）。
结论：
益气解毒化瘀小复方可有效地抑制肾小球系膜细胞增殖，有效地促进系膜细胞凋亡，并优于单味药，从而达到抗肾小球硬化，保护肾脏的目的。实验二：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞FN，Col-IV胶原蛋白影响的研究
目的：
探讨益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞细胞外基质FN，Col-IV胶原蛋白的影响。
材料与方法：
主要材料：血清IV型胶原酶联免疫测定试剂盒、血清纤维连接蛋白(FN)酶联免疫测定试剂盒、Col-IV型胶原酶。主要方法：采用双抗体ELISA夹心法对系膜细胞上清进行测定，取培养液100ul，按血清按照血清FN、Col-IV酶联免疫试剂操作说明书进行检测，结果为酶标仪测定的OD值，减去正常对照组的OD值
结果：
血管紧张素组可明显增高FN，Col-IV胶原蛋白水平，与空白对照组比较有极显著性差异（P<0.01），说明血管紧张素能够促进ECM合成增加，加速肾小球硬化形成；与血管紧张素组对比，替米沙坦组，丹参组，黄芪组，白花蛇舌草组，复方组均有显著性差异（P<0.05），说明上述中药能明显降低FN，Col-IV胶原蛋白水平，抑制ECM合成，防止肾小球硬化；各组降低FN，Col-IV胶原蛋白水平比较，复方组最为明显，与替米沙坦组，黄芪组，丹参组，白花蛇舌草组比较有显著性差异（P<0.05），以上结果显示复方组能够下调FN，Col-IV胶原蛋白水平，降低细胞外基质的沉积，延缓肾小球硬化的进展，并且优越于各单味药组。
结论：
益气解毒化瘀小复方可有效减少FN，Col-IV胶原蛋白含量，降低系膜细胞外基质（ECM）合成，并优于单味药，达到防治肾小球硬化的目的。
实验三：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞MMP3，TIMP1影响研究
目的：
探讨益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞MMP3，TIMP1的基因表达影响，初步明确其抗肾小球硬化的作用机理。
材料与方法：
主要材料与仪器：Trizol、异丙醇、氯仿、DNA-MARKER、RT-PCR试剂盒、MMP-3，TIMP-1。超低温冰箱；超纯水机；电动玻璃匀浆机；超声波细胞粉碎机；蛋白核酸分析仪低温高速离心机；低温冰箱；洁净工作台；快速混匀器；PCR仪；电泳仪；电泳槽；微波炉仪固液相分子杂交仪。
方法：RT-PCR法检测各组含药血清对大鼠肾小球系膜细胞MMP3，TIMP1mRNA的表达影响。
结果：
各药物组对大鼠肾小球系膜细胞MMP3mRNA，TIMP1mRNA表达影响的研究：各组肾小球系膜细胞MMP-3mRNA，TIMP1mRNA明显不均一。各治疗组组均能不同程度地促进MMP3mRNA的表达，降低TIMP1mRNA的表达，而以中药复方最为显著，说明中药复方降低ECM合成，抗肾小球硬化作用与促进MMP3mRNA的表达，降低TIMP1mRNA表达有关。
结论：
益气解毒化瘀小复方抑制系膜细胞增殖，降低细胞外基质合成，与降低MMP3mRNA的表达，促进TIMP1mRNA表达有关。
实验四：益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞Smad3，Smad7mRNA的影响研究
目的：
探讨益气解毒化瘀小复方及其单味药对大鼠肾小球系膜细胞信号通路Smad3，Smad7mRNA的基因表达影响，初步明确其抗肾小球硬化的作用机理。
材料与方法：
Smad3，Smad7试剂。
RT-PCR法检测各组含药血清对大鼠肾小球系膜细胞细胞内信号传递Smad3/7检测：分子Smad3/7mRNA的表达影响
结果：各药物组对大鼠肾小球系膜细胞细胞内信号通路Smad3/Smad7mRNA表达影响：采取益气解毒化瘀小复方及个单味药干预后，可以明显抑制Smad3的表达，促进Smad7的表达，并且益气解毒化瘀复方优越于各单味药组。所以益气解毒化瘀小复方中药可以抑制大鼠肾小球系膜细胞TGF-β/smad信号通路的过程，从而延缓肾小球硬化。这一结论从基因水平阐述了益气解毒化瘀小复方抑制肾小球系膜细胞增殖及细胞外基质（ECM）积聚，保护肾脏的作用机理。
结论：
益气解毒化瘀小复方中药可以抑制大鼠肾小球系膜细胞TGF-β/smad信号通路的过程，从而延缓肾小球硬化。从基因和蛋白水平阐述了益气解毒化瘀小复方抑制肾小球系膜细胞增殖及抑制ECM积聚，保护肾脏的作用机理
Glomerular sclerosis is the main pathological basis of chronic renal failureand the final outcome of various glomerular diseases, with chronic renal failureas the clinical performance. And the main pathological features include theproliferation of mesangial cell (MC) and excessive accumulation of extracellularmatrix (ECM), which can lead to the formation of glomerular sclerosis. Anincreasing number of basic and clinical researches regarding traditional Chinesemedicine and western medicine treatments for glomerular sclerosis have obtainedgreat achievements, mainly focusing on the glomerular sclerosis mechanisms.There is little evidence about the treatment. Many scholars are trying to explorevarious measures for preventing glomerular sclerosis, delaying diseasedevelopment, and retarding renal failure. Single Chinese herbs, single herbalextracts and Chinese herbal compound prescription can inhibit MC proliferation,reduce ECM accumulation, antagonize adverse effects caused by cytokines, preventand treat glomerular sclerosis.
Previous studies of our research group have investigated the change of MCsin rats after treated with separated prescriptions of Yushen granules, includingone for benefiting qi, removing stasis and relieving toxicity, one for benefitingqi and removing stasis, and one for benefiting qi and relieving toxicity.Preliminary results showed that, compound No.1that was consisted of Astragalus,Danshen Root and Oldenlandia could significantly inhibit the MC proliferation,decrease the ECM synthesis, and prevent glomerular sclerosis. According to ahypothesis that “stagnant toxin blocks kidney channel" raised by ProfessorZhang Jun, the pathogenesis of various preliminary and secondary glomerulardiseases attributes to the deficiency-and toxin-caused blood stasis. This studyaims to observe the efficacy and mechanisms underlying small compoundprescriptions (Astragalus, Danshen Root and Oldenlandia) for benefiting qi,detoxification and removing stasis on the glomerular sclerosis, in a broader attempt to provide theoretical and practical significance for the preventionand treatment of glomerular sclerosis.
Experiment1: Influence of compound prescription and itssingle herbs on MC proliferation and apoptosis in ratsPurpose:
The aim of this study was to discuss the effect of compound prescriptionand its single herbs on the angiotensin II (Ang II)-induced MC proliferationand apoptosis in rats.
Material and method:
1. Main materials: Sixty Wistar rats, weighing200±20g； rat MCs； Chineseherbal medicine compound consisting of Astragalus, Danshen Root and Oldenlandia；three single herb groups: Astragalus, Danshen Root and Oldenlandia,respectively；western medicine control group: telmisartan；Dulbecco's modifiedEagle's medium (DMEM) powder, fetal bovine serum, Ang II, MTT, AnnexinV-FITC,trypan blue.
2. Main instruments: Superclean bench； CO2thermostatic incubator； invertedfluorescence microscope； Benehmark microplate reader； electronic precisebalance； ultrasonic cell grinder； flow cytometry； protein nucleic acidspectrophotometer； electrophoresis system； EPS-300(Tanon, Shanghai, China)；gelgraph analyzing system； high-speed freezing centrifuge； DHG series heatingand drying oven； glass Dounce homogenizer； cell culture flask； cell cultureplate (96-well,12-well)； centrifuge tube, frozen pipe, microporous filter.
3. Serum pharmacological method: Traditional Chinese medicine preparations:decoction pieces of Chinese herbs were immersed in cold water for30minutesand boiled in strong fire for30minutes, then the herbal liquid was filteredand the herbs were boiling with water for additional20minutes. High-doseprescriptions can be obtained after three times of decoctions. Astragalusdecoction was condensed to0.7g/mL, Danshen Root decoction was condensed to 0.25g/mL, Oldenlandia decoction was condensed to0.3g/mL, compound decoctionwas condensed1.25g/mL, and telmisartan concentration was adjusted into0.6mg/mL.
Drug serum preparation: Sixty Wistar rats were randomly divided into six groups,with ten rats in each group, namely normal group, compound group, Astragalusgroup, Danshen Root group, Oldenlandia group, and western medicine control group(telmisartan). Rats of the normal group, Chinese herb groups, and telmisartangroup were respectively administrated with4mL of distilled water andcorresponding drug liquids, twice per day, for3consecutive days. At1hourafter the last administration, the abdominal aorta blood was sampled andcentrifuged at1500r/min for5minutes, to obtain blood serum. The complementwas deactivated in56℃water bath for30minutes, filtered with0.22-μm sterilefilter, stored at-20℃for further use.
4. MC culture: The rat MC morphology was observed under inverted microscope.Results showed that, MCs were well growing and spindle-shaped, with a clear ovalnucleus in the center, cells adhered to the wall of culture flask and the majorityof cells were fused.1/2-2/3of the culture medium was replenished with DMEMcontaining10%fetal bovine serum, and the cells were incubated at37℃,5%CO2humidity. The culture medium was changed every2-3days.
5. Random grouping:
Ang II group: Culture medium was added with10-6mol/L Ang II and DMEM containing10%fetal bovine serum；
Normal serum group: Culture medium was added with10-6mol/L Ang II and10%normalrat serum with no Chinese herb.
Compound prescription group: Culture medium was added with10-6mol/L Ang IIand10%serum of rats treated with compound prescription.Astragalus group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Astragalus.
Danshen Root group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Danshen Root. Oldenlandia group: Culture medium was added with10-6mol/L Ang II and10%serumof rats treated with Oldenlandia.Western medicine control group: Culture medium was added with10-6mol/L AngII and10%serum of rats treated with telmisartan.6. Detection of MC proliferation and apoptosis: The absorbance value at570nmwas determined with ELISA colorimetric assay, using MTT method. The rat MCs at1×104/mL were seeded onto96-well culture plate, with200μL in each well. Therewere six parallel wells in each group. The adherent cells were cultured withserum-free DMEM for additional24hours, and the majority of cells weresynchronized in G0/G1phase and cultured with10%normal rat serum and10%serumcontaining drugs,200μL in each well, for48hours. Then culture medium wasremoved and each well was supplemented with20μL MTT6hours prior to thecompletion of culture. After the cultivation was completed, the MTT solutionwas removed, and each well was added with150μL dimethyl sulfoxide, shakenat37oC microporous oscillator for10minutes. After all crystallizations werecompletely dissolved, the absorbance value was read with ELISA assay at570nm.Flow cytometry was used to determine the effect of drug-contained serum on theMC proliferation and apoptosis.
Results:
The proliferation and apoptosis of rat MCs showed extremely significantdifferences in Ang II group comparison with normal control group (P<0.01),suggesting that Ang II can promote abnormal proliferation of rat MCs, and MCproliferation model is successfully established. There were significantdifferences between Ang II group and various drug groups (P<0.01), whichindicated that all the drug interventions can inhibit the Ang II-induced MCproliferation. The compound group also showed significant differences incomparison to other drug groups (P<0.01or P<0.05). This is evidence that thecompound prescription has the strong inhibition effect on MC proliferation. Nosignificant difference was found between any of other drug groups. The efficacy of drug-contained serum was similar at24hours and48hours, which clearlydemonstrated that the inhibition effect on the MC proliferation was not dependenton the time. Flow cytometry results showed that, the apoptosis rate was increasedsignificantly compared with normal control group and Ang II group (P<0.05), withno significant difference between drug groups (P>0.05).
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasisis superior to single Chinese herbs on inhibiting the MC proliferation andpromoting the MC apoptosis, thus can prevent and treat glomerular sclerosis,as well as protect the kidney.
Experiment2: Influence of compound prescription and itssingle herbs on fibronectin and collagen type IV proteinlevels in rat MCs
Purpose:
The aim of this study was to observe the effect of compound prescription andits single herbs on fibronectin and collagen type IV protein levels inextracellular matrix of rat MCs.
Material and method:
1. Main materials: Serum collagen type IV ELISA kit (Beijing Bangding TaikeBiotechnology Co., Ltd., Beijing, China), serum fibronectin ELISA kit (BeijingBangding Taike Biotechnology Co., Ltd., Beijing, China), collagenase type IV (Sigma,USA).
2. Main methods: The MC supernatant was determined using double antibody ELISAsandwich assay.100μL culture medium was taken out and the absorbance valuewas detected with a microplate reader according to the instructions of ELISAkit for serum fibronectin and collagen type IV. The absorbance value=(absorbance value of tested well–absorbance value of normal controlled well).
Results:
The serum fibronectin and collagen type IV protein levels had extremelysignificant differences between Ang II group and normal control group (P <0.01),indicating Ang II can lead to a significantly increased level of fibronectinand collagen type IV proteins, promote ECM synthesis, and accelerate glomerularsclerosis formation. There were also significant differences between Ang IIgroup and drug groups (P <0.05), which suggested that Chinese herbal medicineprescriptions can significantly decrease level of fibronectin and collagen typeIV proteins, inhibit the ECM synthesis, and prevent glomerular sclerosis. Thechanges in the compound prescription group were significantly different withthose in telmisartan group and other single herbal groups (P <0.05). The compoundprescription has the strongest effect to down-regulate the serum levels offibronectin and collagen type IV protein, and delay glomerular sclerosis.
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasiscan effectively reduce levels of fibronectin and collagen type IV protein anddecrease ECM synthesis in MCs, and the compound is superior to single herbs onthe prevention of glomerular sclerosis.Experiment3: Influence of compound prescription and itssingle herbs on matrix metalloproteinases (MMPs) and tissueinhibitor of metalloproteinases (TIMPs) in rat MCs
Purpose:
The aim of this study was to investigate the effect of compound prescriptionand its single herbs on MMP3and TIMP1genetic expression in rat MCs, and topreliminarily reveal the underlying mechanism.
Material and method:
1. Main materials and equipments: Trizol, isopropyl alcohol, chloroform, DNA-MARKER, RT-PCR kit, MMP-3, TIMP-1. Ultra-cold freezer； ultra-pure water；electric glass homogenate machine； ultrasonic cell grinder； protein nucleicacid spectrophotometer； deep-freeze refrigerator； superclean bench； swiftmixing； PCR instrument； electrophoresis system； electrophoresis tank；microwave； solid-liquid phase hybridization instrument.
2. Methods: RT-PCR assay was used for the detection of each drug-contained serumon the MMP3and TIMP1mRNA expression in rats.
Results:
The MMP-3and TIMP1mRNA expression greatly varied in different groups. All drugtreatments could significantly promote the MMP3mRNA expression and decreasethe TIMP1mRNA expression, especially the compound prescription group. This isevidence that the compound prescription can reduce ECM synthesis and preventglomerular sclerosis, and the effect is associated with the increased MMP3mRNAexpression and the decreased TIMP1mRNA expression.
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasisinhibits the MC proliferation and reduces the ECM synthesis, which contributesto the decrease of MMP3mRNA expression and the increase of TIMP1mRNA expression.Experiment4: Influence of compound prescription and itssingle herbs on rat MCs
Purpose:
The aim of this study was to investigate the effect of compound prescriptionand its single herbs on MC signaling pathway Smad3/Smad7mRNA genetic expressionin rats, and preliminarily reveal the underlying mechanism.
Material and method:
1. Main materials: Smad3and Smad7reagent.
2Main methods: The signal transmission of Smad3/7in MCs was detected. TheSmad3/7mRNA expression was also determined. Results:
The effect of each drug treatment on the intercellular signaling pathwaySmad3/Smad7mRNA expression in rats was investigated. Results showed that, thecompound prescription and its single herbs can significantly inhibit Smad3expression and promote Smad7expression, and the compound was superior to singleherbs. So compound prescription can suppress the TGF-beta/Smad signaling pathwayin the MCs, thus delay glomerular sclerosis formation. This is the geneticexplanation for the mechanism underlying compound prescription inhibits MCproliferation and ECM accumulation, as well as protects the kidney.
Conclusion:
The compound prescription of benefiting qi, detoxification and removing stasissuppresses the TGF-beta/Smad signaling pathway in the MCs, thereby delaying theformation of glomerular sclerosis. We have revealed the underlying mechanismsassociated with compound prescription inhibiting MC proliferation and ECMaccumulation, as well as protecting the kidney in the gene and protein levels.
引文
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