杨梅花色苷对胰岛细胞氧化应激损伤的保护作用及其机制探讨
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摘要
糖尿病是多基因遗传与环境因素长期共同作用所导致的一组以慢性高血糖为特征的代谢性疾病,由于体内胰岛素相对或绝对分泌不足或靶细胞对胰岛素敏感性降低所引起的内分泌代谢性疾病。胰岛移植是治疗1型及部分2型糖尿病极有前景的治疗手段,能够生理条件下动态调控血糖水平,脱离胰岛素依赖,延缓糖尿病相关并发症的发生。然而在移植过程中,胰岛在分离、纯化及移植操作中数量的损失及功能、活性的下降,移植后受炎症、排斥反应的打击损伤,使得需要更多的胰岛来达到受体脱离胰岛素的目标,而供体的短缺,使得胰岛移植在临床的广泛开展应用极大受限。因而,在供体缺乏的情况下,在移植前减少应激反应对胰岛细胞的损伤,最大程度的保护胰岛细胞的活性和功能对胰岛移植的成功具有重要意义。
胰岛在分离过程中胶原酶对其的毒性,分离纯化过程中的机械性损伤,离体后缺血、缺氧等应激均可导致胰岛细胞内过量活性氧自由簇(ROS)的产生,而胰岛本身其胞内抗氧化酶表达程度低,ROS介导的氧化应激在这些不利因素导致细胞损伤过程中扮演重要角色。过量及长时的ROS将会破坏细胞,自由基将破坏细胞蛋白,膜脂以及核酸,最终导致细胞凋亡及坏死。研究发现在胰岛分离培养过程中加入一些抗氧化剂可以减轻细胞损伤。如在胰岛分离过程中加入烟酰胺能够提高胰岛得率及保护胰岛细胞降低H202诱导的细胞死亡。在培养基中加入谷胱甘肽可以减少胰岛分离后MCP-1和TF的分泌。近年,越来越多的研究者将目光转向天然的、特别是来源于植物的抗氧化剂,一些黄酮类物质(如姜黄素)能够减少细胞因子刺激引起的胰岛细胞的凋亡；移植前预处理还可延长移植物的生存；某些植物组分还具有降低糖尿病鼠血糖,减少胰岛的损失及促进残余细胞胰岛素分泌的作用。
花色苷是一种广泛分布于自然界的水溶性天然食用色素,属于黄酮多酚类化合物,构成了花、果实、蔬菜中许多品种的蓝色、紫色、红色和黄色等,其在浆果类(edible berry)及其他有色植物中含量丰富。研究表明花色苷具有一系列的生理活性,如抗氧化,保持基因组DNA的完整性、抗炎症、抗肿瘤、降血脂等。富含花色苷的浆果类提取物能够增加红细胞对体外氧化应激的抵抗力,长期食用花色苷能够延缓糖尿病相关并发症的发生。Bollec等发现山茱萸花色苷可增加胰岛素释放功能。
杨梅是我国特有的亚热带水果,因含有花色苷、杨梅素、二氢杨梅素等大量生物活性物质而具有较高的营养和保健价值。既往研究表明,杨梅果实黄酮类化合物中,花色苷占较高比例。提取花色苷对其进行HPLC分析后发现,矢车菊素-3-葡萄糖苷(C3G)是最主要组分,占90%以上。杨梅果实提取物具有较强的氧自由基清除活性,抗氧化能力与C3G含量呈正相关。研究发现在高脂喂养的C57BL／6小鼠食用C3G能够明显降低血脂、血糖水平。杨梅花色苷具有良好的保健、医药开发前景。目前关于杨梅花色苷在生物医学方面的研究甚少,而对于其在胰岛功能保护领域的研究国内外均未见报道。
本研究属国家自然科学基金资助项目(编号：30872531),旨在探讨杨梅果实提取物(杨梅花色苷)对胰岛细胞氧化应激损伤的保护效应及其分子机制,探索其在胰岛移植领域的可能应用前景,为杨梅花色苷成为一种潜在的胰岛保护剂奠定相应理论基础。我们同时还初步探讨了自噬在胰岛体外氧化应激损伤及体内移植后的发生。
研究方法
1、杨梅花色苷对胰岛细胞的保护效应
(1)杨梅果实提取物(花色苷)的制备、鉴定及抗氧化活性的测定
杨梅(荸荠种)果实用含盐酸的甲醇溶液浸提,用旋转蒸发仪进行浓缩。提取物中总酚类化合物的含量采用Folin-Ciocalteau比色法测定,总黄酮类化合物的含量采用比色法测定,花色苷含量采用pH示差分光光度法测定。
采用HPLC对杨梅花色苷组成进行鉴定及含量测定；采用DPPH方法测定杨梅花色苷自由基清除活性。MTT法观察其对胰岛细胞系INS-1的细胞毒性。
(2)杨梅花色苷对H2O2诱导的胰岛细胞凋亡的影响
采用MTT法观察花色苷预处理对胰岛细胞系的保护效应,用台盼蓝据染实验及LDH释放试验进一步验证。
采用ROS特异荧光探针DCF-DA检测胞内ROS的产生。Hoechst33258染色观察细胞凋亡情况,流式细胞术观察细胞凋亡及死亡变化；提取细胞蛋白,western blot观察相关凋亡蛋白(cleaved) caspase-3,-9及Bcl-2的表达。
(3)杨梅花色苷对H2O2诱导的胰岛细胞自噬的影响
将：mRFP-GFP-LC3质粒转染INS-1细胞后,给予H2O2刺激,荧光显微镜下观察细胞内LC3的点聚情况。电镜观察H2O2刺激后细胞内自噬泡的形成。Western blot观察H2O2刺激不同时间后,LC3和BECN1的表达变化。
采用BECN1 siRNA下调BECN1表达,western blot观察自噬相关蛋白LC3和凋亡相关蛋白(cleaved) caspase-9的变化,并进一步用MTT法及LDH释放试验验证。
吖啶橙(AO)和单丹磺酰戊二胺(MDC)染色观察花色苷预处理是否可减少H2O2诱导的自噬的发生。Western blot观察LC3和BECN1蛋白的表达。
(4)杨梅花色苷预处理在肾包膜下移植后早期对细胞移植物的影响
将INS-1细胞移植入糖尿病受体小鼠肾包膜下,72h后取出移植物,部分用4%多聚甲醛固定,部分用戊二醛固定以备做电镜,部分OCT包埋剂包埋后液氮速冻,-80℃保存以备冰冻切片。石蜡切片行H&E、insulin和BECN-1免疫组化染色及TUNEL染色。冰冻切片行LC3免疫荧光染色,电镜观察移植物超微结构变化。
将INS-1细胞在移植前给予花色苷预处理24后肾包膜下移植,72h后取出移植侧肾脏,切片,行cleaved caspase-3、LC3免疫荧光染色及BECN-1免疫组化染色。
2.花色苷对胰岛细胞系保护机制的探讨
采用RT-PCR及定量PCR检测花色苷处理后INS-1细胞HO-1 mRNA的表达。western blot方法检测HO-1的蛋白表达水平。
分离小鼠原代胰岛,给予花色苷处理,RT-PCR和定量PCR观察HO-1的表达。将处理后的胰岛冰冻切片并进行Insulin和HO-1免疫荧光双染验证HO-1的表达。
用HO-1 siRNA下调HO-1的表达后,给予1uM花色苷24h, western blot检测HO-1表达变化。MTT法检测HO-1下调后花色苷对胰岛细胞的保护效应。Western blot检测caspase-3,-9及LC3的表达。
构建HO-1高表达的INS-1细胞。给予空载体及pLNCX2-HO1转染后的细胞H202刺激,MTT法检测两组细胞活性。免疫荧光观察两组细胞H202刺激后LC3的点聚情况。
给予INS-1细胞花色苷处理后,western blot观察pAkt及Akt, pERK1/2及ERK1/2,pJNK及JNK1/2蛋白的表达。用PD98059或LY294002分别预先处理INS-1细胞,再给予花色苷孵育,western blot检测HO-1蛋白表达情况。细胞经PD98059或LY294002预处理1h,花色苷孵育24h后,再给予H202,MTT法检测细胞活性改变。
给予INS-1细胞花色苷处理后,激光共聚焦显微镜方法检测Nrf2核转位情况,提取细胞核蛋白,western blot检测细胞核内Nrf2的表达。
研究结果
1.杨梅花色苷对胰岛细胞的保护效应
(1)杨梅花色苷中最主要的成分为矢车菊素-3-葡萄糖苷
杨梅果实提取物中花色苷为总黄酮类化合物中主要组成部分。对杨梅花色苷进行HPLC分析,发现矢车菊素-3-葡萄糖苷为杨梅花色苷最主要成分,比例在90%以上。DPPH法检测结果提示杨梅果实提取物具有较强的氧自由基清除活性。杨梅花色苷浓度在5uM以内时,对INS-1细胞的活性无明显影响。
(2)杨梅花色苷减少了H2O2诱导的胰岛细胞的凋亡和坏死
H2O2刺激导致细胞活性下降,并呈浓度和时间依赖性,MTT结果花色苷预处理能够显著提高细胞活性,降低H2O2对其的损伤。台盼蓝据染实验和LDH释放实验进一步证明1uM花色苷预处理24h可明显保护胰岛细胞系抵抗H2O2损伤。PI单染流式检测显示花色苷预处理明显减少了H2O2诱导的细胞坏死。
荧光显微镜及流式细胞仪检测提示花色苷预处理减少了H2O2诱导的胞内过量ROS的产生。流式细胞仪检测结果提示花色苷预处理减少了H2O2诱导的细胞凋亡及坏死。Western blot显示花色苷预处理减少了H2O2诱导的caspase-3,-9的剪切体的产生,并上调了Bcl-2的表达,进一步支持花色苷可减少H2O2诱导的凋
(3)杨梅花色苷减少了H2O2诱导的胰岛细胞的自噬
mRFP-GFP-LC3质粒转染INS-1细胞,给予H2O2刺激后,荧光显微镜观察到胞浆内出现LC3的点聚。电镜观察发现在H2O2处理组细胞胞浆内出现较多明显的空泡,有肿胀的线粒体及含胞内容物的自噬泡。Western blot结果提示1mMH2O2作用0.5,1,2及4h后,LC3B及BECN1的表达逐渐上调,且在2h处表达量最高。
采用BECN-1 siRNA转染INS-1细胞下调BECN-1的表达,降低了H2O2诱导的自噬的发生。Western blot结果显示BECN1 siRNA转染后H2O2诱导的caspase-9剪切体表达下降。MTT实验及LDH释放实验提示抑制自噬减少了H2O2刺激导致的细胞的损伤。
AO及MDC染色结果提示花色苷预处理减少了H2O2刺激导致的胞内自噬泡及酸性溶酶体的产生。Western blot结果显示H2O2刺激2h后LC3Ⅱ表达明显增加,LC3II/LC3I值增大,而花色苷预处理则降低了LC3Ⅱ的表达和LC3II/LC3I的比例。
(4)杨梅花色苷预处理对移植后早期细胞移植物的影响
INS-1细胞移植入肾包膜下72h后,取出移植物,insulin免疫组化染色证实为胰岛细胞系。TUNEL染色显示部分细胞移植物在72h时出现凋亡。免疫组化染色提示BECN1强阳性,LC3免疫荧光提示移植物胞浆内出现LC3的点聚,电镜检查显示移植物胞浆内出现含胞内容物的自噬泡,支持自噬的发生。
Cleaved caspase-3免疫荧光结果显示花色苷预处理组细胞移植物激活型caspase-3的表达低于未处理组。同时,与对照组相比,花色苷预处理组其BECN1的表达水平降低,胞浆内LC3的点聚程度也降低。
2.杨梅花色苷保护胰岛的机制探讨
(1)杨梅花色苷浓度及时间依赖型的上调HO-1的表达
定量PCR及western blot结果提示花色苷浓度及时间依赖的上调了INS-1细胞HO-1的表达。在小鼠胰岛细胞系beta-TC-6,花色苷同样可浓度依赖型的上调HO-1的表达。
RT-PCR及定量PCR结果显示花色苷孵育也明显上调了原代胰岛HO-1基因表达水平。胰岛冰冻切片Insulin和HO-1免疫荧光双染提示花色苷处理组胰岛细胞HO-1的表达水平高于未处理组。
(2)HO-1的上调表达参与了花色苷的保护效应
MTT结果显示HO-1 siRNA转染组细胞,H2O2刺激后,活性明显下降,并且降低了花色苷对INS-1细胞的保护效应。(?)Vestern blot结果显示,与单纯H2O2处理组相比,HO-1 siRNA转染组细胞caspase-3,-9前体表达水平下降,cleaved caspase-9表达增加,同时LC3Ⅱ表达及LC3Ⅱ/LC3Ⅰ值增加。
给予空载体与pLNCX2-HO1转染组INS-1细胞H2O2刺激,MTT结果显示HO-1过表达细胞其活性较对照组高。LC3免疫荧光染色显示HO-1过表达细胞胞内LC3点聚程度低于对照组。
(3)花色苷通过ERK1／2及P13K／Akt通路介导HO-1的上调表达Western blot结果提示花色苷浓度及时间依赖性的上调pERK1/2及pAkt的表达,而JNK通路则未被激活。PD98059及LY294002预处理抑制了花色苷对HO-1蛋白的上调表达。MTT结果显示与单一花色苷处理组相比,PD98059及LY294002预处理组细胞活性下降,降低了花色苷对胰岛细胞的保护效应。
(4)花色苷诱导转录因子Nrf2向细胞核内转移
激光共聚焦扫描结果表明,与未处理组相比,花色苷诱导了部分Nrf2向细胞核内的转移。提取细胞核蛋白,western blot结果提示1uM的花色苷诱导了Nrf2在细胞核内的表达。
小结：
1.杨梅花色苷中最主要成分为矢车菊素-3-葡萄糖苷,其比例占至90%以上。杨梅花色苷具有较强的氧自由基清除能力；其浓度在5uM以内时,对胰岛细胞系INS-1细胞无明显毒性。
2.花色苷预处理可以明显降低H202刺激导致的细胞内过量ROS的产生,进而减少氧化应激损伤所致的细胞凋亡和坏死。
3.H2O2刺激除可导致细胞凋亡和坏死外,还可引起细胞发生自噬,且发生的自噬促进细胞死亡(即为“自噬性细胞死亡”)。花色苷预处理可以减少H2O2诱导细胞自噬的发生。
4.细胞移植入肾包膜下早期(72h),在各类刺激下可发生凋亡和自噬。在移植前予以花色苷预处理,可降低细胞移植后凋亡和自噬的发生程度。
5.花色苷可上调胰岛细胞(系)HO-1的表达,且存在明显的时间-效应关系和剂量-效应关系。HO-1的上调表达可减少H202诱导的自噬和凋亡,参与了花色苷对胰岛细胞的保护作用。花色苷通过ERK1/2及PI3K/Akt通路介导了HO-1的表达上调。同时,花色苷还可诱导核转录因子Nrf2向胞核转移,增加其转录活性。
Diabetes is a metabolic disease, possibly caused by both of polygenic inheritance and environmental factors. It is characterized by chronic hyperglycemia, mainly due to absolutely or relatively insufficient insulin release from beta-cells or insulin resistance of surrounding tissues. Islet transplantation is a promising therapy for all of type 1 and part of type 2 diabetic patients, which could regulate blood glucose manipulating physiological condition, achieve insulin independence, and delay the occurrence of diabetes-associated complications. However, during the process of islet isolation, purification and transplantation, there exists notable decrease in the number, function and viability of islet grafts. After transplantation, non-specific inflammation and immune rejection also cause the injury to the grafts. All these factors cause the situation that more than one donor are needed for one receipt to achieve insulin independence. However, the inadequate of donor exacerbates the obstacles limiting the development of islet transplantation in clinics. Improved islet isolation and better viability/function preservation before transplantation have been the keys to the evolution of this procedure.
During isolation procedure, islets are exposed to the cytotoxicity of collagenase. mechanical trauma, ischemia and hypoxia, all of which contribute to the excessive production of reactive oxygen species (ROS). ROS-mediated oxidative stress plays an important role in the process of cell injury caused by these stimulations. Pancreatic beta-cells usually express low levels of antioxidant enzymes, the long-term exposure of excessive ROS will damage the intracellular proteins, membrane lipid and nucleic acid, eventually leading to apoptosis and necrosis. Some researchers found that adding some antioxidants during islet isolation and culture process could alleviate cellular injury. Nicotinamide (NA) supplementation of the processing medium during islet isolation significantly improved islet yields and protected beta-cells from cell death (both necrosis and apoptosis) induced by H2O2. Addition of glutathione in culture medium significantly reduced CCL2/MCP-1 and tissue factor (TF) production. Recently, many researchers focused on the protective role of naturally existed antioxidant, especially from plant. Some flavonoids (such as curcumin) could protect islets against cytokines-induced apoptosis; pretreatment of islets with some antioxidants could prolong the survival of graft after transplantation. Some plants themselves have hypoglycemia effect in diabetic rodent model, reduce the extent of islet loss, and promote the insulin release from the left islets.
Anthocyanins, one type of flavoids, are naturally occurring water soluble polyphenolic compounds in the plant foods and widely distributed in fruits, vegetables, and pigmented cereals, imparting color (blue, purple, red, yellow etc) to plants. Anthocyanin is rich in edible berries and other colorful plants. Many studies have shown that anthocyanins exhibits an array of pharmacological properties, such as keeping the integrity of genome DNA, antioxidative. anti-inflammatory, antitumor and antiatherosclerotic activities. Berry extracts rich in anthocyanins could improve resistance of red blood cell against oxidative stress in vitro. Long-term administration of berry-derived supplements including anthocyanins could inhibit the development of the early stages of some diabetic complications without adverse drug reactions. Bollec et al suggested that bioactive anthocyanins from Cornus fruits could increase insulin release of INS-1 cells.
Chinese bayberry, one of six myrica species native to China, is a fruit with high nutrition and health values due to the content of various bioactive substances such as anthocyanins, myricetin and dihydro-myricetin. Our previous study demonstrated that anthocyanins occupies the major portion of total flavonoid compounds. HPLC analysis of Chinese bayberry extract showed that cyanidin-3-O-glucoside (C3G) was the major anthocyanin component, accounting for 95% of the total anthocyanins. Chinese bayberry extracts possess notable radical scavenging activities indicated by DPPH and ABTS cation assays, which is correlated to the content of C3G. C3G was observed to exert hypolipemia and hypoglycemia effects in high-fat-diet (HFD)-feed C57BL/6 mice. Up to now, the application of anthocyanins in CBE has not been fully investigated. Whether anthocyanins has protective effect for islet has not been reported before.
The present topic, supported by the National Natural Science Foundation (serial number:30872531), is to explore whether Chinese bayberry extract could protect against oxidative stress-induced beta-cell injury and its possible mechanism, and is to investigate the possible application of anthocyanins as an islet-protective agent in the field of islet transplantation. We also discuss the occurrence of autophagy in beta-cells induced by oxidative stress in vitro or after transplantation.
Experimental Procedures
1. The protective effect of anthocyanins to pancreatic beta-cells
(1) Chinese bayberry extract preparation, HPLC analysis and antioxidant ability assessment.
Chinese bayberry fruits (Biqi) were ground and extracted in methanol acidified with HCl, and concentrated through rotary evaporation. Total phenolics content was estimated using the Folin-Ciocalteau colorimetric method. Total flavonoids were determined by colorimetric method after reaction with NaNO2 and Al(NO3)3. Anthocyanin quantitation was performed by the pH differential method. HPLC analysis was performed to identify the composition. Free radical scavenging activity of fruit extracts was measured according to the DPPH method. MTT assay was performed to observe the cytotoxicity of extract to INS-1 cells.
(2) The effect of anthocyanins on H2O2-induced apoptosis of pancreatic beta-cells. INS-1 cells were pre-incubated with anthocyanins followed by H2O2 stimulation. Cell viability was evaluated by MTT assay. The protective effect of anthocyains was further confirmed by trypan blue exclusion method and LDH release assay.
A ROS-specific probe, DCF-DA, was used to detect intracellular ROS production in INS-1 cells after various treatments. INS-1 cells stimulated by various agents were stained with Hoechst 33258 to observe the apoptosis. Flow cytometry analysis was adopted to quantify the apoptosis and necrosis. The expression of apoptosis-related proteins (caspase-3,-9 and Bcl-2) was assessed by western blot.
(3) The effect of anthocyains on H2O2-induced autophagy in pancreatic beta-cells
INS-1 cells, transfected with mRFP-GFP-LC3 plasmid, were treated with H2O2 and autophagy was evaluated by observing the formation of intracellular LC3 punctuate dots. After experimental manipulations, cells were fixed by glutaraldehyde and osmic acid, and were further observed by transmission electron microscopy. The expression of autophagic-related proteins (LC3 and BECN1) was determined by western blot to confirm the occurrence of autophagy.
After transfected with BECN1 siRNA, INS-1 cells were exposed to H2O2 for 2h, and the expressions of LC3 and caspase-9 were evaluated. MTT assay and LDH release assay were performed to observe the changes of cell injury after downregulating autophagy.
INS-1 cells, treated with various protocols, were stained by AO and MDC to assess the degree of lysosomes and autophagosome formation. The expression of LC3 and BECN1 were evaluated.
(4) The effect of anthocyanins on cell graft at the early phase of posttransplantation beneath renal capsue
INS-1 cells were transplanted under the left renal capsule of diabetic mice. Seventy-two hours later, the beta-cell grafts were harvested, snap frozen in liquid nitrogen or fixed with 4% paraformaldehyde, or were fixed in glutaraldehyde for transmission electronic microscopic analyzation.
Immunohistochemistry staining for BECN1, immunofluorescence staining for LC3, TUNEL staining and transmission electronic microscopic analyzation were performed.
INS-1 cells pretreated with or without anthocyanins were transplanted under renal capsule of diabetic mice. The cell grafts were harvest after 72h. Immunofluorescence for cleaved caspase-3 and LC3 and immunohistochemistry staining for BECN1 were carried out.
2. Possible mechanism of anthocyanins'protective effect.
RNA was extracted from INS-1 cells treated with luM anthocyanins for various time or incubated with various concentrations of anthocyanins for 12h. The mRNA level of HO-1 was determined by reverse transcription PCR and real-time PCR. The expression of HO-1 protein was evaluated by western blot.
Primary islets were isolated from mice.100-200 islets were incubated in culture medium with or without 2uM anthocyanins for 6h. The expression of HO-1 mRNA in islets was determined by real-time PCR. Dual immunofluorescence for insulin and HO-1 of islets was performed to further observe the HO-1 expression.
The expression of HO-1 in INS-1 cells was downregulated by siRNA transfection. After transfected with HO-1 or control siRNA. INS-1 cells were incubated with luM anthocyanins for 24h, then were exposed to H2O2 for 2h, MTT assay was performed to evaluate the cellular viability. The expression of caspase-3,-9 and LC3 proteins were determined.
The pLNCX2-HO1 vector was constructed and transfected to INS-1 cells. INS-1 cells transfected with pLNCX2-HO1 or empty vector were exposed to H2O2. MTT assay was carried out to assess the viability between two groups. Immunofluorescence for LC3 was performed to evaluate the intracellular LC3 punctuate dot formation in both type of cells treated with H2O2.
INS-1 cells were treated with various concentrations of anthocyanins for 24h, or 1uM anthocyanins for different time. The expressions of pAkt, Akt, pERK1/2, ERK1/2, pJNK and JNK protein were evaluated by western blot. After pretreated with PD98059 (ERK1/2 inhibitor) or LY294002 (PI3K inhibitor) for 1h, INS-1 cells were further incubated with anthocyanins. HO-1 protein expression was evaluated. INS-1 cells were preincubated with PD98059 or LY294002 for 1h, treated with anthocyanins for 24h, followed by H2O2 stimulation. MTT assay was performed to evaluate the viability.
INS-1 cells were incubated with anthocyanins, and Immunofluorescence for Nrf2 was performed and observed under confocal microscopy. Nuclear protein fraction was extracted from cells treated as above, and immunoblotting for Nrf2 and LaminA/C was performed.
Results
1. The protective effect anthocyanins on pancreatic beta-cells.
(1) The major portion of anthocyanins in Chinese bayberry extract is cyanidin-3-glucoside (C3G).
Anthocyanin is the major portion of total flavonoids in Chinese bayberry extract. HPLC analysis of Chinese bayberry extract showed that cyanidin-3-O-glucoside (C3G) was the major anthocyanin component, accounting for more than 90% of the total anthocyanins. DPPH assay indicated the strong antioxidant capacity of CBE, and C3G is the dominant attributor. There is no cytotoxicity to INS-1 cells below the concentration of 5uM.
(2) Anthocyanins alleviated H2O2-induced apoptosis and necrosis of INS-1 cells
H2O2 injured INS-1 cells in a dose-and time-dependent manner. Pretreatment of INS-1 cells with anthocyanins resulted in the prevention of cell death by H2O2. Trypan blue exclusion assay and LDH release assay further confirmed that treatment with 1 uM anthocyanins for 24h could protect bete-cells against H2O2-induced injury. FCM analysis also displayed that anthocyanins decreased H2O2-induced cell necrosis.
Compared to normal cells, H2O2 caused excessive ROS production. Anthocyanins preincubation also decreased oxidative stress-induced intracellular ROS generation. FCM demonstrated anthocyanins incubation reduced the extent of both cellular apoptosis and necrosis induced by H2O2 stimulation. Treatment of INS-1 cells with 1mM H2O2 caused the activation of caspase-9 and-3, which were mitigated by pretreatment with anthocyanins in a concentration dependent manner.
(3) Anthocyanins decreased H2O2-induced autophagy in pancreatic beta-cells
GFP-mRFP-LC3 fluorescence was diffusely distributed in untreated group, whereas in cells treated with H2O2 there were detectable the punctate dots of LC3 in the cytoplasm. Under TEM, cells stimulated by H2O2 showed the presence of swelling mitochondrial and some autophagosomes with cytoplasmic contents, which were rarely seen in control INS-1 cells. Western blot analysis showed that the expression of LC3B and BECN1 increased in H2O2-treated INS-1 cells in a time-dependent manner.
Cells transfected with BECN1 siRNA displayed decreased expression of LC3Ⅱafter H2O2 treatment, compared with control siRNA transfection group. Downregulation of BECN1 also decreased ROS-induced activation of caspase-9. The results of MTT assay and LDH release assay demonstrated that H2O2-induced cell death was mitigated after inhibition of autophagy.
Cells pretreated with anthocyanins exhibited decreased cytoplasmic AO and MDC staining. Immunoblotting analysis demonstrated decreased LC3II generation and the ratio of LC3Ⅱto LC3Ⅰin the presence of anthocyanins, compared with cells treated with H2O2 alone.
(4) Anthocyanins pretreatment improved the graft's tolerance in beta-cell transplantation model.
INS-1 cells were transplanted under left renal subcapsue.72h later, the graft was resected. Beta-cell grafts were confirmed by immunohistochemical staining for insulin. TEM examination showed that autophagic vacuolization was observed in transplanted cells. Notable immunopositive for BECN1 and detectable the punctate dots of LC3 in grafts further supported the occurrence of autophagy at the early phase of transplantation. In addition, TUNEL positive staining verified that some cells underwent apoptosis.
Beta-cell grafts pretreated with anthocyanins for 24h displayed less positive for active caspase-3, BECN1 and decreased extent of punctate dots of LC3.
2. The possible mechanism of anthocyanins'protective effect to beta-cells
(1) Anthocyanins increased HO-1 expression in beta-cell lines and islets
INS-1 cells treated with anthocyanins resulted in a dose-dependent increment in HO-1 mRNA(12h) and protein expression(24h). In Beta-TC-6 cells, a mouse insulinoma cell line, anthocyanins also cause the upregulation of HO-1 in a dose dependent manner.
Real-time PCR analysis demonstrated that the expression of HO-1 was significantly upregulated in primary islets treated with anthocyanins (6.2±1.9 fold increase compared to control, p<0.01). Immunocytochemical analysis of HO-1 in islets further supported this observation.
(2) Increased HO-1 expression contributed to the protective effect of anthocyanins
Compared to control siRNA transfection group, INS-1 cells transfected with HO-1 siRNA displayed more notable cell death after H2O2 stimulation. Reducing HO-1 expression by siRNA attenuated the protective effect of anthocyanidins against H2O2-induced cytotoxity, as reflected by MTT assay. Additionally, downregulating HO-1 in INS-1 cells displayed decreased of pro-caspase-3 and-9, but increased expression of cleaved caspase-9 and LC3II protein.
Overexpression of HO-1 in INS-1 cells reduced H2O2-induced injury. Similarly, immunofluorescence of MAP LC3 indicated that INS-1 overexpressing HO-1 displayed decreased autophagic cell death.
(3) Anthocyanins upregulate HO-1 expression via ERK1/2 and PI3K/Akt pathways
The results showed that anthocyanins dose-and time-dependently induced the activation of Akt and ERK1/2 via induction of phosphorylation. Inhibition of PI3K/Akt and ERK1/2 signaling with LY294002 and PD98059 attenuated the anthocyanins-induced HO-1 expression. The results of MTT assay showed that pre-incubating with PD98059 or LY294002 compromised the protective effect of anthocyanins compared with anthocyanins treating alone.
(4) Anthocyanins induced the nuclear translocation of transcription factor Nrf2
Compared to control group, INS-1 cells treated with anthocyanins displayed more notable Nrf2 accumulation in nucleus. Cellular protein of nuclear fraction was extracted, and immunoblotting for Nrf2 demonstrated that anthocyanins increased the expression of Nrf2 in nuleus.
Conclusions:
1. Cyanidin-3-O-glucoside (C3G) was indentified as a major anthocyanin component, which accounted for 95% of the total anthocyanins in Chinese bayberry fruit.
2. Anthocyanins pre-incubation decreased oxidative stress-induced intracellular ROS generation, and reduced ROS-mediated apoptosis and necrosis of beta cells.
3. Besides of apoptosis and necrosis, H2O2 exposure caused autophagic cell death in beta-cells and pretreated with anthocyanins attenuated such type of cell death.
4. Under various stimulations, cell grafts underwent apoptosis and autophagy at the early phase of posttransplantation. Anthocyanins pretreatment improved the graft's tolerance in beta-cell transplantation model.
5. Anthocyanins could time-and dose-dependently upregulate HO-1 expression in beta-cells. Overexpression of HO-1 decreased H2O2-induced apoptosis and autophagy, contributing to the protective effect of anthocyanins. Anthocyanins increased the HO-1 expression via ERK1/2 and PI3K/Akt pathways. Additionally, anthocyanins could induce transcription factor Nrf2 translocating into nucleus to increase its transcriptive activity.
胰岛在分离过程中胶原酶对其的毒性,分离纯化过程中的机械性损伤,离体后缺血、缺氧等应激均可导致胰岛细胞内过量活性氧自由簇(ROS)的产生,而胰岛本身其胞内抗氧化酶表达程度低,ROS介导的氧化应激在这些不利因素导致细胞损伤过程中扮演重要角色。过量及长时的ROS将会破坏细胞,自由基将破坏细胞蛋白,膜脂以及核酸,最终导致细胞凋亡及坏死。研究发现在胰岛分离培养过程中加入一些抗氧化剂可以减轻细胞损伤。如在胰岛分离过程中加入烟酰胺能够提高胰岛得率及保护胰岛细胞降低H202诱导的细胞死亡。在培养基中加入谷胱甘肽可以减少胰岛分离后MCP-1和TF的分泌。近年,越来越多的研究者将目光转向天然的、特别是来源于植物的抗氧化剂,一些黄酮类物质(如姜黄素)能够减少细胞因子刺激引起的胰岛细胞的凋亡；移植前预处理还可延长移植物的生存；某些植物组分还具有降低糖尿病鼠血糖,减少胰岛的损失及促进残余细胞胰岛素分泌的作用。
花色苷是一种广泛分布于自然界的水溶性天然食用色素,属于黄酮多酚类化合物,构成了花、果实、蔬菜中许多品种的蓝色、紫色、红色和黄色等,其在浆果类(edible berry)及其他有色植物中含量丰富。研究表明花色苷具有一系列的生理活性,如抗氧化,保持基因组DNA的完整性、抗炎症、抗肿瘤、降血脂等。富含花色苷的浆果类提取物能够增加红细胞对体外氧化应激的抵抗力,长期食用花色苷能够延缓糖尿病相关并发症的发生。Bollec等发现山茱萸花色苷可增加胰岛素释放功能。
杨梅是我国特有的亚热带水果,因含有花色苷、杨梅素、二氢杨梅素等大量生物活性物质而具有较高的营养和保健价值。既往研究表明,杨梅果实黄酮类化合物中,花色苷占较高比例。提取花色苷对其进行HPLC分析后发现,矢车菊素-3-葡萄糖苷(C3G)是最主要组分,占90%以上。杨梅果实提取物具有较强的氧自由基清除活性,抗氧化能力与C3G含量呈正相关。研究发现在高脂喂养的C57BL／6小鼠食用C3G能够明显降低血脂、血糖水平。杨梅花色苷具有良好的保健、医药开发前景。目前关于杨梅花色苷在生物医学方面的研究甚少,而对于其在胰岛功能保护领域的研究国内外均未见报道。
本研究属国家自然科学基金资助项目(编号：30872531),旨在探讨杨梅果实提取物(杨梅花色苷)对胰岛细胞氧化应激损伤的保护效应及其分子机制,探索其在胰岛移植领域的可能应用前景,为杨梅花色苷成为一种潜在的胰岛保护剂奠定相应理论基础。我们同时还初步探讨了自噬在胰岛体外氧化应激损伤及体内移植后的发生。
研究方法
1、杨梅花色苷对胰岛细胞的保护效应
(1)杨梅果实提取物(花色苷)的制备、鉴定及抗氧化活性的测定
杨梅(荸荠种)果实用含盐酸的甲醇溶液浸提,用旋转蒸发仪进行浓缩。提取物中总酚类化合物的含量采用Folin-Ciocalteau比色法测定,总黄酮类化合物的含量采用比色法测定,花色苷含量采用pH示差分光光度法测定。
采用HPLC对杨梅花色苷组成进行鉴定及含量测定；采用DPPH方法测定杨梅花色苷自由基清除活性。MTT法观察其对胰岛细胞系INS-1的细胞毒性。
(2)杨梅花色苷对H2O2诱导的胰岛细胞凋亡的影响
采用MTT法观察花色苷预处理对胰岛细胞系的保护效应,用台盼蓝据染实验及LDH释放试验进一步验证。
采用ROS特异荧光探针DCF-DA检测胞内ROS的产生。Hoechst33258染色观察细胞凋亡情况,流式细胞术观察细胞凋亡及死亡变化；提取细胞蛋白,western blot观察相关凋亡蛋白(cleaved) caspase-3,-9及Bcl-2的表达。
(3)杨梅花色苷对H2O2诱导的胰岛细胞自噬的影响
将：mRFP-GFP-LC3质粒转染INS-1细胞后,给予H2O2刺激,荧光显微镜下观察细胞内LC3的点聚情况。电镜观察H2O2刺激后细胞内自噬泡的形成。Western blot观察H2O2刺激不同时间后,LC3和BECN1的表达变化。
采用BECN1 siRNA下调BECN1表达,western blot观察自噬相关蛋白LC3和凋亡相关蛋白(cleaved) caspase-9的变化,并进一步用MTT法及LDH释放试验验证。
吖啶橙(AO)和单丹磺酰戊二胺(MDC)染色观察花色苷预处理是否可减少H2O2诱导的自噬的发生。Western blot观察LC3和BECN1蛋白的表达。
(4)杨梅花色苷预处理在肾包膜下移植后早期对细胞移植物的影响
将INS-1细胞移植入糖尿病受体小鼠肾包膜下,72h后取出移植物,部分用4%多聚甲醛固定,部分用戊二醛固定以备做电镜,部分OCT包埋剂包埋后液氮速冻,-80℃保存以备冰冻切片。石蜡切片行H&E、insulin和BECN-1免疫组化染色及TUNEL染色。冰冻切片行LC3免疫荧光染色,电镜观察移植物超微结构变化。
将INS-1细胞在移植前给予花色苷预处理24后肾包膜下移植,72h后取出移植侧肾脏,切片,行cleaved caspase-3、LC3免疫荧光染色及BECN-1免疫组化染色。
2.花色苷对胰岛细胞系保护机制的探讨
采用RT-PCR及定量PCR检测花色苷处理后INS-1细胞HO-1 mRNA的表达。western blot方法检测HO-1的蛋白表达水平。
分离小鼠原代胰岛,给予花色苷处理,RT-PCR和定量PCR观察HO-1的表达。将处理后的胰岛冰冻切片并进行Insulin和HO-1免疫荧光双染验证HO-1的表达。
用HO-1 siRNA下调HO-1的表达后,给予1uM花色苷24h, western blot检测HO-1表达变化。MTT法检测HO-1下调后花色苷对胰岛细胞的保护效应。Western blot检测caspase-3,-9及LC3的表达。
构建HO-1高表达的INS-1细胞。给予空载体及pLNCX2-HO1转染后的细胞H202刺激,MTT法检测两组细胞活性。免疫荧光观察两组细胞H202刺激后LC3的点聚情况。
给予INS-1细胞花色苷处理后,western blot观察pAkt及Akt, pERK1/2及ERK1/2,pJNK及JNK1/2蛋白的表达。用PD98059或LY294002分别预先处理INS-1细胞,再给予花色苷孵育,western blot检测HO-1蛋白表达情况。细胞经PD98059或LY294002预处理1h,花色苷孵育24h后,再给予H202,MTT法检测细胞活性改变。
给予INS-1细胞花色苷处理后,激光共聚焦显微镜方法检测Nrf2核转位情况,提取细胞核蛋白,western blot检测细胞核内Nrf2的表达。
研究结果
1.杨梅花色苷对胰岛细胞的保护效应
(1)杨梅花色苷中最主要的成分为矢车菊素-3-葡萄糖苷
杨梅果实提取物中花色苷为总黄酮类化合物中主要组成部分。对杨梅花色苷进行HPLC分析,发现矢车菊素-3-葡萄糖苷为杨梅花色苷最主要成分,比例在90%以上。DPPH法检测结果提示杨梅果实提取物具有较强的氧自由基清除活性。杨梅花色苷浓度在5uM以内时,对INS-1细胞的活性无明显影响。
(2)杨梅花色苷减少了H2O2诱导的胰岛细胞的凋亡和坏死
H2O2刺激导致细胞活性下降,并呈浓度和时间依赖性,MTT结果花色苷预处理能够显著提高细胞活性,降低H2O2对其的损伤。台盼蓝据染实验和LDH释放实验进一步证明1uM花色苷预处理24h可明显保护胰岛细胞系抵抗H2O2损伤。PI单染流式检测显示花色苷预处理明显减少了H2O2诱导的细胞坏死。
荧光显微镜及流式细胞仪检测提示花色苷预处理减少了H2O2诱导的胞内过量ROS的产生。流式细胞仪检测结果提示花色苷预处理减少了H2O2诱导的细胞凋亡及坏死。Western blot显示花色苷预处理减少了H2O2诱导的caspase-3,-9的剪切体的产生,并上调了Bcl-2的表达,进一步支持花色苷可减少H2O2诱导的凋
(3)杨梅花色苷减少了H2O2诱导的胰岛细胞的自噬
mRFP-GFP-LC3质粒转染INS-1细胞,给予H2O2刺激后,荧光显微镜观察到胞浆内出现LC3的点聚。电镜观察发现在H2O2处理组细胞胞浆内出现较多明显的空泡,有肿胀的线粒体及含胞内容物的自噬泡。Western blot结果提示1mMH2O2作用0.5,1,2及4h后,LC3B及BECN1的表达逐渐上调,且在2h处表达量最高。
采用BECN-1 siRNA转染INS-1细胞下调BECN-1的表达,降低了H2O2诱导的自噬的发生。Western blot结果显示BECN1 siRNA转染后H2O2诱导的caspase-9剪切体表达下降。MTT实验及LDH释放实验提示抑制自噬减少了H2O2刺激导致的细胞的损伤。
AO及MDC染色结果提示花色苷预处理减少了H2O2刺激导致的胞内自噬泡及酸性溶酶体的产生。Western blot结果显示H2O2刺激2h后LC3Ⅱ表达明显增加,LC3II/LC3I值增大,而花色苷预处理则降低了LC3Ⅱ的表达和LC3II/LC3I的比例。
(4)杨梅花色苷预处理对移植后早期细胞移植物的影响
INS-1细胞移植入肾包膜下72h后,取出移植物,insulin免疫组化染色证实为胰岛细胞系。TUNEL染色显示部分细胞移植物在72h时出现凋亡。免疫组化染色提示BECN1强阳性,LC3免疫荧光提示移植物胞浆内出现LC3的点聚,电镜检查显示移植物胞浆内出现含胞内容物的自噬泡,支持自噬的发生。
Cleaved caspase-3免疫荧光结果显示花色苷预处理组细胞移植物激活型caspase-3的表达低于未处理组。同时,与对照组相比,花色苷预处理组其BECN1的表达水平降低,胞浆内LC3的点聚程度也降低。
2.杨梅花色苷保护胰岛的机制探讨
(1)杨梅花色苷浓度及时间依赖型的上调HO-1的表达
定量PCR及western blot结果提示花色苷浓度及时间依赖的上调了INS-1细胞HO-1的表达。在小鼠胰岛细胞系beta-TC-6,花色苷同样可浓度依赖型的上调HO-1的表达。
RT-PCR及定量PCR结果显示花色苷孵育也明显上调了原代胰岛HO-1基因表达水平。胰岛冰冻切片Insulin和HO-1免疫荧光双染提示花色苷处理组胰岛细胞HO-1的表达水平高于未处理组。
(2)HO-1的上调表达参与了花色苷的保护效应
MTT结果显示HO-1 siRNA转染组细胞,H2O2刺激后,活性明显下降,并且降低了花色苷对INS-1细胞的保护效应。(?)Vestern blot结果显示,与单纯H2O2处理组相比,HO-1 siRNA转染组细胞caspase-3,-9前体表达水平下降,cleaved caspase-9表达增加,同时LC3Ⅱ表达及LC3Ⅱ/LC3Ⅰ值增加。
给予空载体与pLNCX2-HO1转染组INS-1细胞H2O2刺激,MTT结果显示HO-1过表达细胞其活性较对照组高。LC3免疫荧光染色显示HO-1过表达细胞胞内LC3点聚程度低于对照组。
(3)花色苷通过ERK1／2及P13K／Akt通路介导HO-1的上调表达Western blot结果提示花色苷浓度及时间依赖性的上调pERK1/2及pAkt的表达,而JNK通路则未被激活。PD98059及LY294002预处理抑制了花色苷对HO-1蛋白的上调表达。MTT结果显示与单一花色苷处理组相比,PD98059及LY294002预处理组细胞活性下降,降低了花色苷对胰岛细胞的保护效应。
(4)花色苷诱导转录因子Nrf2向细胞核内转移
激光共聚焦扫描结果表明,与未处理组相比,花色苷诱导了部分Nrf2向细胞核内的转移。提取细胞核蛋白,western blot结果提示1uM的花色苷诱导了Nrf2在细胞核内的表达。
小结：
1.杨梅花色苷中最主要成分为矢车菊素-3-葡萄糖苷,其比例占至90%以上。杨梅花色苷具有较强的氧自由基清除能力；其浓度在5uM以内时,对胰岛细胞系INS-1细胞无明显毒性。
2.花色苷预处理可以明显降低H202刺激导致的细胞内过量ROS的产生,进而减少氧化应激损伤所致的细胞凋亡和坏死。
3.H2O2刺激除可导致细胞凋亡和坏死外,还可引起细胞发生自噬,且发生的自噬促进细胞死亡(即为“自噬性细胞死亡”)。花色苷预处理可以减少H2O2诱导细胞自噬的发生。
4.细胞移植入肾包膜下早期(72h),在各类刺激下可发生凋亡和自噬。在移植前予以花色苷预处理,可降低细胞移植后凋亡和自噬的发生程度。
5.花色苷可上调胰岛细胞(系)HO-1的表达,且存在明显的时间-效应关系和剂量-效应关系。HO-1的上调表达可减少H202诱导的自噬和凋亡,参与了花色苷对胰岛细胞的保护作用。花色苷通过ERK1/2及PI3K/Akt通路介导了HO-1的表达上调。同时,花色苷还可诱导核转录因子Nrf2向胞核转移,增加其转录活性。
Diabetes is a metabolic disease, possibly caused by both of polygenic inheritance and environmental factors. It is characterized by chronic hyperglycemia, mainly due to absolutely or relatively insufficient insulin release from beta-cells or insulin resistance of surrounding tissues. Islet transplantation is a promising therapy for all of type 1 and part of type 2 diabetic patients, which could regulate blood glucose manipulating physiological condition, achieve insulin independence, and delay the occurrence of diabetes-associated complications. However, during the process of islet isolation, purification and transplantation, there exists notable decrease in the number, function and viability of islet grafts. After transplantation, non-specific inflammation and immune rejection also cause the injury to the grafts. All these factors cause the situation that more than one donor are needed for one receipt to achieve insulin independence. However, the inadequate of donor exacerbates the obstacles limiting the development of islet transplantation in clinics. Improved islet isolation and better viability/function preservation before transplantation have been the keys to the evolution of this procedure.
During isolation procedure, islets are exposed to the cytotoxicity of collagenase. mechanical trauma, ischemia and hypoxia, all of which contribute to the excessive production of reactive oxygen species (ROS). ROS-mediated oxidative stress plays an important role in the process of cell injury caused by these stimulations. Pancreatic beta-cells usually express low levels of antioxidant enzymes, the long-term exposure of excessive ROS will damage the intracellular proteins, membrane lipid and nucleic acid, eventually leading to apoptosis and necrosis. Some researchers found that adding some antioxidants during islet isolation and culture process could alleviate cellular injury. Nicotinamide (NA) supplementation of the processing medium during islet isolation significantly improved islet yields and protected beta-cells from cell death (both necrosis and apoptosis) induced by H2O2. Addition of glutathione in culture medium significantly reduced CCL2/MCP-1 and tissue factor (TF) production. Recently, many researchers focused on the protective role of naturally existed antioxidant, especially from plant. Some flavonoids (such as curcumin) could protect islets against cytokines-induced apoptosis; pretreatment of islets with some antioxidants could prolong the survival of graft after transplantation. Some plants themselves have hypoglycemia effect in diabetic rodent model, reduce the extent of islet loss, and promote the insulin release from the left islets.
Anthocyanins, one type of flavoids, are naturally occurring water soluble polyphenolic compounds in the plant foods and widely distributed in fruits, vegetables, and pigmented cereals, imparting color (blue, purple, red, yellow etc) to plants. Anthocyanin is rich in edible berries and other colorful plants. Many studies have shown that anthocyanins exhibits an array of pharmacological properties, such as keeping the integrity of genome DNA, antioxidative. anti-inflammatory, antitumor and antiatherosclerotic activities. Berry extracts rich in anthocyanins could improve resistance of red blood cell against oxidative stress in vitro. Long-term administration of berry-derived supplements including anthocyanins could inhibit the development of the early stages of some diabetic complications without adverse drug reactions. Bollec et al suggested that bioactive anthocyanins from Cornus fruits could increase insulin release of INS-1 cells.
Chinese bayberry, one of six myrica species native to China, is a fruit with high nutrition and health values due to the content of various bioactive substances such as anthocyanins, myricetin and dihydro-myricetin. Our previous study demonstrated that anthocyanins occupies the major portion of total flavonoid compounds. HPLC analysis of Chinese bayberry extract showed that cyanidin-3-O-glucoside (C3G) was the major anthocyanin component, accounting for 95% of the total anthocyanins. Chinese bayberry extracts possess notable radical scavenging activities indicated by DPPH and ABTS cation assays, which is correlated to the content of C3G. C3G was observed to exert hypolipemia and hypoglycemia effects in high-fat-diet (HFD)-feed C57BL/6 mice. Up to now, the application of anthocyanins in CBE has not been fully investigated. Whether anthocyanins has protective effect for islet has not been reported before.
The present topic, supported by the National Natural Science Foundation (serial number:30872531), is to explore whether Chinese bayberry extract could protect against oxidative stress-induced beta-cell injury and its possible mechanism, and is to investigate the possible application of anthocyanins as an islet-protective agent in the field of islet transplantation. We also discuss the occurrence of autophagy in beta-cells induced by oxidative stress in vitro or after transplantation.
Experimental Procedures
1. The protective effect of anthocyanins to pancreatic beta-cells
(1) Chinese bayberry extract preparation, HPLC analysis and antioxidant ability assessment.
Chinese bayberry fruits (Biqi) were ground and extracted in methanol acidified with HCl, and concentrated through rotary evaporation. Total phenolics content was estimated using the Folin-Ciocalteau colorimetric method. Total flavonoids were determined by colorimetric method after reaction with NaNO2 and Al(NO3)3. Anthocyanin quantitation was performed by the pH differential method. HPLC analysis was performed to identify the composition. Free radical scavenging activity of fruit extracts was measured according to the DPPH method. MTT assay was performed to observe the cytotoxicity of extract to INS-1 cells.
(2) The effect of anthocyanins on H2O2-induced apoptosis of pancreatic beta-cells. INS-1 cells were pre-incubated with anthocyanins followed by H2O2 stimulation. Cell viability was evaluated by MTT assay. The protective effect of anthocyains was further confirmed by trypan blue exclusion method and LDH release assay.
A ROS-specific probe, DCF-DA, was used to detect intracellular ROS production in INS-1 cells after various treatments. INS-1 cells stimulated by various agents were stained with Hoechst 33258 to observe the apoptosis. Flow cytometry analysis was adopted to quantify the apoptosis and necrosis. The expression of apoptosis-related proteins (caspase-3,-9 and Bcl-2) was assessed by western blot.
(3) The effect of anthocyains on H2O2-induced autophagy in pancreatic beta-cells
INS-1 cells, transfected with mRFP-GFP-LC3 plasmid, were treated with H2O2 and autophagy was evaluated by observing the formation of intracellular LC3 punctuate dots. After experimental manipulations, cells were fixed by glutaraldehyde and osmic acid, and were further observed by transmission electron microscopy. The expression of autophagic-related proteins (LC3 and BECN1) was determined by western blot to confirm the occurrence of autophagy.
After transfected with BECN1 siRNA, INS-1 cells were exposed to H2O2 for 2h, and the expressions of LC3 and caspase-9 were evaluated. MTT assay and LDH release assay were performed to observe the changes of cell injury after downregulating autophagy.
INS-1 cells, treated with various protocols, were stained by AO and MDC to assess the degree of lysosomes and autophagosome formation. The expression of LC3 and BECN1 were evaluated.
(4) The effect of anthocyanins on cell graft at the early phase of posttransplantation beneath renal capsue
INS-1 cells were transplanted under the left renal capsule of diabetic mice. Seventy-two hours later, the beta-cell grafts were harvested, snap frozen in liquid nitrogen or fixed with 4% paraformaldehyde, or were fixed in glutaraldehyde for transmission electronic microscopic analyzation.
Immunohistochemistry staining for BECN1, immunofluorescence staining for LC3, TUNEL staining and transmission electronic microscopic analyzation were performed.
INS-1 cells pretreated with or without anthocyanins were transplanted under renal capsule of diabetic mice. The cell grafts were harvest after 72h. Immunofluorescence for cleaved caspase-3 and LC3 and immunohistochemistry staining for BECN1 were carried out.
2. Possible mechanism of anthocyanins'protective effect.
RNA was extracted from INS-1 cells treated with luM anthocyanins for various time or incubated with various concentrations of anthocyanins for 12h. The mRNA level of HO-1 was determined by reverse transcription PCR and real-time PCR. The expression of HO-1 protein was evaluated by western blot.
Primary islets were isolated from mice.100-200 islets were incubated in culture medium with or without 2uM anthocyanins for 6h. The expression of HO-1 mRNA in islets was determined by real-time PCR. Dual immunofluorescence for insulin and HO-1 of islets was performed to further observe the HO-1 expression.
The expression of HO-1 in INS-1 cells was downregulated by siRNA transfection. After transfected with HO-1 or control siRNA. INS-1 cells were incubated with luM anthocyanins for 24h, then were exposed to H2O2 for 2h, MTT assay was performed to evaluate the cellular viability. The expression of caspase-3,-9 and LC3 proteins were determined.
The pLNCX2-HO1 vector was constructed and transfected to INS-1 cells. INS-1 cells transfected with pLNCX2-HO1 or empty vector were exposed to H2O2. MTT assay was carried out to assess the viability between two groups. Immunofluorescence for LC3 was performed to evaluate the intracellular LC3 punctuate dot formation in both type of cells treated with H2O2.
INS-1 cells were treated with various concentrations of anthocyanins for 24h, or 1uM anthocyanins for different time. The expressions of pAkt, Akt, pERK1/2, ERK1/2, pJNK and JNK protein were evaluated by western blot. After pretreated with PD98059 (ERK1/2 inhibitor) or LY294002 (PI3K inhibitor) for 1h, INS-1 cells were further incubated with anthocyanins. HO-1 protein expression was evaluated. INS-1 cells were preincubated with PD98059 or LY294002 for 1h, treated with anthocyanins for 24h, followed by H2O2 stimulation. MTT assay was performed to evaluate the viability.
INS-1 cells were incubated with anthocyanins, and Immunofluorescence for Nrf2 was performed and observed under confocal microscopy. Nuclear protein fraction was extracted from cells treated as above, and immunoblotting for Nrf2 and LaminA/C was performed.
Results
1. The protective effect anthocyanins on pancreatic beta-cells.
(1) The major portion of anthocyanins in Chinese bayberry extract is cyanidin-3-glucoside (C3G).
Anthocyanin is the major portion of total flavonoids in Chinese bayberry extract. HPLC analysis of Chinese bayberry extract showed that cyanidin-3-O-glucoside (C3G) was the major anthocyanin component, accounting for more than 90% of the total anthocyanins. DPPH assay indicated the strong antioxidant capacity of CBE, and C3G is the dominant attributor. There is no cytotoxicity to INS-1 cells below the concentration of 5uM.
(2) Anthocyanins alleviated H2O2-induced apoptosis and necrosis of INS-1 cells
H2O2 injured INS-1 cells in a dose-and time-dependent manner. Pretreatment of INS-1 cells with anthocyanins resulted in the prevention of cell death by H2O2. Trypan blue exclusion assay and LDH release assay further confirmed that treatment with 1 uM anthocyanins for 24h could protect bete-cells against H2O2-induced injury. FCM analysis also displayed that anthocyanins decreased H2O2-induced cell necrosis.
Compared to normal cells, H2O2 caused excessive ROS production. Anthocyanins preincubation also decreased oxidative stress-induced intracellular ROS generation. FCM demonstrated anthocyanins incubation reduced the extent of both cellular apoptosis and necrosis induced by H2O2 stimulation. Treatment of INS-1 cells with 1mM H2O2 caused the activation of caspase-9 and-3, which were mitigated by pretreatment with anthocyanins in a concentration dependent manner.
(3) Anthocyanins decreased H2O2-induced autophagy in pancreatic beta-cells
GFP-mRFP-LC3 fluorescence was diffusely distributed in untreated group, whereas in cells treated with H2O2 there were detectable the punctate dots of LC3 in the cytoplasm. Under TEM, cells stimulated by H2O2 showed the presence of swelling mitochondrial and some autophagosomes with cytoplasmic contents, which were rarely seen in control INS-1 cells. Western blot analysis showed that the expression of LC3B and BECN1 increased in H2O2-treated INS-1 cells in a time-dependent manner.
Cells transfected with BECN1 siRNA displayed decreased expression of LC3Ⅱafter H2O2 treatment, compared with control siRNA transfection group. Downregulation of BECN1 also decreased ROS-induced activation of caspase-9. The results of MTT assay and LDH release assay demonstrated that H2O2-induced cell death was mitigated after inhibition of autophagy.
Cells pretreated with anthocyanins exhibited decreased cytoplasmic AO and MDC staining. Immunoblotting analysis demonstrated decreased LC3II generation and the ratio of LC3Ⅱto LC3Ⅰin the presence of anthocyanins, compared with cells treated with H2O2 alone.
(4) Anthocyanins pretreatment improved the graft's tolerance in beta-cell transplantation model.
INS-1 cells were transplanted under left renal subcapsue.72h later, the graft was resected. Beta-cell grafts were confirmed by immunohistochemical staining for insulin. TEM examination showed that autophagic vacuolization was observed in transplanted cells. Notable immunopositive for BECN1 and detectable the punctate dots of LC3 in grafts further supported the occurrence of autophagy at the early phase of transplantation. In addition, TUNEL positive staining verified that some cells underwent apoptosis.
Beta-cell grafts pretreated with anthocyanins for 24h displayed less positive for active caspase-3, BECN1 and decreased extent of punctate dots of LC3.
2. The possible mechanism of anthocyanins'protective effect to beta-cells
(1) Anthocyanins increased HO-1 expression in beta-cell lines and islets
INS-1 cells treated with anthocyanins resulted in a dose-dependent increment in HO-1 mRNA(12h) and protein expression(24h). In Beta-TC-6 cells, a mouse insulinoma cell line, anthocyanins also cause the upregulation of HO-1 in a dose dependent manner.
Real-time PCR analysis demonstrated that the expression of HO-1 was significantly upregulated in primary islets treated with anthocyanins (6.2±1.9 fold increase compared to control, p<0.01). Immunocytochemical analysis of HO-1 in islets further supported this observation.
(2) Increased HO-1 expression contributed to the protective effect of anthocyanins
Compared to control siRNA transfection group, INS-1 cells transfected with HO-1 siRNA displayed more notable cell death after H2O2 stimulation. Reducing HO-1 expression by siRNA attenuated the protective effect of anthocyanidins against H2O2-induced cytotoxity, as reflected by MTT assay. Additionally, downregulating HO-1 in INS-1 cells displayed decreased of pro-caspase-3 and-9, but increased expression of cleaved caspase-9 and LC3II protein.
Overexpression of HO-1 in INS-1 cells reduced H2O2-induced injury. Similarly, immunofluorescence of MAP LC3 indicated that INS-1 overexpressing HO-1 displayed decreased autophagic cell death.
(3) Anthocyanins upregulate HO-1 expression via ERK1/2 and PI3K/Akt pathways
The results showed that anthocyanins dose-and time-dependently induced the activation of Akt and ERK1/2 via induction of phosphorylation. Inhibition of PI3K/Akt and ERK1/2 signaling with LY294002 and PD98059 attenuated the anthocyanins-induced HO-1 expression. The results of MTT assay showed that pre-incubating with PD98059 or LY294002 compromised the protective effect of anthocyanins compared with anthocyanins treating alone.
(4) Anthocyanins induced the nuclear translocation of transcription factor Nrf2
Compared to control group, INS-1 cells treated with anthocyanins displayed more notable Nrf2 accumulation in nucleus. Cellular protein of nuclear fraction was extracted, and immunoblotting for Nrf2 demonstrated that anthocyanins increased the expression of Nrf2 in nuleus.
Conclusions:
1. Cyanidin-3-O-glucoside (C3G) was indentified as a major anthocyanin component, which accounted for 95% of the total anthocyanins in Chinese bayberry fruit.
2. Anthocyanins pre-incubation decreased oxidative stress-induced intracellular ROS generation, and reduced ROS-mediated apoptosis and necrosis of beta cells.
3. Besides of apoptosis and necrosis, H2O2 exposure caused autophagic cell death in beta-cells and pretreated with anthocyanins attenuated such type of cell death.
4. Under various stimulations, cell grafts underwent apoptosis and autophagy at the early phase of posttransplantation. Anthocyanins pretreatment improved the graft's tolerance in beta-cell transplantation model.
5. Anthocyanins could time-and dose-dependently upregulate HO-1 expression in beta-cells. Overexpression of HO-1 decreased H2O2-induced apoptosis and autophagy, contributing to the protective effect of anthocyanins. Anthocyanins increased the HO-1 expression via ERK1/2 and PI3K/Akt pathways. Additionally, anthocyanins could induce transcription factor Nrf2 translocating into nucleus to increase its transcriptive activity.
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