辛伐他汀和血脂康对糖尿病大鼠肾脏细胞凋亡的影响及其可能机制
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摘要
随着人们生活水平的提高,人均寿命的延长,糖尿病及其慢性并发症对患者的健康和生命造成严重威胁,是导致糖尿病致死率和致残率增高的最主要原因。糖尿病肾病是糖尿病最常见和严重的微血管并发症之一,也是导致终末期肾功能衰竭的最主要原因之一。糖尿病肾病的发病机制还没有完全明确,随着对细胞凋亡的研究的不断深入,人们逐渐把细胞凋亡和糖尿病肾病联系起来,认为肾脏细胞的缺失可能是糖尿病肾病的早期表现。
他汀类药物是3-羟-3-甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,临床上主要用于治疗脂代谢异常。他汀类药物在临床上应用十分广泛,糖尿病患者常伴有血脂调节异常,且血脂控制要求较非糖尿病患者更为严格,因此在糖尿病患者中他汀类药物的使用尤为普遍。众多研究表明他汀类药物具有肾脏保护作用,可以延缓糖尿病肾病的进展。辛伐他汀是他汀类药物的一种,临床应用较为广泛,大规模的临床研究证实辛伐他汀能够延缓糖尿病患者肾小球滤过率(GFR)的下降。血脂康是一种天然他汀类调脂药,由特制红曲精炼而成,主要包含洛伐他汀在内的13种天然他汀及他汀同系物,还含有多种人体必需的氨基酸和不饱和脂肪酸及多糖等,基础和临床研究提示血脂康具有调节氧化应激、改善内皮细胞功能及抑制炎症反应等作用,Meta分析显示血脂康能够降低24小时尿蛋白及尿微量白蛋白排泄率。
本研究通过链脲佐菌素(STZ)腹腔注射诱导糖尿病大鼠模型,分别给予辛伐他汀和血脂康治疗,拟观察糖尿病时大鼠肾脏的结构改变和组成细胞的凋亡情况,以及辛伐他汀和血脂康对肾脏结构功能和细胞凋亡的影响,并进一步探讨其可能的作用机制。
1材料与方法
1.1试剂及材料
STZ购自Sigma公司,辛伐他汀由默沙东公司馈赠,血脂康由北大维信馈赠,PAS、Masson染色试剂盒购自珠海贝索,TUNEL原位凋亡染色试剂盒购自Roche公司,Caspase-9、Bax抗体购自Santa cruz, Bcl-2抗体购自武汉博士德,细胞色素c抗体购自BD公司。
1.2实验动物模型建立及分组
清洁级雄性Sprague Dawley(SD)大鼠90只,体重180-220克(购自浙江大学医学院动物实验中心),饲以标准饲料块,随意充分饮水,室内保持通风和相对恒温,保持正常昼夜节律。适应性饲养1周后随机选取70只采用STZ一次性腹腔注射方法建立糖尿病模型,建模前禁食12小时,注射剂量60mg／kg(溶于0.1mol／L柠檬酸缓冲液,pH 4.2),注射后48小时及72小时分别尾静脉采血,血糖≥16.7mmol／L者为造模成功,正常对照组注射相应体积的柠檬酸缓冲液。糖尿病模型确立后将糖尿病大鼠随机分为3组：1.辛伐他汀治疗组(SIM组,n=22)：每天予辛伐他汀灌胃,给药剂量20mg／kg；2.血脂康治疗组(XZK组,n=22)：每天予血脂康灌胃,给药剂量1200mg／kg；3.糖尿病组(DM组,n=22)：每天予相应体积生理盐水灌胃。另20只设为正常对照组(SD组,n=20),每天予相应体积生理盐水灌胃。建模开始后第7天开始给药。
1.3生化指标检测
给药3月后每组分别取半数大鼠处死,给药6月后处死剩余大鼠。各大鼠处死前放入代谢笼收集24小时尿,离心后采用散射比浊法(Beckman CoulterImmage800)测定尿白蛋白。腹腔注射麻醉后称体重,经腹主动脉采血,BeckmanLX20全自动生化仪检测血糖及血脂。
1.4肾脏形态学观察及细胞凋亡情况
用4℃生理盐水注射液灌洗大鼠腹主动脉后,迅速取出双侧肾脏,右肾去包膜用滤纸吸干血迹后称重,右肾重量(g)／体重(kg)比值为肾重指数。将右肾对半剖开,其中一半10%中性福尔马林固定,石蜡包埋后切片,行PAS染色和Masson染色检测肾组织内糖原及胶原纤维沉积情况,TUNEL法原位检测凋亡细胞。每例切片高倍镜下观察5个视野并进行半定量分析。
1.5肾皮质Bax、Bcl-2、Caspase-9、细胞色素c的表达
左肾分离皮质,-80℃冷冻保存,提取总蛋白行Western Blot检测肾皮质Bax、Bcl-2和活化的Caspase-9的表达：右肾剩余一半置于冰上,取皮质去除线粒体后提取细胞浆蛋白,Western Blot方法测定细胞色素c表达。同时采用免疫组化方法检测肾组织内Bax及Bcl-2的定位表达,并进行半定量分析。免疫组化图像分析采用Image Pro Plus (IPP)6.0图像分析软件进行；Western Blot图像采集及分析采用化学发光荧光影像分析仪及配套软件进行。
1.6统计方法
实验数据以平均值±标准差表示,使用SPSS 16.0软件进行统计分析。多组数据平均值比较采用方差分析(ANOVA),组间比较用Bonferroni分析进行。所有检验均为双侧检验,有显著性统计学差异定义为P<0.05。
2实验结果
2.1大鼠一般情况
共70只大鼠接受糖尿病造模,其中66只血糖≥16.7mmol／L,确认为造模成功,4只血糖未达到标准。66只糖尿病大鼠随机分为3组,饲养过程中监测血糖,其中7只血糖不达标排除,另有4只死亡,最终各组大鼠数量为：SD组3月,n=10；SD组6月,n=10；DM组3月,n=9；DM组6月,n=10；SIM组3月,n=10；SIM组6月,n=9；XZK组3月,n=9；XZK组6月,n=8。
正常对照组大鼠血糖均低于7mmol／L,尿糖阴性,注射STZ后48小时及72小时大鼠血糖明显升高(≥16.7mmol／L),实验期间监测血糖维持造模标准,辛伐他汀和血脂康对糖尿病大鼠的血糖没有改善作用；实验期间正常SD大鼠体重逐渐增加,没有多饮多尿多食症状,而糖尿病大鼠体重增长缓慢或不增长,出现较明显的三多一少等糖尿病常见临床表现,辛伐他汀和血脂康治疗均未能恢复糖尿病大鼠的体重。各实验组大鼠血脂水平未见显著性差异。
2.2肾重指数及24小时尿白蛋白排泄量
糖尿病组3月和6月后肾重指数出现明显增加(P<0.01),辛伐他汀和血脂康治疗后各时间点肾重指数均较糖尿病组呈下降趋势(P<0.05或P<0.01),但仍高于正常对照组(P<0.01),两种药物之间未见明显统计学差异。
正常对照组尿白蛋白排泄量较少,糖尿病造模3月后尿白蛋白排泄明显增多6月后进一步加重(P<0.01)：辛伐他汀和血脂康治疗3月和6月后尿白蛋白排泄量较同时间点的糖尿病组减少(P<0.01或P<0.05),但仍高于同一时间点的正常对照组(P<0.05或P<0.01)；辛伐他汀治疗组和血脂康治疗组间尿白蛋白排泄量无统计学差异。
2.3肾组织PAS和Masson染色结果
PAS染色见正常大鼠肾小球及肾小管结构清晰,肾小管上皮细胞排列整齐,糖尿病模型组大鼠3月后可见肾小球系膜区扩张,肾小管基底膜不规则增厚,肾小球系膜及基底膜区PAS阳性物质增多,较正常对照组有显著性差异(P＜0.01),6月后上述区域PAS阳性物质沉积进一步增多。辛伐他汀治疗能逆转大鼠肾脏PAS阳性物质的沉积增加(P＜0.01),且与正常对照组未见明显差异(P＞0.05)；血脂康治疗亦能减少糖尿病时肾脏PAS阳性物质的沉积(P＜0.01),但仍高于同一时间点的正常对照组(P＜0.05)。给药3月时血脂康治疗组肾组织内PAS阳性物质的沉积多于辛伐他汀治疗组(P＜0.05),6月后两组间未见明显差异。
Masson染色显示3月后糖尿病模型组大鼠肾小球及肾小管基底膜区、肾小球系膜区胶原纤维沉积较正常对照组明显增多,6月后进一步加重(P＜0.01)。辛伐他汀治疗3月或6月后肾组织内胶原沉积较相应时间点的糖尿病模型组均明显减少(P＜0.01),但仍较正常对照组增多(P＜0.05)。血脂康治疗6月后肾组织内胶原沉积较糖尿病模型组显著减少(P<0.01),但治疗3月时血脂康组与糖尿病组未见明显差异,治疗3月或6月后与正常对照组均存在显著差异(P＜0.01)。辛伐他汀治疗组胶原沉积少于血脂康治疗组,3月和6月时两组间均存在显著性差异(P<0.01或P<0.05)。
2.4肾组织细胞凋亡检测结果
TUNEL原位凋亡染色显示正常对照组大鼠肾脏内凋亡细胞很少,主要位于远端肾小管上皮细胞,糖尿病模型组大鼠肾脏凋亡细胞明显增多(P＜0.01),主要集中于肾远曲小管、近曲小管,肾小球亦可见凋亡细胞,数量较肾小管区域少,随糖尿病病程延长肾脏细胞凋亡逐渐增加。辛伐他汀治疗3月和6月后肾脏凋亡细胞较糖尿病组均明显减少(P＜0.01),3月时与正常对照组间未见明显差异,6月时肾脏凋亡细胞较正常对照组增多(P＜0.05)。血脂康治疗3月和6月后肾脏细胞凋亡较相应时间点的糖尿病组明显减少(P＜0.01),但均多于正常对照组(P<0.05)。
2.5肾组织内Caspase-9和细胞色素c的表达情况
Western Blot显示正常对照组活化的Caspase-9和细胞色素c的表达较少,糖尿病组活化的Caspase-9和细胞浆内细胞色素c的表达明显增加,辛伐他汀和血脂康治疗组活化的Caspase-9和细胞浆内细胞色素c表达水平较糖尿病组降低,但仍高于正常对照组。
2.6肾组织内Bax和Bcl-2的表达情况
免疫组化结果和Western Blot结果显示,正常对照组大鼠肾组织中Bax和Bcl-2表达量较少,糖尿病模型组Bax表达显著增加(P<0.01),随着病程延长逐渐增加；糖尿病组3月时肾组织中Bcl-2表达较正常对照组稍有增加,但未见明显差异,6月时Bcl-2表达较正常对照组显著下降(P＜0.01),糖尿病组Bax/Bcl-2比值高于正常对照组(P＜0.01),随病程进展进一步升高。辛伐他汀和血脂康治疗可以明显抑制糖尿病肾脏组织中Bax表达的增加,上调Bcl-2的表达,从而降低Bax/Bcl-2比值(P<0.01)。
3结论
3.1糖尿病肾病时肾脏细胞出现明显凋亡,且随病程延长逐步进展,辛伐他汀和血脂康能改善糖尿病时肾脏的病理改变,减少肾脏细胞的凋亡,其作用不依赖于他汀类药物的降脂效应；
3.2辛伐他汀和血脂康能减少肾皮质内Caspase-9的活化以及细胞浆内的细胞色素c水平,可以通过抑制线粒体凋亡通路的活化减少肾脏细胞的凋亡；
3.3辛伐他汀和血脂康能降低Bax/Bcl-2的比值,可能通过调节Bcl-2蛋白家族的表达减少细胞凋亡。
The prevalence of diabetes is increasing at an alarming rate, regardless of the population studied. Due to rapid economic growth, an increase in life expectancy and changes in lifestyle, diabetes and its complications have become great threats to global health in the 21st century. Diabetic nephropathy, common but severe microangiopathy in diabetes, is becoming one of the major reasons that lead to end stage renal dysfunction. The mechanism of diabetic nephropathy is not clear yet. Researchers have hooked renal cell apoptosis with diabetic nephropathy recently, as the studies on cell apoptosis going deeper, suggesting renal cell apoptosis an early manifestation of diabetic nephropathy.
Statins, HMG-CoA reductase inhibitors, are a class of drugs used to lower plasma cholesterol levels. Statins are used widely in clinical works, especially in diabetic patients, since diabetic patients often suffer from lipid dysfunction and the criterion of lipid control is stricter for diabetic patients. Studies have demonstrated a protective effect of statins on renal function that statins could ameliorate the progress of diabetic nephropathy. Simvastatin, used widely clinically, is confirmed to attenuate the decline of glomerular filtration rate in diabetic patients according to large scale clinical trials. Xuezhikang is a natural statin extract from red yeast rice, including thirteen natural statins and homologues of statin. Xuezhikang is suggested to have effect on oxidative stress, endothelial cell function and inflammation according to basic and clinical researches. Meta analysis also demonstrated that xuezhikang could lower urinary protein and albumin extretion.
Streptozocin (STZ) induced diabetic rats were used in this study, giving simvastatin and xuezhikang treatment after establishment of diabetic model. Renal histological changes and renal cell apoptosis in each group (control group, diabetic group, diabetic group with simvastatin treatment and diabetic group with xuezhikang treatment) were observed, and the possible mechanism how statins affect cell apoptosis was also investigated.
1 Materials and methods
1.1 Reagents and materials
STZ was from Sigma, simvastatin from Merck and Co. China, xuezhikang from Beijing WBL Peking University Biotech Co., PAS and Masson staining kits from Baso Diagnostics Inc. Zhuhai, TUNEL apoptosis assay kit from Roche, Caspase-9 and Bax antibodies from Santa cruz, Bcl-2 antibody from Boster (Wuhan), and cytochrome c antibody from BD Inc.
1.2 Induction of diabetes and experimental groups
Ninety male Sprague-Dawley (SD) rats, of 180-220g, were purchased from the Experimental Animal Center of Zhejiang University and raised with standard food, adequate water and normal light/dark cycle. Diabetes was induced by a single intraperitoneal injection of 1% STZ (in cold 0.1M citrate buffer. pH 4.2) at 60mg/kg body weight. Blood samples for glucose measurements were taken from the tail vein 48-72 hours after STZ injection and rats with blood glucose of 16.7mmol/L or higher were considered diabetic rats. The control rats were injected with equal volume of citrate buffer instead. The experimental groups included (1) normal control rats (group SD); (2) diabetic control group (group DM); (3) diabetic rats treated with simvastatin (20mg/kg body weight gastric perfusion everyday, group SIM); (4) diabetic rats treated with xuezhikang (1200mg/kg body weight gastric perfusion everyday, group XZK). Treatments started 7 days after STZ injection. Rats from group SD and DM received equal volume of saline instead.
1.3 Detection of clinical measurements
Half the rats from each group were sacrificed after three months'treatments and the rest after six months'treatments. Twenty-four hours'urine was collected using metabolic cages and urinary albumin excretion was measured by rate nephelometry (Beckman Coulter Immage800). After weighing the body weight, blood sample was collected from abdominal aorta for glucose and lipid detection by Beckman Coulter LX20 biochemical analyzer.
1.4 Histological examination and apoptosis detection
After lavaging the abdominal aorta by cold saline, both kidneys were taken out. The ratio of right kidney weight to body weight was recorded as kidney index. The right kidney was cut into two pieces, half of which was fixed by formalin and embedded by paraffin. Paraffin-embedded tissue section (3μm thick) were deparaffinized. rehydrated. and stained with PAS and Masson's trichrome for saccharides and collagen detection, and TUNEL for apoptosis detection.
1.5 Expression of Bax, Bcl-2, Caspase-9 and Cytochrome c
Renal cortex was separated and stored in-80℃. After extracting the total protein, Bax, Bcl-2 and active Caspase-9 expression in renal cortex were detected by western blotting. Half of the right kidney was used for detection of cytochrome c expression after removing mitochondria. Bax and Bcl-2 expression were also detected by immunohistochemistry.
1.6 Statistical analysis
All data were presented as mean±SD of the mean. When multiple comparisons were made, statistical significance was determined using one-way ANOVA followed by Bonferroni's test. All statistical analyses were performed using the software package SPSS 16.0 and P<0.05 was considered statistically significant.
2 Results
2.1 General data
Seventy SD rats underwent STZ injection, sixty-six of which had blood glucose of 16.7mmol/L or higher were considered diabetic rats and divided into three groups randomly. Except those died during experiment and blood glucose lower than 16.7mmol/L, the final number of rats in each group was as follows:SD (3 months). n=10; SD (6 months), n=10; DM (3 months), n=9; DM (6 months), n=10; SIM (3 months), n=10; SIM (6 months), n=9; XZK (3 months), n=9; XZK (6 months), n=8.
The blood glucose was less than 7mmol/L in control group and increased dramatically (≥16.7mmol/L) 48-72 hours after STZ injection; neither simvastatin or xuezhikang treatment showed significant differences from group DM in blood glucose level. The STZ-induced diabetic rats showed classic diabetic symptoms of polyuria, polydipsia and weight loss:simvastatin and xuezhikang did not help to retrieve the body weight. No significant difference was found in lipid level among each group.
2.2 Kidney index and 24 hours'urinary albumin excretion
Kidney index of group DM increased significantly after three and six months(P< 0.01). Simvastatin and xuezhikang treatment could decrease kidney index compare to that of group DM at each time point (P< 0.05或P< 0.01), but kidney indexes of group SIM and XZK were higher compare to that of group SD (P<0.01). There was no significant difference between the two drugs.
Urinary albumin excretion was low in group SD, which increased in group DM after three months and progressed after six months (P< 0.01). Three and six months' treatment of simvastatin and xuezhikang could reduce urinary albumin excretion compare to that of group DM (P< 0.01或P< 0.05). There was no difference between group SIM and group XZK in urinary albumin excretion.
2.3 Results of PAS and Masson stain
Pathological sections stained with PAS showed that control rats had normal glomerulus and renal tubule, with tubular cells arraying in order. However, diabetic rats showed dilation of glomerular mesangial region, irregular thickening of tubular basement, as well as increased deposition of PAS positive substance after three and six months (P< 0.01). Simvastatin treatment could totally alleviate the deposition of PAS positive substance in diabetic kidney (P<0.01). PAS positive substance was reduced significantly after xuezhikang treatment (P< 0.01), but more than that in control rats at each time point (P< 0.05). There was difference between group SIM and group XZK at three months (P< 0.05), which disappeared six months later.
Masson's trichrome stain showed increased accumulation of collagen in glomerular mesangial region and basement as well as in tubular basement in diabetic rats after three and six months (P< 0.01). Treatment of simvastatin decreased collagen accumulation in diabetic rats at each time point (P< 0.01). Xuezhikang treatment could decrease collagen deposition in diabetic kidney after six months (P< 0.01), but no difference was observed after three months'treatment. Neither simvastatin nor xuezhikang treatment could totally reverse collagen deposition compared to control rats (P< 0.01 or P< 0.05). Collagen deposition was less in group SIM compare to that in group XZK at both time points (P< 0.01 or P< 0.05).
2.4 Renal cell apoptosis
TUNEL apoptosis detection found few apoptotic cells in kidney of control rats, mainly distal tubular epithelial cells. More apoptotic cells were observed in diabetic rats (P< 0.01), mainly proximal and distal tubular cells, as well as some glomerular cells, the number of which was fewer. Simvastatin and xuezhikang treatment could reduce renal cell apoptosis compared to diabetic rats at each point (P< 0.01), but not completely as compared to control rats (P< 0.05).
2.5 Expression of Caspase-9 and cytoplasmic Cytochrome c
Expression of renal Caspase-9 and cytochrome c were detected by western blotting. Active Caspase-9 and cytochrome c expression were low in control rats. Western blotting revealed that expression of active Caspase-9 and cytoplasmic cytochrome c increased significantly in diabetic rats, which was ameliorated by both simvastatin and xuezhikang treatment, but still higher than that in control group.
2.6 Expression of Bax and Bcl-2
Renal expression of Bax and Bcl-2 were detected by both immunohistochemisty and western blotting. Bax and Bcl-2 expression was low in control group. Bax expression was higher in diabetic group and progressed as diabetic duration prolonged (P< 0.01), while Bcl-2 expression increased a little after three months, without significant difference, but decreased significantly after six months (P< 0.01), thus Bax/Bcl-2 ratio increased dramatically in diabetic rats (P<0.01). Both simvastatin and xuezhikang treatment could suppress the increment of Bax and up-regulate the expression of Bcl-2, leading to the suppression of Bax/Bcl-2 ratio (P< 0.01).
3 Conclusions
3.1 Obvious renal cell apoptosis was observed in diabetic kidney, which progressed as diabetic duration prolonged. Simvastatin and xuezhikang treatment could attenuate pathological changes in diabetic kidney, reduce renal cell apoptosis. The effect of statins did not depend on their cholesterol-lowering effect.
3.2 Simvastatin and xuezhikang could reduce the activation of Caspase-9 and the expression of cytochrome c in cytoplasm, suggesting that suppression of mitochondria pathway was involved in statins' effect against cell apoptosis.
3.3 Simvastatin and xuezhikang could suppress Bax/Bcl-2 ratio, suggesting that statins could reduce cell apoptosis by regulating the expression of Bcl-2 family.
他汀类药物是3-羟-3-甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂,临床上主要用于治疗脂代谢异常。他汀类药物在临床上应用十分广泛,糖尿病患者常伴有血脂调节异常,且血脂控制要求较非糖尿病患者更为严格,因此在糖尿病患者中他汀类药物的使用尤为普遍。众多研究表明他汀类药物具有肾脏保护作用,可以延缓糖尿病肾病的进展。辛伐他汀是他汀类药物的一种,临床应用较为广泛,大规模的临床研究证实辛伐他汀能够延缓糖尿病患者肾小球滤过率(GFR)的下降。血脂康是一种天然他汀类调脂药,由特制红曲精炼而成,主要包含洛伐他汀在内的13种天然他汀及他汀同系物,还含有多种人体必需的氨基酸和不饱和脂肪酸及多糖等,基础和临床研究提示血脂康具有调节氧化应激、改善内皮细胞功能及抑制炎症反应等作用,Meta分析显示血脂康能够降低24小时尿蛋白及尿微量白蛋白排泄率。
本研究通过链脲佐菌素(STZ)腹腔注射诱导糖尿病大鼠模型,分别给予辛伐他汀和血脂康治疗,拟观察糖尿病时大鼠肾脏的结构改变和组成细胞的凋亡情况,以及辛伐他汀和血脂康对肾脏结构功能和细胞凋亡的影响,并进一步探讨其可能的作用机制。
1材料与方法
1.1试剂及材料
STZ购自Sigma公司,辛伐他汀由默沙东公司馈赠,血脂康由北大维信馈赠,PAS、Masson染色试剂盒购自珠海贝索,TUNEL原位凋亡染色试剂盒购自Roche公司,Caspase-9、Bax抗体购自Santa cruz, Bcl-2抗体购自武汉博士德,细胞色素c抗体购自BD公司。
1.2实验动物模型建立及分组
清洁级雄性Sprague Dawley(SD)大鼠90只,体重180-220克(购自浙江大学医学院动物实验中心),饲以标准饲料块,随意充分饮水,室内保持通风和相对恒温,保持正常昼夜节律。适应性饲养1周后随机选取70只采用STZ一次性腹腔注射方法建立糖尿病模型,建模前禁食12小时,注射剂量60mg／kg(溶于0.1mol／L柠檬酸缓冲液,pH 4.2),注射后48小时及72小时分别尾静脉采血,血糖≥16.7mmol／L者为造模成功,正常对照组注射相应体积的柠檬酸缓冲液。糖尿病模型确立后将糖尿病大鼠随机分为3组：1.辛伐他汀治疗组(SIM组,n=22)：每天予辛伐他汀灌胃,给药剂量20mg／kg；2.血脂康治疗组(XZK组,n=22)：每天予血脂康灌胃,给药剂量1200mg／kg；3.糖尿病组(DM组,n=22)：每天予相应体积生理盐水灌胃。另20只设为正常对照组(SD组,n=20),每天予相应体积生理盐水灌胃。建模开始后第7天开始给药。
1.3生化指标检测
给药3月后每组分别取半数大鼠处死,给药6月后处死剩余大鼠。各大鼠处死前放入代谢笼收集24小时尿,离心后采用散射比浊法(Beckman CoulterImmage800)测定尿白蛋白。腹腔注射麻醉后称体重,经腹主动脉采血,BeckmanLX20全自动生化仪检测血糖及血脂。
1.4肾脏形态学观察及细胞凋亡情况
用4℃生理盐水注射液灌洗大鼠腹主动脉后,迅速取出双侧肾脏,右肾去包膜用滤纸吸干血迹后称重,右肾重量(g)／体重(kg)比值为肾重指数。将右肾对半剖开,其中一半10%中性福尔马林固定,石蜡包埋后切片,行PAS染色和Masson染色检测肾组织内糖原及胶原纤维沉积情况,TUNEL法原位检测凋亡细胞。每例切片高倍镜下观察5个视野并进行半定量分析。
1.5肾皮质Bax、Bcl-2、Caspase-9、细胞色素c的表达
左肾分离皮质,-80℃冷冻保存,提取总蛋白行Western Blot检测肾皮质Bax、Bcl-2和活化的Caspase-9的表达：右肾剩余一半置于冰上,取皮质去除线粒体后提取细胞浆蛋白,Western Blot方法测定细胞色素c表达。同时采用免疫组化方法检测肾组织内Bax及Bcl-2的定位表达,并进行半定量分析。免疫组化图像分析采用Image Pro Plus (IPP)6.0图像分析软件进行；Western Blot图像采集及分析采用化学发光荧光影像分析仪及配套软件进行。
1.6统计方法
实验数据以平均值±标准差表示,使用SPSS 16.0软件进行统计分析。多组数据平均值比较采用方差分析(ANOVA),组间比较用Bonferroni分析进行。所有检验均为双侧检验,有显著性统计学差异定义为P<0.05。
2实验结果
2.1大鼠一般情况
共70只大鼠接受糖尿病造模,其中66只血糖≥16.7mmol／L,确认为造模成功,4只血糖未达到标准。66只糖尿病大鼠随机分为3组,饲养过程中监测血糖,其中7只血糖不达标排除,另有4只死亡,最终各组大鼠数量为：SD组3月,n=10；SD组6月,n=10；DM组3月,n=9；DM组6月,n=10；SIM组3月,n=10；SIM组6月,n=9；XZK组3月,n=9；XZK组6月,n=8。
正常对照组大鼠血糖均低于7mmol／L,尿糖阴性,注射STZ后48小时及72小时大鼠血糖明显升高(≥16.7mmol／L),实验期间监测血糖维持造模标准,辛伐他汀和血脂康对糖尿病大鼠的血糖没有改善作用；实验期间正常SD大鼠体重逐渐增加,没有多饮多尿多食症状,而糖尿病大鼠体重增长缓慢或不增长,出现较明显的三多一少等糖尿病常见临床表现,辛伐他汀和血脂康治疗均未能恢复糖尿病大鼠的体重。各实验组大鼠血脂水平未见显著性差异。
2.2肾重指数及24小时尿白蛋白排泄量
糖尿病组3月和6月后肾重指数出现明显增加(P<0.01),辛伐他汀和血脂康治疗后各时间点肾重指数均较糖尿病组呈下降趋势(P<0.05或P<0.01),但仍高于正常对照组(P<0.01),两种药物之间未见明显统计学差异。
正常对照组尿白蛋白排泄量较少,糖尿病造模3月后尿白蛋白排泄明显增多6月后进一步加重(P<0.01)：辛伐他汀和血脂康治疗3月和6月后尿白蛋白排泄量较同时间点的糖尿病组减少(P<0.01或P<0.05),但仍高于同一时间点的正常对照组(P<0.05或P<0.01)；辛伐他汀治疗组和血脂康治疗组间尿白蛋白排泄量无统计学差异。
2.3肾组织PAS和Masson染色结果
PAS染色见正常大鼠肾小球及肾小管结构清晰,肾小管上皮细胞排列整齐,糖尿病模型组大鼠3月后可见肾小球系膜区扩张,肾小管基底膜不规则增厚,肾小球系膜及基底膜区PAS阳性物质增多,较正常对照组有显著性差异(P＜0.01),6月后上述区域PAS阳性物质沉积进一步增多。辛伐他汀治疗能逆转大鼠肾脏PAS阳性物质的沉积增加(P＜0.01),且与正常对照组未见明显差异(P＞0.05)；血脂康治疗亦能减少糖尿病时肾脏PAS阳性物质的沉积(P＜0.01),但仍高于同一时间点的正常对照组(P＜0.05)。给药3月时血脂康治疗组肾组织内PAS阳性物质的沉积多于辛伐他汀治疗组(P＜0.05),6月后两组间未见明显差异。
Masson染色显示3月后糖尿病模型组大鼠肾小球及肾小管基底膜区、肾小球系膜区胶原纤维沉积较正常对照组明显增多,6月后进一步加重(P＜0.01)。辛伐他汀治疗3月或6月后肾组织内胶原沉积较相应时间点的糖尿病模型组均明显减少(P＜0.01),但仍较正常对照组增多(P＜0.05)。血脂康治疗6月后肾组织内胶原沉积较糖尿病模型组显著减少(P<0.01),但治疗3月时血脂康组与糖尿病组未见明显差异,治疗3月或6月后与正常对照组均存在显著差异(P＜0.01)。辛伐他汀治疗组胶原沉积少于血脂康治疗组,3月和6月时两组间均存在显著性差异(P<0.01或P<0.05)。
2.4肾组织细胞凋亡检测结果
TUNEL原位凋亡染色显示正常对照组大鼠肾脏内凋亡细胞很少,主要位于远端肾小管上皮细胞,糖尿病模型组大鼠肾脏凋亡细胞明显增多(P＜0.01),主要集中于肾远曲小管、近曲小管,肾小球亦可见凋亡细胞,数量较肾小管区域少,随糖尿病病程延长肾脏细胞凋亡逐渐增加。辛伐他汀治疗3月和6月后肾脏凋亡细胞较糖尿病组均明显减少(P＜0.01),3月时与正常对照组间未见明显差异,6月时肾脏凋亡细胞较正常对照组增多(P＜0.05)。血脂康治疗3月和6月后肾脏细胞凋亡较相应时间点的糖尿病组明显减少(P＜0.01),但均多于正常对照组(P<0.05)。
2.5肾组织内Caspase-9和细胞色素c的表达情况
Western Blot显示正常对照组活化的Caspase-9和细胞色素c的表达较少,糖尿病组活化的Caspase-9和细胞浆内细胞色素c的表达明显增加,辛伐他汀和血脂康治疗组活化的Caspase-9和细胞浆内细胞色素c表达水平较糖尿病组降低,但仍高于正常对照组。
2.6肾组织内Bax和Bcl-2的表达情况
免疫组化结果和Western Blot结果显示,正常对照组大鼠肾组织中Bax和Bcl-2表达量较少,糖尿病模型组Bax表达显著增加(P<0.01),随着病程延长逐渐增加；糖尿病组3月时肾组织中Bcl-2表达较正常对照组稍有增加,但未见明显差异,6月时Bcl-2表达较正常对照组显著下降(P＜0.01),糖尿病组Bax/Bcl-2比值高于正常对照组(P＜0.01),随病程进展进一步升高。辛伐他汀和血脂康治疗可以明显抑制糖尿病肾脏组织中Bax表达的增加,上调Bcl-2的表达,从而降低Bax/Bcl-2比值(P<0.01)。
3结论
3.1糖尿病肾病时肾脏细胞出现明显凋亡,且随病程延长逐步进展,辛伐他汀和血脂康能改善糖尿病时肾脏的病理改变,减少肾脏细胞的凋亡,其作用不依赖于他汀类药物的降脂效应；
3.2辛伐他汀和血脂康能减少肾皮质内Caspase-9的活化以及细胞浆内的细胞色素c水平,可以通过抑制线粒体凋亡通路的活化减少肾脏细胞的凋亡；
3.3辛伐他汀和血脂康能降低Bax/Bcl-2的比值,可能通过调节Bcl-2蛋白家族的表达减少细胞凋亡。
The prevalence of diabetes is increasing at an alarming rate, regardless of the population studied. Due to rapid economic growth, an increase in life expectancy and changes in lifestyle, diabetes and its complications have become great threats to global health in the 21st century. Diabetic nephropathy, common but severe microangiopathy in diabetes, is becoming one of the major reasons that lead to end stage renal dysfunction. The mechanism of diabetic nephropathy is not clear yet. Researchers have hooked renal cell apoptosis with diabetic nephropathy recently, as the studies on cell apoptosis going deeper, suggesting renal cell apoptosis an early manifestation of diabetic nephropathy.
Statins, HMG-CoA reductase inhibitors, are a class of drugs used to lower plasma cholesterol levels. Statins are used widely in clinical works, especially in diabetic patients, since diabetic patients often suffer from lipid dysfunction and the criterion of lipid control is stricter for diabetic patients. Studies have demonstrated a protective effect of statins on renal function that statins could ameliorate the progress of diabetic nephropathy. Simvastatin, used widely clinically, is confirmed to attenuate the decline of glomerular filtration rate in diabetic patients according to large scale clinical trials. Xuezhikang is a natural statin extract from red yeast rice, including thirteen natural statins and homologues of statin. Xuezhikang is suggested to have effect on oxidative stress, endothelial cell function and inflammation according to basic and clinical researches. Meta analysis also demonstrated that xuezhikang could lower urinary protein and albumin extretion.
Streptozocin (STZ) induced diabetic rats were used in this study, giving simvastatin and xuezhikang treatment after establishment of diabetic model. Renal histological changes and renal cell apoptosis in each group (control group, diabetic group, diabetic group with simvastatin treatment and diabetic group with xuezhikang treatment) were observed, and the possible mechanism how statins affect cell apoptosis was also investigated.
1 Materials and methods
1.1 Reagents and materials
STZ was from Sigma, simvastatin from Merck and Co. China, xuezhikang from Beijing WBL Peking University Biotech Co., PAS and Masson staining kits from Baso Diagnostics Inc. Zhuhai, TUNEL apoptosis assay kit from Roche, Caspase-9 and Bax antibodies from Santa cruz, Bcl-2 antibody from Boster (Wuhan), and cytochrome c antibody from BD Inc.
1.2 Induction of diabetes and experimental groups
Ninety male Sprague-Dawley (SD) rats, of 180-220g, were purchased from the Experimental Animal Center of Zhejiang University and raised with standard food, adequate water and normal light/dark cycle. Diabetes was induced by a single intraperitoneal injection of 1% STZ (in cold 0.1M citrate buffer. pH 4.2) at 60mg/kg body weight. Blood samples for glucose measurements were taken from the tail vein 48-72 hours after STZ injection and rats with blood glucose of 16.7mmol/L or higher were considered diabetic rats. The control rats were injected with equal volume of citrate buffer instead. The experimental groups included (1) normal control rats (group SD); (2) diabetic control group (group DM); (3) diabetic rats treated with simvastatin (20mg/kg body weight gastric perfusion everyday, group SIM); (4) diabetic rats treated with xuezhikang (1200mg/kg body weight gastric perfusion everyday, group XZK). Treatments started 7 days after STZ injection. Rats from group SD and DM received equal volume of saline instead.
1.3 Detection of clinical measurements
Half the rats from each group were sacrificed after three months'treatments and the rest after six months'treatments. Twenty-four hours'urine was collected using metabolic cages and urinary albumin excretion was measured by rate nephelometry (Beckman Coulter Immage800). After weighing the body weight, blood sample was collected from abdominal aorta for glucose and lipid detection by Beckman Coulter LX20 biochemical analyzer.
1.4 Histological examination and apoptosis detection
After lavaging the abdominal aorta by cold saline, both kidneys were taken out. The ratio of right kidney weight to body weight was recorded as kidney index. The right kidney was cut into two pieces, half of which was fixed by formalin and embedded by paraffin. Paraffin-embedded tissue section (3μm thick) were deparaffinized. rehydrated. and stained with PAS and Masson's trichrome for saccharides and collagen detection, and TUNEL for apoptosis detection.
1.5 Expression of Bax, Bcl-2, Caspase-9 and Cytochrome c
Renal cortex was separated and stored in-80℃. After extracting the total protein, Bax, Bcl-2 and active Caspase-9 expression in renal cortex were detected by western blotting. Half of the right kidney was used for detection of cytochrome c expression after removing mitochondria. Bax and Bcl-2 expression were also detected by immunohistochemistry.
1.6 Statistical analysis
All data were presented as mean±SD of the mean. When multiple comparisons were made, statistical significance was determined using one-way ANOVA followed by Bonferroni's test. All statistical analyses were performed using the software package SPSS 16.0 and P<0.05 was considered statistically significant.
2 Results
2.1 General data
Seventy SD rats underwent STZ injection, sixty-six of which had blood glucose of 16.7mmol/L or higher were considered diabetic rats and divided into three groups randomly. Except those died during experiment and blood glucose lower than 16.7mmol/L, the final number of rats in each group was as follows:SD (3 months). n=10; SD (6 months), n=10; DM (3 months), n=9; DM (6 months), n=10; SIM (3 months), n=10; SIM (6 months), n=9; XZK (3 months), n=9; XZK (6 months), n=8.
The blood glucose was less than 7mmol/L in control group and increased dramatically (≥16.7mmol/L) 48-72 hours after STZ injection; neither simvastatin or xuezhikang treatment showed significant differences from group DM in blood glucose level. The STZ-induced diabetic rats showed classic diabetic symptoms of polyuria, polydipsia and weight loss:simvastatin and xuezhikang did not help to retrieve the body weight. No significant difference was found in lipid level among each group.
2.2 Kidney index and 24 hours'urinary albumin excretion
Kidney index of group DM increased significantly after three and six months(P< 0.01). Simvastatin and xuezhikang treatment could decrease kidney index compare to that of group DM at each time point (P< 0.05或P< 0.01), but kidney indexes of group SIM and XZK were higher compare to that of group SD (P<0.01). There was no significant difference between the two drugs.
Urinary albumin excretion was low in group SD, which increased in group DM after three months and progressed after six months (P< 0.01). Three and six months' treatment of simvastatin and xuezhikang could reduce urinary albumin excretion compare to that of group DM (P< 0.01或P< 0.05). There was no difference between group SIM and group XZK in urinary albumin excretion.
2.3 Results of PAS and Masson stain
Pathological sections stained with PAS showed that control rats had normal glomerulus and renal tubule, with tubular cells arraying in order. However, diabetic rats showed dilation of glomerular mesangial region, irregular thickening of tubular basement, as well as increased deposition of PAS positive substance after three and six months (P< 0.01). Simvastatin treatment could totally alleviate the deposition of PAS positive substance in diabetic kidney (P<0.01). PAS positive substance was reduced significantly after xuezhikang treatment (P< 0.01), but more than that in control rats at each time point (P< 0.05). There was difference between group SIM and group XZK at three months (P< 0.05), which disappeared six months later.
Masson's trichrome stain showed increased accumulation of collagen in glomerular mesangial region and basement as well as in tubular basement in diabetic rats after three and six months (P< 0.01). Treatment of simvastatin decreased collagen accumulation in diabetic rats at each time point (P< 0.01). Xuezhikang treatment could decrease collagen deposition in diabetic kidney after six months (P< 0.01), but no difference was observed after three months'treatment. Neither simvastatin nor xuezhikang treatment could totally reverse collagen deposition compared to control rats (P< 0.01 or P< 0.05). Collagen deposition was less in group SIM compare to that in group XZK at both time points (P< 0.01 or P< 0.05).
2.4 Renal cell apoptosis
TUNEL apoptosis detection found few apoptotic cells in kidney of control rats, mainly distal tubular epithelial cells. More apoptotic cells were observed in diabetic rats (P< 0.01), mainly proximal and distal tubular cells, as well as some glomerular cells, the number of which was fewer. Simvastatin and xuezhikang treatment could reduce renal cell apoptosis compared to diabetic rats at each point (P< 0.01), but not completely as compared to control rats (P< 0.05).
2.5 Expression of Caspase-9 and cytoplasmic Cytochrome c
Expression of renal Caspase-9 and cytochrome c were detected by western blotting. Active Caspase-9 and cytochrome c expression were low in control rats. Western blotting revealed that expression of active Caspase-9 and cytoplasmic cytochrome c increased significantly in diabetic rats, which was ameliorated by both simvastatin and xuezhikang treatment, but still higher than that in control group.
2.6 Expression of Bax and Bcl-2
Renal expression of Bax and Bcl-2 were detected by both immunohistochemisty and western blotting. Bax and Bcl-2 expression was low in control group. Bax expression was higher in diabetic group and progressed as diabetic duration prolonged (P< 0.01), while Bcl-2 expression increased a little after three months, without significant difference, but decreased significantly after six months (P< 0.01), thus Bax/Bcl-2 ratio increased dramatically in diabetic rats (P<0.01). Both simvastatin and xuezhikang treatment could suppress the increment of Bax and up-regulate the expression of Bcl-2, leading to the suppression of Bax/Bcl-2 ratio (P< 0.01).
3 Conclusions
3.1 Obvious renal cell apoptosis was observed in diabetic kidney, which progressed as diabetic duration prolonged. Simvastatin and xuezhikang treatment could attenuate pathological changes in diabetic kidney, reduce renal cell apoptosis. The effect of statins did not depend on their cholesterol-lowering effect.
3.2 Simvastatin and xuezhikang could reduce the activation of Caspase-9 and the expression of cytochrome c in cytoplasm, suggesting that suppression of mitochondria pathway was involved in statins' effect against cell apoptosis.
3.3 Simvastatin and xuezhikang could suppress Bax/Bcl-2 ratio, suggesting that statins could reduce cell apoptosis by regulating the expression of Bcl-2 family.
引文
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