【作者】 林京莲；

【导师】 鲁盈；

【作者基本信息】 浙江中医药大学 ， 中医学 中医内科学， 2016， 博士

【摘要】 目的:观察沉默信息调节因子1(SIRT1)在P-gp介导的狼疮肾炎小鼠激素耐药中的调控作用及三七总皂苷的干预效应。方法:1.NZBWF1狼疮小鼠用小剂量甲泼尼龙片(0.8mg/kg)灌胃4周,诱导激素耐药模型。2.同时给予三七总皂苷低、高剂量联合大剂量甲泼尼龙(12mg/kg)干预,并以单纯大剂量甲泼尼龙、SIRT1-siRNA联合大剂量甲泼尼龙、SIRT1-siRNA阴性对照联合大剂量甲泼尼龙等作为对照。3.分别于实验前、实验第2周及实验第4周检测小鼠24小时尿蛋白;并于实验结束时取血清检测血肌酐、尿素氮、血清白蛋白水平;ELISA法检测小鼠血清ANA、dsDNA、C3等免疫学指标含量;肾脏病理行HE、Masson染色及免疫荧光检测IgG、C3、C1q沉积观察肾病理损伤等临床指标。4.激素耐药效应通过流式细胞术检测狼疮鼠脾淋巴细胞耐药蛋白P-gp表达和转运底物罗丹明123累积;免疫组化检测肾组织中P-gp蛋白表达来体现。5.通过荧光定量RT-PCR检测狼疮鼠脾淋巴细胞SIRT1 mRNA表达和Western blot法检测小鼠脾淋巴细胞SIRT1蛋白表达揭示三七总皂苷逆转P-gp介导的狼疮肾炎激素耐药的机制。结果:1.在实验前、实验第2周及第4周,激素耐药模型组狼疮小鼠24小时尿蛋白均比正常组显著增加(6.78±2.05 vs 2.33±0.65;8.87±3.57 vs2.24±0.77;11.09±4.67 vs 2.56±0.98,P<0.01),并随着时间延长,尿蛋白明显增多,但与狼疮鼠无统计学差异,各治疗及对照组小鼠尿蛋白均有下降,以三七总皂苷大剂量组(4.33±1.79 vs 2.24±0.77,6.78±2.78 vs 2.56±0.98,P<0.01)及SIRT1-siRNA组(4.41±2.01 vs 2.24±0.77,6.56±2.45 vs 2.56±0.98,P<0.01)下降最明显。与模型组比较,三七总皂苷各治疗组及各对照组小鼠血清白蛋白也有所升高(32.35±6.77,34.34±6.87,32.03±6.65,33.23±7.12,31,45±6.56 vs 29.37±6.12,P<0.05),但各组小鼠血肌酐、尿素氮水平无明显下降趋势(43.69±4.66、44.02±4.23、43.78±4.01、43.86±-4.32、44.45±3.98 vs 44.21±3.32;7.89±2.50、7.68±2.45、8.34±3.11、8.22±2.67、8.43±3.31 vs 8.49±2.56,P>0.05)。ELISA结果显示:三七总皂苷各治疗组及对照组小鼠血清ANA、dsDNA、C3与模型组相比均有不同程度下降(15.08±4.89、14.34±4.56、16.98±5.34、14.23±5.01、16.67±5.87 vs 17.83±5.79,P<0.05),且三七总皂苷低,高剂量组与SIRT1-siRNA组疗效相当(P>0.05)。肾脏病理HE、Masson染色显示:治疗组及SIRT1-siRNA组较激素耐药模型组在肾小球系膜增殖、硬化及间质炎细胞浸润及纤维化程度等病理损伤程度明显减轻,免疫荧光病理也提示三七总皂苷治疗组IgG、C3表达减少,但C1q表达无差异。2.小剂量甲泼尼龙灌胃4周后,激素耐药模型组狼疮鼠脾淋巴细胞P-gp表达较正常鼠及NZBWF1狼疮鼠明显升高(37.6±6.8%vs 18.9±4.1%、25.5±4.6%,P<0.01),而罗丹明123累积明显下降(31.7±6.7%vs 57.8±11.2%、45.6±7.6%,P<0.01),提示NZBWF1狼疮鼠激素耐药模型构建成功,而三七总皂苷低、高剂量治疗组及各对照组P-gp、罗丹明123表达均有不同程度改善(26.4±4.7%、23.5±3.5%、31.6±6.7%、21.4±3.2%、33.7±5.8%vs 模型组37.6±6.8%,P<0.05;36.8±4.9%、44.3±5.7%、38.5±4.3%、46.6±6.6%、33.2±4.7%vs模型组31.7±6.7%,P<0.05),提示三七总皂苷治疗组和对照组均有一定的逆转激素耐药作用,以三七总皂苷高剂量组和SIRT1-siRNA组效果最为显著。肾组织免疫组化显示:三七总皂苷治疗组及SIRT1-siRNA组小鼠肾组织P-gp阳性表达指数较激素耐药模型组均明显下降(26.24±5.84、23.58±5.05、23.14±4.72 vs 38.66±8.78,P<0.05),但各组间无显著性差异。3.与模型组比较,三七总皂苷低、高剂量治疗组及SIRT1-siRNA对照组均能下调 NZBWF1 狼疮鼠脾淋巴细胞 SIRT1-mRNA 表达(0.56±0.11、0.49±0.07、0.43±0.12vs 1.00,P<0.05),但各组之间差异并无统计学意义(P>0.05),提示三七总皂苷低、高剂量及SIRT1-siRNA具有相似的抑制激素耐药狼疮鼠脾淋巴细胞SIRT1基因表达的作用。小剂量激素诱导耐药后,狼疮鼠脾淋巴细胞SIRT1蛋白水平显著上调(1.95±0.03 vs正常组0.98±0.13、狼疮鼠1.34±0.07,P<0.05),而三七总皂苷能下调SIRT1蛋白表达(1.11±0.02、1.20±0.07 vs模型组1.95±0.03,P<0.05),但与剂量不相关,且与对照组比较无统计学差异。结论:1.小剂量甲泼尼龙可诱导NZBWF1狼疮小鼠激素耐药,成功构建小鼠激素耐药模型。2.三七总皂苷能协同大剂量甲泼尼龙降低NZBWF1狼疮鼠脾淋巴细胞和肾组织P-gp表达,逆转激素耐药;改善激素耐药型狼疮肾炎小鼠尿蛋白、血清白蛋白、肾病理损伤等临床指标,起肾保护作用,且作用与SIRT1-siRNA相仿。3.三七总皂苷可通过SIRT1对P-gp表达调控作用途径发挥逆转狼疮肾炎激素耐药及肾保护作用,为活血化瘀中药提高狼疮临床疗效提供了科学依据。更多还原

【Abstract】 Objectives To observe the regulatory effect of silent information regulation factor 1(SIRT1)on steroid resistance in lupus nephritis induced by P-gp and the intervention effect of Panax Notoginseng Saponins(PNS)Methods 1.NZBWF1 mice was gavaged with a small dose of Methylprednisolone(0.8mg/kg)for 4 weeks to induce steroid resistance model.2.While giving the low and high dose PNS combined with high dose Methylprednisolone(12mg/kg)to intervene,and to take the simple large dose of Methylprednisolone,SIRT1-siRNA combined with large dose of Methylprednisolone,SIRT1-siRNA negative control combined with high dose Methylprednisolone as control.3.Respectively before experiment,the second week and the fourth week of the test mice were detected in 24 hour urine protein;and at the end of the experiment from serum creatinine,blood urea nitrogen,serum albumin;ELISA method to detect the serum immunological indicators content of ANA,dsDNA and C3;renal pathological HE staining,Masson staining and immunofluorescence detection of IgG,C3,Clq deposits to observe renal pathological injury.4.Steroid resistance effect was detected by the expression of drug resistance protein P-gp and transport substrate R-123 accumulation in lupus mouse spleen lymphocytes detected by flow cytometry.The expression of P-gp protein in renal tissue was detected by immunohistochemistry.5.The expression of SIRT1 mRNA in mouse spleen lymphocytes was detected by fluorescence quantitative RT-PCR and protein by Western blot to reveal the mechanism of PNS reversing steroid resistance induced by P-gp in lupus nephritis.Results 1.Before experiment,the second week and the fourth week the 24 hours urine protein of the mice in the steroid resistant model group was significantly higher than that in the normal group(6.78±2.05 vs 2.33±0.65;8.87±3.57 vs2.24±0.77;11.09±4.67 vs 2.56±0.98,P<0.01),and urinary protein were increased with the extension of time,but without significant difference with lupus mice,the treatment and the control group mice urinary protein were decreased,PNS high dose group(4.33±1.79 vs 2.24±0.77,6.78±2.78 vs 2.56±0.98,P<0.01)and SIRT1-siRNA group(4.41±2.01 vs 2.24±0.77,6.56±2.45 vs 2.56±0.98,P<0.01)decreased the most obviously.Compared with the model group,each PNS treatment groups and the control groups mice serum albumin was also increased(32.35±6.77,34.34±6.87,32.03±6.65,33.23±7.12,31,45±6.56 vs 29.37±6.12,P<0.05),but the mice serum creatinine,blood urea nitrogen level decreased not significantly(43.69±4.66、44.02±4.23、43.78±4.01、43.86±4.32、44.45±3.98 vs 44.21±3.32;7.89±2.50、7.68±2.45、8.34±3.11、8.22±2.67、8.43±3.31 vs 8.49±2.56,P>0.05).ELISA results showed that:PNS in each treatment groups and control groups of mice serum ANA,dsDNA,C3,ANA had different degrees of declinecompared to model group,and the low,the high dose group of PNS and the SIRT1-siRNA group(15.08±4.89、14.34±4.56、16.98±5.34、14.23±5.01、16.67±5.87 vs 17.83±5.79,P<0.05)decreased the most(P>0.05).Renal pathological HE staining,Masson staining showed that the treatment groups and SIRT1-siRNA group pathological injury of glomerular mesangial proliferation,glomerular sclerosis,Interstitial inflammatory cell infiltration,Interstitial fibrosis reduced than the model group,Immunofluorescence assay also showed that the expression of IgG and C3 in the treatment group was decreased,but the expression of C1q was not different between the each other groups.2.After 4 weeks small doses of methylprednisolone was gavaged,steroid resistant mice spleen lymphocyte P-gp expression was significantly higher than that of normal mice and NZBWF1 mice(37.6±6.8%vs 18.9±4.1%、25.5±4.6%,P<0.01),while R-123 accumulation decreased significantly(31.7±6.7%vs 57.8±11.2%、45.6±7.6%,P<0.01).It suggested that steroid resistance model in NZBWF1 mice was constructed successfully.The PNS low,high dose treatment groups and the control group had different degrees of change on the expression of P-gp and R-123(26.4±4.7%、23.5±3.5%、31.6±6.7%、21.4±3.2%、33.7±5.8%vs model 37.6±6.8%,P<0.05;36.8±4.9%、44.3±5.7%、38.5±4.3%、46.6±6.6%、33.2±4.7%vs model 31.7±6.7%,P<0.05),It suggested that the PNS treatment groups and control groups could reverse steroid resistance and the PNS high dose group and SIRT1-siRNA group had the best effect.The immunohistochemistry showed that:the positive expression rate of P-gp of the PNS treatment groups and SIRT1-siRNA group were significantly decreased(26.24±5.84、23.58±5.05、23.14±4.72 vs 38.66±8.78,P<0.05),but there was no significant difference between groups.3.Compared with the model group,the low and high dose PNS treatment group and SIRT1-siRNA control group can cut down the expression of NZBWF1 lupus mice spleen lymphocytes SIRT1 mRNA(0.56±0.11、0.49±0.07、0.43±0.12vs 1.00,P<0.05),But the difference between groups was not significant(P>0.05).It suggested that the low and high dose PNS treatment group and SIRT1-siRNA control group had similar effect on inhibiting SIRT1 gene expression on steroid resistant lupus mice of spleen lymphocyte.After low dose hormone induced drug resistance,lupus mice spleen lymphocyte SIRT1 protein levels were significantly up-regulated(1.95±0.03 vs normal mice0.98±0.13、lupus micel.34±0.07,P<0.05),PNS can decrease the expression of SIRT1 protein(1.11±0.02、1.20±0.07 vs model1.95±0.03,P<0.05),but it was not correlated with dose and not significant different between with groups.Conclusion 1.Small doses of Methylprednisolone can induce steroid resistance in NZBWF1 mice and constructe steroid resistance model.2.Panax Notoginseng Saponins can reverse steroid resistance of lupus mice induced by P-gp cooperated with large dose of methylprednisolone and improve clinical indicators in lupus nephritis mice of steroid resistance.It was similar to SIRT1-siRNA in renal protective effect.3.Panax Notoginseng Saponins can take effect on reversing steroid resistance and protecting renal in lupus nephritis mice through SIRT1 regulating P-gp.It provided scientific basises for traditional chinese medicine promoting blood circulation to remove blood to improve the clinical effect on lupus.更多还原