| Objective Through the study of the effects of PPARgamma and SIRT1 feedback to clarify the inner relation of lupus nephritis steroid resistance and the lipid metabolism disorders,and to clarify the double effect of reversing steroid resistance in lupus nephritis and improving lipid metabolism disorder of panax notoginseng saponins.Methods(1)fill the stomach of NZB/WF1 lupus mice with small doses of a prednisolone(0.8mg/kg)for 8 weeks,to induce steroid resistance model,while giving the low and high doses of PNS intervention,we set up SIRT1-si RNA,SIRT1-si RNA negative,lupus mice and normal ICR mice as control.(2)urine protein of 24 hours and urine sugar levels were detected at the4 th and 8th week during the experiment.At the end of the experiment,we killed the mice and select the blood sample to measure the serum creatinine,urea nitrogen and total cholesterol,triglyceride,low density lipoprotein and high-density lipoprotein levels.ELISA was adopted to detect the serum CRP and e NOS levels.(3)The pathological changes of renal and abdominal aorta were observed.The immunohistochemistry of ACAT1 in the liver and VCAM-1 in the abdominal aorta were used to observe the expression of these two target proteins.The apoptosis rate of endothelial cells in abdominal aorta was detected by flow cytometry.All above were used to elucidate the internal correlation between steroid resistance and lipid metabolism disorder and to observe the effect of PNS in the progression of atherosclerosis and renal damage in lupus mice.(4)The establishment of steroid resistance modal and the reverse effect of PNS was verified by detecting the expression of P-gp with flow cytometry.PPAR and SIRT1 immunoprecipitation in splenic lymphocytes revealed the presence of PPAR gamma-SIRT1 feedback pathway.By using real time RT-PCR to detect the PPARgamma and SIRT1 m RNA expression levels in the splenic lymphocytes and liver tissues in lupus mice.Western blotting method was used to detect the expression of PPARgamma,SIRT1 in the splenic lymphocytes and liver tissues of lupus mice to verify the mechanism of PNS reversing P-gp mediated steroid-resistance and improving the lipid metabolism disorders in lupus mice.Results(1)At the point of before,4th and 8th weeks of the experiment,urinary protein of 24 hours of steroid resistance model mice are increased significantly than the ICR mice(6.59±0.38 vs2.12±0.32,8.12±0.57 vs 2.22±0.30,9.98±0.70 vs 2.49±0.36,P<0.01),but there is no obvious difference compared with lupus mice;At the end of the experiment,the urine protein levels of lupus mice,steroid-resistant model mice and SIRT1-si RNA negative control mice were significantly higher(10.39±0.60vs6.91±0.63,9.98±0.70vs6.59±0.38,10.74±0.54vs6.56±0.61,P<0.05).Serum creatinine levels in lupus mice and steroid resistance model mice(46.28±2.03,45.25±2.58 vs 41.12±2.13,P<0.05)were higher than the ICR mice,but there is no significant difference in blood urea nitrogen levels compared with the ICR mice.Serum creatinine levels(44.27±2.18,45.67±3.41 vs 41.12±2.13,P<0.05)and urea nitrogen levels in SIRT1-si RNA group and SIRT1-si RNA negative control mice(45.51±2.69,47.47±2.34 vs41.98 ± 1.23,P < 0.05)are both higher than the ICR mice.After the experiment,the total cholesterol in the Lupus mice,the steroid-resistant model mice,the SIRT1-si RNA group,SIRT1-si RNA negative control group,low dose of PNS intervention group and high dose of PNS intervention group are higher than the ICR mice(5.92±0.23,6.02±0.18,5.06±0.26,5.71±0.41,5.68±0.20,5.40±0.24vs3.36±0.33,P<0.05),as well as the triglycerides(1.43±0.03,1.45±0.06,1.31±0.03,1.36±0.11,1.24±0.05,1.22±0.04vs1.16±0.04,P<0.05)and the low density lipoprotein(0.44±0.02,0.46±0.02,0.37±0.04,0.50±0.06,0.34±0.07,0.32±0.06vs0.26±0.03,P<0.05),among those lupus mice group and steroid resistance model are the most obvious.At the end of the experiment,the urine sugar level of NZB/WF1 lupus mice is higher than ICR mice(1.05±0.57 vs 0.77±0.17,1.09±0.37 vs 0.86±0.18,P<0.01),but no difference compared with the steroid-resistant lupus mice,the urine sugar level of SIRT1-si RNA group and low dose of PNS intervention group,high dose of PNS intervention group(0.95±0.26,0.87±0.22,0.90±0.28 vs 1.29±0.32,P<0.05)are lower than the lupus mice group.Level of CRP in each group is higher than ICR mice(11.12±0.52,10.68±0.57,7.93±0.32,9.85±0.98,7.38±0.65,8.15±0.28 vs 5.92±0.45,P<0.05),the CRP level of SIRT1-si RNA group and the low and high dose of PNS intervention group mice are lower than lupus mice group(7.93±0.32,7.38±0.65,8.15± 0.28 vs 11.12 ± 0.52,P< 0.05)and the steroid resistance model group(7.93 ±0.32,7.38±0.65,8.15±0.28 vs 10.68±0.57,P<0.05).The relative quantity expression of CYP7A1 protein of Lupus mice,steroid resistance in the liver tissue are lower than the ICR mice(0.56±0.21,0.21±0.14 vs 0.93±0.02,P<0.05),however,the expression of CETP of the steroid-resistant mice was increased(1.15±0.23vs0.87±0.06,P<0.05);Among them,the levels of CYP7A1 expression of SIRT1-si RNA group,low dose of PNS intervention group and high dose of PNS intervention group mice are increased than lupus mice(0.95±0.25,1.03±0.19,0.19±0.24 vs 0.56±0.21,P<0.05)and steroid resistance model mice(0.95±0.25,1.03±0.19,0.19±0.24 vs 0.58±0.14,P<0.05).(2)Pathological HE staining of kidney indicated that SIRT1-si RNA and PNS were involved in the improvement of renal lesion in lupus mice.Pathological HE staining of abdominal aorta showed that the endothelial injury was more serious in mice with lupus,steroid-resistant mice and SIRT1-si RNA negative control mice.Liver immunohistochemical results show that the expression of ACAT1 protein in the liver of lupus mice and steroid resistant mice are higher than ICR mice(49.75±6.83,48.66±6.42 vs 22.66±5.64,P<0.05),after the intervention with SIRT1-si RNA and low and high dose of PNS,the expression of ACAT1 are lower(36.14±5.95,36.24±6.41,39.58±5.89 vs 49.75±6.83,P<0.05).Aorta immunohistochemical results show that the expression levels of VCAM-1 protein in abdominal aorta of lupus mice,steroid resistance model mice and SIRT1-si RNA negative control group mice are the most obvious,those of SIRT1-si RNA group and the low and high dose of PNS intervention group are lower,however they are all higher than the ICR mice(0.75±0.05,0.66±0.04,0.77±0.03 vs 0.44±0.02,0.54±0.05,0.58±0.03 vs 0.36±0.03,P<0.05).The analysis of endothelial cell apoptosis rate of abdominal aortic indicates that ICR mice did not show the apoptosis.but the abdominal aortic endothelial cell apoptosis of lupus mice,steroid resistant lupus mice and the SIRT1-si RNA negative control group of mice are the most pronounced(0.67±0.03% vs 30.31±0.05% vs79.45±0.06% vs 1.76±0.02% vs 70.88±0.06% vs 3.42±0.03% vs 1.84 ±0.02%,P<0.05).(3)After inducing steroid-resistance with small dose of methyl prednisolone lavage,the P-gp protein expression in splenic lymphocytes of lupus mice were obviously higher than the ICR mice and the NZB/WF1 lupus modal mice(3.19±0.08% vs 0.24±0.03%,0.39±0.02%,P<0.01).However,the P-gp expression of the low,high-dose intervention group and the SIRT1-si RNA group was lower than that of the steroid-resistance group,and the intervention effect of high dose of PNS and SIRT1-si RNA were better(0.78±0.01% vs 0.56±0.02% vs 0.48±0.03% vs0.48±0.08%,P<0.05).The immunoprecipitation results of PPARgamma and SIRT1 in spleen lymphocytes of mice show that there exist interplay between them.RT-PCR results show that the PPARgamma m RNA of the SIRT1-si RNA group,low dose of PNS intervention group and high dose of PNS intervention group are higher than the lupus mice and the steroid-resistant mice(4.82±0.11,4.35±0.18 and 0.18±0.14 vs 0.75±0.12 vs 0.54±0.14,P<0.05)in splenic lymphocyte and in the liver tissue(1.23±0.15,1.17±0.15 and 0.15±0.13 vs 0.73±0.14 vs0.62±0.13,P<0.05).The expression level of SIRT1 m RNA in the SIRT1-si RNA group,low dose of PNS intervention group and high dose of PNS intervention group are lower than the lupus mice and the steroid-resistant mice in the splenic lymphocytes(1.44±0.19,1.90±0.17 and 0.17±0.21vs2.28±0.11vs5.13±0.16,P<0.05)and in the liver tissue(2.00±0.18,2.82±0.21 and 2.55±0.19 vs 2.98±0.12 vs 5.03±0.17,P<0.05).The expression level of PPARgamma m RNA in lupus mice is higher than the steroid-resistant model mice,while the SIRT1 m RNA is lower.Western Blotting results show that the expression levels of PPARgamma of SIRT1-si RNA group and the low and high dose of PNS intervention group in splenic lymphocyte are increased in the steroid resistance model mice(0.62±0.02,0.71±0.03,0.82±0.08 vs 0.55±0.06,P<0.05),while the expression levels of SIRT1 are decreased(0.87±0.23,0.98± 0.13,0.87± 0.24 vs 2.08± 0.26,P < 0.05);the expression levels of SIRT1 of SIRT1-si RNA group and the low and high dose of PNS intervention group in the liver tissue are decreased in the steroid resistance model mice(0.87±0.21,0.81±0.13,0.52±0.03 vs 1.58±0.27,P<0.05),and PPARgamma protein expression of steroid-resistant mice is lower than the lupus mice,while the SIRT1 expression is higher in the liver tissue.Conclusion1.There is a feedback loop effect of PPARgamma and SIRT1 in the steroid-resistant lupus nephritis mice.2.PNS has the effect of both regulating the lipid metabolic disorder and improving atherosclerosis in lupus nephritis mice.3.PNS has the effect of reversing steroid resistance and protecting the renal function in lupus nephritis mice.4.The mechanism of PNS playing the dual role above is closely related to the regulation of the "PPAR gamma-SIRT1 feedback pathway". |
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