| 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. |
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