| Objective To explore the mechanism of peroxidase proliferator activated receptor gamma(PPAR gamma)and silent information regulator 1 enzymes(SIRT1)feedback loop in spleen lymphocytes with steroid resistance of NZB/WF1 lupus nephritis mice on lipid metabolism disorder,and to clarify the relationship between steroid resistance and lipid metabolism disorder;confirm Panax Notoginseng Saponins on a dual effect regulation of PPAR gamma and SIRT1 feedback loop to reverse glucocorticoid resistance and improve the intracellular lipid metabolism disorder.The purpose was to research the effect and mechanism of Chinese herbs in reversing steroid resistance of lupus nephritis,and providing a new idea and useful scientific basis for Traditional Chinese medicine,and to improve the clinical curative effect of refractory lupus nephritis.Methods 1.It was used by the density gradient separation of spleen lymphocytes in NZB/WF1 lupus nephritis mice with low dose methylprednisolone(final concentration2?g/m L,72h culture)induced steroid resistance for cell model construction,detection of separation of cells with flow cytometry and automatic biochemical analyzer;detection of separation of cells viability with trypan blue staining;detection of P-glycoprotein expression and transport substrate rhodamine-123 in cell model with flow cytometry.2.Spleen lymphocytes of steroid resistance of NZB/WF1 lupus nephritis mice were divided into model group,PPAR gamma plasmid transfection group,PPAR gamma si RNA group,SIRT1 si RNA group,SIRT1 plasmid transfection group,and set with normal mice group and NZB/WF1 lupus nephritis mice group.Each group was cultured 72h respectively.Detection of the transfection efficiency of si RNA and lentivirus plasmids with fluorescence microscopy and flow cytometry.Detection of the interaction between PPAR gamma and SIRT expression with co-immunoprecipitation and super-shift-emsa assay.Detection of PPAR gamma and SIRT1m RNA with fluorescence quantitative PCR real-time assay.Detection of PPAR gamma and SIRT1 protein with western blotting assay.In order to explore the relationship and mechanism of PPAR gamma and SIRT1 between the steroid resistance of NZB/WF1 lupus nephritis mice and lipid metabolism disorder.3.Spleen lymphocytes of steroid resistance of NZB/WF1 lupus nephritis mice were divided into model group,PPAR gamma si RNA with Panax Notoginseng Saponins group,PPAR gamma plasmid transfection group,Panax Notoginseng Saponins group,and set with NZB/WF1 lupus mice group.Each group was cultured 72h respectively.Detection of PPAR gamma and SIRT1m RNA with fluorescence quantitative PCR real-time assay.Detection of PPAR gamma and SIRT1 protein with western blotting assay.Detection of determination of cholesterol ester content of splenic lymphocytes with fluorescence spectrophotometric method.Detection of cholesterol efflux rate of splenic lymphocytes with fluorescence Elisa method.Detection of the expression of ATP binding cassette transporter A1(ABCA1)protein with flow cytometry.In order to observe the intervention effects of Panax Notoginseng Saponins on steroid resistance and intracellular lipid metabolism disorder.Results 1.The ratio of lymphocyte was 97.2%(7.4×10~9/L)with automatic biochemical analyzer,and the ratio of lymphocyte with flow cytometry was 94.5%.The number of living cells were more than 97%by using trypan blue staining.The expression of P-gp was significantly up-regulated in spleen lymphocytes of NZB/WF1 lupus nephritis mice with low dose methylprednisolone induction(38.6±2.2%vs NZB/WF1 lupus nephritis mice group 7.8±1.4%,P<0.05),rhodamine-123 was significantly decreased(33.20±1.7%vs NZB/WF1 lupus nephritis mice 44.90±1.8%,P<0.05).2.SIRT1si RNA transfection rate was 92±2.2%with flow cytometry,PPAR gamma si RNA transfection rate was 90±1.8%(P<0.05).SIRT1 lentivirus plasmid transfection rate was 66±3.1%,PPAR gamma lentivirus plasmid transfection rate was 70±2.5%(P<0.05).There was achieved a good transfection effect.There was the interaction between PPAR gamma and SIRT1 with CO-IP and Super-shift-EMSA detection in steroid resistance of NZB/WF1 lupus nephritis mice and NZB/WF1 lupus nephritis mice(P<0.05).Compared with the normal mice group,the PPAR gamma m RNA of model group,PPAR gamma si RNA group,SIRT1 si RNA group,SIRT1 plasmid transfection group and lupus nephritis mice group were significantly decreased(1±0.0028vs0.0284±0.0032,0.0084±0.0044,0.3231±0.0037,0.0148±0.0023,0.1386±0.0018,P<0.01),the PPAR gamma plasmid transfection group was significantly up-regulated(1±0.0028vs7.674±0.0047,P<0.01);compared with the model group,the PPAR gamma m RNA of PPAR gamma plasmid transfection group,lupus nephritis mice group,SIRT1si RNA group and PPAR gamma m RNA were increased(0.0284±0.0032vs7.674±0.0047,0.1386±0.0018,0.3231±0.0037,P<0.05),PPAR gamma si RNA group and SIRT1 plasmid transfection group were decreased(0.0284±0.0032vs0.0084±0.0044,0.0148±0.0023,P<0.05).Compared with the normal group,model group,the SIRT1 m RNA of PPAR si RNA group,PPAR plasmid transfection group,SIRT1 plasmid transfection group,LN mice group were increased(1±0.0030vs3.2490±0.0028,1.5052±0.0036,4.2871±0.0021,5.5790±0.0042,2.028±0.0024,P<0.05),SIRT1si RNA group was reduced(1±0.0030vs0.0458±0.0033,P<0.01);compared with the model group,the SIRT1m RNA of SIRT1 plasmid transfection group,PPAR si RNA group were increased(3.2490±0.0028vs5.5790±0.0042,4.2871±0.0021,P<0.05),PPAR gamma plasmid transfection group,SIRT1si RNA group,LN mice group were decreased(3.2490±0.0028vs1.5052±0.0036 0.0458±0.0033,2.0280±0.0024,P<0.05).Compared with the normal mice,the expression of PPAR gamma protein of model group,PPAR gamma si RNA group,SIRT1 si RNA group,SIRT1 plasmid transfection group and LN group were decreased(0.89±0.05vs0.55±0.08,0.18±0.09,0.72±0.05,0.32±0.04,0.62±0.02,P<0.05),PPAR gamma transfection plasmid group was increased(0.89±0.05vs0.99±0.17,P<0.05);compared with the model group,the expression of PPAR gamma protein of PPAR plasmid transfection group,LN mice group,SIRT1si RNA group were increased(0.55±0.08vs0.99+0.17,0.62±0.02,0.72±0.05,P<0.05),PPAR gamma si RNA group,SIRT1plasmid transfection group were reduced(0.55±0.08vs0.18±0.09,0.32±0.04,P<0.05).Compared with the normal mice group,the SIRT1 protein of model group,PPAR gamma plasmid transfection group,PPAR gamma si RNA group,SIRT1 plasmid transfection group,LN group were increased(0.41±0.03vs0.78±0.02,0.52±0.04,0.80±0.05,0.88±0.13,0.71±0.06,P<0.05),SIRT1si RNA group was reduced(0.41±0.03vs0.20±0.08,P<0.01);compared with the model group,the expression of SIRT1 protein of SIRT1 plasmid transfection group,PPAR si RNA group were increased(0.78±0.02vs0.88±0.13,0.80±0.05,P<0.05),PPAR g a m m a p l a s m i d t r a n s f e c t i o n g r o u p,S I RT 1 s i R N A g r o u p,L N g r o u p w e r e decreased(0.78±0.02vs0.52±0.04,0.20±0.08,0.69±0.06,P<0.05).Compared with the LN mice group,the expression of PPAR gamma m RNA and protein were decreased in the model group(0.1386±0.0018vs0.0284±0.0032,0.62±0.02vs0.55±0.08,P<0.05),and the expression of SIRT1m RNA and protein were increased in the model group(2.028±0.0024vs3.2490±0.0028,0.69±0.06vs0.78±0.02,P<0.05).After the up-regulation of PPAR gamma m RNA,the expression of SIRT1m RNA and protein were decreased(P<0.05),and the expression of PPAR m RNA and protein were increased after silencing SIRT1m RNA(P<0.05).After the up-regulation of SIRT1m RNA,the expression of PPAR gamma m RNA and protein were all decreased(P<0.05),and the expression of SIRT1m RNA and protein were increased after silencing PPAR gamma m RNA(P<0.05).3.Compared with LN mice group,SIRT1 PPAR gamma m RNA of model group,PPAR gamma si RNA with PNS group were decreased(1±0.0053vs0.0083±0.0042,0.0197±0.0085,P<0.05),PPAR gamma plasmid transfection group and PNS group were increased(1±0.0053vs2.9281±0.0035,1.7654±0.0071,P<0.05).Compared with the model group,the PPAR gamma m RNA of PPAR gamma si RNA with PNS group was decreased(0.0083±0.0042vs0.0197±0.0085,P<0.05),PPAR plasmid transfection group and PNS group were increased(0.0083±0.0042vs2.9281±0.0035,1.7654±0.0071,P<0.05).Compared with LN mice group,SIRT1m RNA of model group was increased(1±0.0064vs2.1584±0.0024,P<0.05),PPAR gamma si RNA with PNS group,PPAR gamma plasmid transfection group and PNS group were decreased(1±0.0064vs0.1037±0.0061,0.0097±0.0033,0.0074±0.0092,P<0.05).Compared with the model group,the SIRT1m RNA of PPAR gamma si RNA with PNS group,PPAR gamma plasmid transfection group and PNS group were decreased(2.1584±0.0024vs0.1037±0.0061,0.0097±0.0033,0.0074±0.0092,P<0.05).Compared with LN mice group,the PPAR gamma protein of model group and PPAR gamma si RNA with PNS group were decreased(0.32±0.06vs0.24±0.07,0.22±0.08,P<0.05),PPAR gamma plasmid transfection group and PNS group were increased(0.32±0.06vs0.76±0.06,0.75±0.04,P<0.05).Compared with the model group,the PPAR gamma protein of PPAR gamma plasmid transfection group and PNS group were increased(0.24±0.07vs0.76±0.06,0.75±0.04,P<0.05),and PPAR gamma si RNA with PNS group had no statistical significance(0.24±0.07vs0.22±0.08,P>0.05).Compared with the LN mice group,the SIRT1 protein of model group and PPAR gamma si RNA with PNS group were increased(0.52±0.05vs0.99±0.03,0.87±0.02,P<0.05),PPAR gamma plasmid transfection group and PNS group were decreased(0.52±0.05vs0.42±0.09,0.33±0.06,P<0.05).Compared with the model group,the SIRT1 protein of PPAR gamma si RNA with PNS group,PPAR gamma plasmid transfection group and PNS group were all decreased(0.99±0.03vs0.87±0.02,0.42±0.09,0.33±0.06,P<0.05).Compared with the LN mice group,the content of cholesterol ester in cells of model group,PPAR gamma si RNA with PNS group were reduce(1.413±0.035vs0.762±0.071,0.824±0.045,P<0.05);PPAR gamma plasmid transfection group and PNS group were increased(1.413±0.035vs2.347±0.032,2.036±0.087,P<0.05).Compared with the model group,the content of cholesteryl ester in cells of PPAR gamma plasmid transfection group and PNS group were increased(0.762±0.071vs2.347±0.032,2.036±0.087,P<0.05),but there was no significant difference in PPAR gamma si RNA with PNS group(0.762±0.071vs0.824±0.045,P>0.05).Compared with the LN mice group,the rate of cholesterol efflux of the model group,PPAR gamma si RNA with PNS group,PPAR gamma plasmid transfection group and PNS group were all increased(10.4±2.5%vs34.7±3.4%,26.2±2.3%,20.3±3.2%,15.8±2.2%,P<0.05).Compared with the model group,the rate of cholesterol efflux of PPAR gamma si RNA with PNS group,PPAR gamma plasmid transfection group and PNS group were all decreased,by the way,PNS group was the lowest(34.7±3.4%vs26.2±2.3%,20.3±3.2%,15.8±2.2%,P<0.05).Compared with LN mice group,the expression of ABCA1 protein of model group,PPAR gamma si RNA with PNS group were increased(21.8±2.6%vs45.2±3.2%,34.7±2.5%,P<0.05),PPAR gamma plasmid group and PNS group were reduced(21.8±2.2%vs17.5±2.4%,13.6±3%,P<0.05);compared with the model group,the expression of ABCA1 protein of PPAR gamma si RN with PNS group,PPAR gamma plasmid transfection group and PNS group were all reduced,by the way,PNS group was lowest(45.2±3.2%vs34.7±2.5%,17.5±2.4%,13.6±3.0%,P<0.05).Conclusion 1.A small dose of methylprednisolone can induce spleen lymphocyte of NZB/WF1 lupus nephritis mice to occur hormone resistance.2.It was found and confirmed that there was a negative feedback loop between mutual inhibition of PPAR gamma and SIRT1 in the model of spleen lymphocyte with steroid resistance of NZB/WF1 lupus nephritis mice.The abnormal activation of feedback loop was one of the important mechanisms of spleen lymphocyte with steroid resistant in NZB/WF1lupus nephritis mice combined with SIRT1/Fox O1/MDR1/P-gp signaling pathway in previous studies.At the same time,due to the feedback loop it had led to a more serious steroid resistance.Moreover,it may play an important role in the regulation of intracellular lipid metabolism mediated by acting on PPAR gamma.3.PPAR gamma was a low level in spleen lymphocyte with steroid resistance of NZB/WF1lupus nephritis mice,and there was a disorder of lipid metabolism and abnormal cholesterol outflow state in steroid resistant cells.4.It was confirmed that in vitro cell PNS had a dual role in reversing hormone resistance and improving lipid metabolism disorder by acting on PPAR gamma and SIRT1 feedback loop,and showed that PNS as the representative of the Chinese herbal medicine of activating blood circulation had the multi-level regulation effect.There had important clinical significance for prevention and treatment of atherosclerosis,improve the prognosis of the patients.It can provide scientific evidence for improving the clinical efficacy of refractory LN and exploring the possible target points in treatment. |
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