| Objective:To study the potential mechanism of action of matrine(MAT)on the behavioral characteristics of experimental autoimmune encephalomyelitis(EAE),a classic animal model of multiple sclerosis(MS).We observed and recorded the histopathological effects of MAT on the spinal cord of rats,and detected the apoptosis(TUNEL)of OLIG2(oligodendrocytes)in the white matter of the brain,changes of the apoptosis protease caspase-3,small heat shock protein HSPB5 and the proinflammatory cytokine interleukin IL-1? in rat brain white matter,expression of mitochondrial key protein OLIG2+Cyt c+,expression of autophagy-related proteins OLIG2+Beclin1+and mitochondrial TOMM20+LC3+in oligodendrocytes,determination of glutathione peroxidase GSH-Px and MDA in brain and spinal cord;to observe the histopathological effects of MAT on optic neuritis(ON)in EAE rats,co-expression of Nrf2,SIRT1,PGC1?,TOMM20 and Brn3a in the retina was detected.To explore the mechanism of MAT on mitochondrial autophagy in oligodendrocytes in the brain of EAE rats and the mechanism of mitochondrial biosynthesis in ON of EAE complications.The aim is to find a potential mechanism for the treatment of MS and lay a theoretical foundation for the clinical application of MAT.Methods:1 Induction of EAEGuinea pig spinal cord homogenate(GPSCH)was prepared in a sterile environment,and an equal amount of Freund's complete adjuvant(CFA,containing 6 mg/mL BCG vaccine)was added to prepare a water-in-oil antigen emulsion.Twenty female Wistar rats from 6 to 8 weeks were randomly selected to inject the antigen emulsion subcutaneously on the limb pads.2 Animal grouping and MAT interventionUsing random number table method,20 immunized Wistar rats were randomly divided into two groups(n=10 each group):EAE model group(EAE+Vehicle),and MAT treatment group(EAE+MAT).In addition,healthy non-immunized Wistar rats were in the normal group(Naive).In the EAE+MAT group,rats were given 250 mg/kg(6.7 mL/kg)of MAT daily for intraperitoneal injection on the 11th day after immunization.Rats in the Naive group and EAE+Vehicle group were treated with the same amount of saline at the same time point from the 11th day after immunization.The rats in the three groups intervened until the 17th day.3 Collection of tissue samplesOn day 18 after immunization,the rats were anesthetized,after apical blood collection,the heart was perfused with pre-cooled normal saline,and the eyeball,optic nerve,brain and spinal cord were taken.Half of them are immersed in 4%paraformaldehyde to make paraffin sections,and the other half is frozen in-80?refrigerator for future use.4 Indicator detectionHematoxylin-eosin(HE)staining and LFB staining to observe the infiltration and demyelination of inflammatory cells in the spinal cord and optic nerve of rats.TUNEL staining to detect oligodendrocyte apoptosis.Immunofluorescence double staining was used to detect the expression of OLIG2+Cyt c+,OLIG2+Beclin1+,TOMM20+LC3+,Brn3a+Nrf2+,Brn3a+SIRT1+,Brn3a+PGC1?+and Brn3a+TOMM20+.Western Blot method to detect the expression of caspase-3,HSPB5,IL-1?.Colorimetric method was used to detect the activity of GSH-Px and MDA.The main research results are divided into the following two parts:Part one:Matrine protects oligodendrocytes by inhibiting their apoptosis and enhancing mitochondrial autophagy1 EAE incidenceNo incidence was observed in rats in the Naive group.Eight rats in the EAE+Vehicle group developed disease,with an incidence of 80%,and seven rats in the EAE+MAT group developed disease,the incidence was 70%.The incidence of rats in the Naive group was significantly lower than that of the EAE+Vehicle group and the EAE+MAT group,which was statistically significant(P<0.05);the incidence of the rats in the EAE+MAT group was not significantly different from that of the EAE+Vehicle group,and there was no statistics Significance(P>0.05).2 Weight changes and neurological function scoresThe weight of the three groups of rats decreased slightly during the initial period after immunization.The weight of the rats in the Naive group increased steadily from the third day.The other two groups generally showed a trend of first increase and then decrease.It was significantly lower than the EAE+Vehicle group(P<0.05).The nerve score of rats in the Naive group was always 0.On the 17th day after immunization,the average neurological score in the EAE+ehicle group was significantly higher than that in the EAE+MAT group(P<0.01).3 Histopathological changesThe spinal cord of the rats in the Naive group showed no inflammatory infiltration and demyelination,and the scores were all 0.Compared with the rats in the Naive group,the EAE+Vehicle group had severe inflammatory infiltration and a large number of demyelination.The scores of inflammation infiltration and demyelination in the EAE+MAT group were significantly lower than those in the EAE+Vehicle group,and the differences were statistically significant(P<0.01,P<0.001).4 TUNEL stainingThere was a few oligodendrocyte apoptosis in the central nervous system of rats in the Naive group,while a large number of apoptotic cells appeared in the EAE+Vehicle group compared with the Naive group(P<0.001).Compared with the rats in EAE+Vehicle group,the number of oligodendrocyte apoptosis in the EAE+MAT group decreased significantly(P<0.01).5 Expression of Cyt c,Beclinl,TOMM20 and LC3 in white matterThe expressions of Beclinl,TOMM20 and LC3 in the white matter of rats in EAE+Vehicle group were significantly lower than those in Naive group(P<0.001),while the expressions of Beclinl,TOMM20 and LC3 in EAE+MAT group were significantly higher than those in EAE+Vehicle group(P<0.001).Compared with the Naive group,Cyt c expression in the EAE+Vehicle group was significantly increased(P<0.001),and Cyt c expression in the EAE+MAT group was significantly decreased(P<0.01)compared with the EAE+Vehicle group.6 Expression of caspase-3,HSPB5 and IL-1? in brain and spinal cordThe expressions of caspase-3,HSPB5 and IL-1? in the brain and spinal cord of rats in the EAE+Vehicle group were significantly higher than those in the Naive group(P<0.001).The expression levels of caspase-3,HSPB5 and IL-1? in the EAE+MAT group were significantly lower than those in the EAE+Vehicle group(P<0.001,P<0.001,P<0.01).7 Changes of GSH-Px and MDA in brain and spinal cordCompared with the Naive group,the GSH-Px activity in the EAE+Vehicle group was significantly reduced(P<0.001);compared with the EAE+Vehicle group,the GSH-Px activity in the EAE+MAT group was significantly increased(P<0.001).The change of MDA content is opposite to that of GSH-Px.Part two:Matrine promotes mitochondrial biosynthesis and reduces oxidative stress by SIRT1 activation in experimental optic neuritis1 EAE incidenceNo incidence was observed in rats in the Naive group.Seven rats in the EAE+Vehicle group developed disease,with an incidence of 70%,and eight rats in the EAE+MAT group developed disease,the incidence was 80%.The incidence of rats in the Naive group was significantly lower than that of the EAE+Vehicle group and the EAE+MAT group,which was statistically significant(P<0.05);the incidence of the rats in the EAE+MAT group was not significantly different from that of the EAE+Vehicle group,and there was no statistics Significance(P>0.05).2 Weight changes and neurological function scoresThe weight of the three groups of rats continued to decrease 3 days after immunization.The weight of the rats in the Naive group increased steadily from the 3rd day.The weight of the rats in the EAE+Vehicle group and the EAE+MAT group generally increased first and then decreased.Among them,the weight loss trend of EAE+MAT group was significantly lower than that of EAE+Vehicle group(P<0.05).The average nerve score of the rats in the Naive group was always 0.On the 17th day after immunization,the average neurological score in the EAE+Vehicle group was significantly higher than in the EAE+MAT group(P<0.01).3 Histopathological changesIn the Naive group,no inflammatory infiltration and demyelination occurred in the optic nerve,and the scores were all 0.Compared with the rats in the Naive group,the EAE+Vehicle group had severe inflammatory infiltration and a large number of demyelination.The scores of inflammation infiltration and demyelination in EAE+MAT group were significantly lower than those in EAE+Vehicle group,and the difference was statistically significant(P<0.001).4 The co-expression of Brn3a+SIRT1+,Brn3a+Nrf2+,Brn3a+PGC1?+and Brn3a+TOMM20+in the retinaCompared with rats in the Naive group,the expressions of Brn3a+SIRT1+,Brn3a+Nrf2+,Brn3a+PGC1?+and Brn3a+TOMM20+in the EAE+Vehicle group were significantly reduced(P<0.001,P<0.01,P<0.001,P<0.001);and Compared with the EAE+Vehicle group,Brn3a+SIRT1+,Brn3a+Nrf2+,Brn3a+PGC1?+and Brn3a+TOMM20+were significantly increased in the EAE+MAT group(P<0.01).Conclusion:1 MAT has obvious therapeutic effect on acute EAE rats,probably by inhibiting oligodendrocyte apoptosis,enhancing mitochondrial autophagy,and improving the oxidative stress resistance of mitochondria in oligodendrocytes,thereby reducing oligodendrocyte apoptosis.2 MAT has a good therapeutic effect on EAE complication optic neuritis,which may be achieved by activating SIRT1 and PGC1? deacetylation to promote mitochondrial biosynthesis and Nrf2 regulated antioxidant defense mechanism to prevent retinal ganglion cell loss. |
PDF Full Text Request