Mechanism Of Matrine On Experimental Autoimmune Encephalomyelitis And Its Complications Optic Neuritis

Objective Observe the symptoms associated with the use of matrine(Matrine,MAT)in the treatment of experimental autoimmune encephalomyelitis(EAE)mouse model.For the EAE mouse model,examine the mouse spinal cord tissue.Pathological changes,co-expression of the intermediate factor Olig2+Ki67+ that signifies oligodendrocyte proliferation,and the expression of mature oligodendrocyte marker CC1 and the surface marker PLP of mature myelinating cells,in addition to detection Phosphorylation of PI3K/Akt signaling pathway-related factors Akt,m TOR,PI3 K,and P70S6.To investigate whether MAT can increase the number of oligodendrocytes and promote myelination by activating the PI3K/Akt signal transduction pathway,thereby alleviating neurological dysfunction in EAE mice;for the EAE rat model,observe the treatment of MAT for EAE Effects of rat complications on optic neuritis: By comparing weight changes,neurological function scores,and pathological changes of optic nerve tissue in experimental rats,and detecting Iba1,CD4,and NFs in optic nerve tissue,and using immunofluorescence double staining RGCs + TUNEL were stained in the retina to investigate the effect of MAT on the complications of optic nerve(ON)in EAE rats.The purpose of this study was to explore the mechanism of MAT on multiple sclerosis(MS)and its complications,and to provide a reliable theoretical basis for seeking new effective therapeutic approaches and drug targets for clinical MS treatment.Methods 1 Induction of EAE animal model 1.1 Induction of acute EAE rat model Under sterile conditions,guinea pig spinal cord homogenate(GPSCH)was prepared using a guinea pig spinal cord,and an equal volume of complete freund adjuvant(CFA)containing 6 mg/ml BCG was added and thoroughly mixed.A stable water-in-oil type antigen emulsion prepared as an induction model was immunized to 6-8 week-old female Wistar rats to induce an experimental acute autoimmune encephalomyelitis acute model.2.2 Induction of chronic progressive EAE mouse model Dissolve MOG35-55(10 mg)in 2 ml normal saline and mix it with an equal volume of Freund's complete adjuvant containing 2.5 mg/ml Mycobacterium tuberculosis.Use two glass syringes in the frapped sand(0 °C environment)repeatedly pumping about 1.5h until the mixture is fully mixed to form a water-in-oil type milky white antigen emulsion,and take a drop of water into the clear water,and let stand for about 10 minutes without diffusion.After C57BL/6 mice were anesthetized with 2% pentobarbital,they were subcutaneously injected(0.2 m L/body)subcutaneously at two points directly above and below(left and right)the dorsal midline of the spine.After 0 h and 48 h of immunization,all experimental mice were intraperitoneally injected with 200 ng of PBS solution containing pertussis toxin to induce EAE mouse model.2 Animal groups and MAT interventions 2.1 Rat groups and MAT interventions 20 immunized female Wistar rats were randomly divided into EAE model group(MAT)and MAT treatment group(MAT)with 10 rats in each group.In addition,10 non-immunized healthy female Wistar rats were normal.From the initial stage of disease(9 days after immunization),rats in the MAT group were treated intraperitoneally with 6.7 ml/kg(200 mg/kg)of MAT once a day,while rats in the Normal and Vehicle groups were given equal doses.Normal saline,all three groups of rats were intervened and sacrificed on the 19 th day(19th day after immunization).2.2 Mouse groups and MAT interventions Thirty immunized female C57BL/6 mice were randomly divided into two groups using the principle of random distribution: EAE model group(Vehicle),MAT treatment group(MAT),15 in each group.In addition,15 non-immune healthy female C57BL/6 mice were in the normal group.From the early stage of disease(13 days after immunization),mice in the MAT group were intraperitoneally injected with 6.7 ml/kg(200 mg/kg)of MAT once a day,and mice in the Normal group and Vehicle group were given equal amounts of physiology at the same time.In saline,all three groups of mice were intervened and sacrificed on the 23 rd day after immunization.3 Collect experimental specimens On the 19 th day(rat)and 23 th day(in mice)after immunization,the animals were anesthetized,and preincubated physiological saline was used for cardiac perfusion.The brains and spinal cords of the big mice were collected,and partially fixed with 4% paraformaldehyde.Paraffin-embedded sections were prepared and the remaining portion was placed in a refrigerator at-80°C for use.In addition,rat optic nerve and retina were removed and fixed with 4% paraformaldehyde to make paraffin sections.4 Detection of indicators Hematoxylin Eosin(HE)staining and Luxol Fast Blue(LFB)staining were used to observe the inflammatory infiltrates and myelin sheath degeneration in the spinal cord and rat optic nerves,respectively.Immunofluorescence staining was used to analyze CC1 and PLP in the spinal cord of mice.The expression of Iba1,CD4 and NFs in rat optic nerve was analyzed.Immunofluorescence double staining was used to analyze the expression of Olig2+Ki67 in rat spinal cord and rat TUNEL+RGCs.The expression of p-Akt,pm TOR and p-PI3 K in spinal cord of mice was detected by Western Blot.p-P70S6 content.Divided into the following two parts of the main research results:Part I: Effects of matrine on the Proliferation of Oligodendrocytes in EAE Mice by PI3K/Akt Signaling Pathway 1 Incidence rate There was no incidence observed in mice in the Normal group.Vehicles in the Vehicle group had 13 cases and the incidence rate was 86.67%.There were 12 cases in the MAT group and the incidence rate was 80.00%.The incidence rate in the Normal group was significantly lower than that in the MAT group.In the Vehicle group,the difference was statistically significant(p<0.01);the incidence of mice in the MAT group was not significantly different from that in the Vehicle group and was not statistically significant(p>0.05).2 Neurological function scores From the first day after the immunization,the average neurological function score of the Normal group mice was always 0,and the neurological function scores of the Vehicle group and the MAT group increased first and then decreased.After the MAT treatment,the MAT group of mice The decrease of the mean neurological score was significantly lower than that of the Vehicle group(p<0.05).3 Histopathological changes In the Normal group,the scores of average inflammatory infiltration and demyelination scores of the spinal cord in mice were 0,and inflammation and infiltration of myelin sheaths were observed in the spinal cord of Vehicle and MAT mice.In comparison,both were significantly higher and the difference was statistically significant(p<0.01).The average inflammatory infiltration and demyelination of mice in the Vehicle group were more severe than those in the MAT group(p<0.05).4 Expression of Olig2+Ki67,CC1,and PLP in Spinal Cord Compared with Normal group,the content of Olig2+Ki67 and CC1 in Vehicle and MAT mice was significantly decreased(p<0.01).Compared with the Vehicle group,the expression of Olig2+Ki67,CC1,and PLP in the MAT group was significantly higher(p<0.01).5 Changes in p-Akt,p-m TOR,p-PI3 K,and p-P70S6 levels in the brain Compared with the Normal group,the levels of p-Akt,p-m TOR,p-PI3 K,and p-P70S6 were significantly lower in the Vehicle group.(p<0.01),but the content of p-Akt,p-m TOR and p-PI3 K in MAT group was significantly higher than that in Normal group(p<0.01),and the content of p-P70S6 was not significantly changed(p>0.05).The contents of p-Akt,p-m TOR,p-PI3 K,and p-P70S6 in the MAT group were significantly higher than those in the Vehicle group(p<0.01).Part II: To investigate the therapeutic effect of matrine on complications of optic nerve in EAE rats 1 Incidence rate No symptoms were observed in the Normal group.There were 9 cases in the Vehicle group.The incidence rate was 90.00%.There were 6 cases in the MAT group.The incidence rate was 60.00%.The incidence rate in the Normal group was significantly lower.In the MAT group and the Vehicle group(p<0.01),there was no significant difference in the incidence rate between the MAT group and the Vehicle group(p>0.05).2 Body weight changes and neurological function scores From the first day after immunization,the body weight of Normal rats increased steadily,and the body weights of Vehicle group and MAT group increased first and then decreased.Among them,at the peak of onset(9 days after immunization),MAT treatment was performed.The mean weight loss of rats in the MAT group was significantly lower than that in the Vehicle group(p<0.01).After normal immunization,the normal group had a neurological score of 0.The average neurological function scores of rats in the Vehicle group and the MAT group increased first and then decreased,and the mean neurological scores in the MAT group were significantly lower than those in the Vehicle group.The difference was statistically significant(p<0.05).3 Histopathological changes There was no inflammatory cell infiltration and demyelination within the optic nerve of the Normal group.Both the Vehicle group and the MAT group exhibited inflammatory infiltrates and demyelination in the optic nerve of rats.,both significantly increased,the difference was statistically significant(p<0.01).The average inflammatory infiltration and demyelination of rats in the vehicle group were significantly higher than those in the MAT group(p<0.05).4 Expression of Iba1,CD4,and NFs in optic nerve Compared with Normal group,the expression of Iba1 and CD4 in Vehicle and MAT rats was significantly increased,and the expression of NFs was significantly decreased,and the differences were statistically significant(p<0.05);Compared with the Vehicle group,the expression of Iba1 and CD4 in the MAT group was significantly lower,and the expression of NFs was significantly higher(p<0.05).5 Expression of RGCs in the retina Compared with the Normal group,the expression of RGCs in the Vehicle and MAT groups was significantly decreased(p<0.05),and the number of apoptotic RGCs was significantly increased(p<0.05);In rats,the expression of RGCs was increased compared with Vehicle group,and the number of apoptotic RGCs was significantly decreased(p<0.05).Conclusion 1.MAT has obvious preventive and therapeutic effects on chronic progressive EAE female mice,probably through up-regulation of PI3K/Akt signaling pathway and promotion of proliferation of oligodendrocytes in the central nervous system,thereby stimulating the production of myelin sheath proteins,thereby alleviating EAE progression in mice.2.MAT has a certain mitigation effect on complications of acute EAE female rats with optic neuritis.Its mechanism may be to inhibit the expression of inflammatory cells Iba1 and CD4 in optic nerve tissue and promote the expression of NFs,which in turn increases the expression of RGCs.Play a role in the prevention and treatment of optic neuritis.