Analysis Of Myelin Protection Effect And Mechanism Of Dihydrotanshinone?

Objective:Multiple Sclerosis(MS)is a demyelinating disease of the central nervous system(CNS),which is more common in young adults.There is no clear pathogenesis and effective treatment.There are two main animal models of MS:Experimental autoimmune encephalomyelitis(EAE)and Cuprizone model.EAE is the most commonly used MS model,but it is mainly used to study the mechanism of MS immune disorders and find effective immunomodulatory drugs.Cuprizone mainly acts on cells of oligodendrocyte mass spectrometry,and is an animal model for studying demyelination and remyelination.Some studies believe that activated microglia(MG)remove myelin debris and have a protective effect of myelin,but other studies believe that the inflammatory substances released by inflammatory MG have a myelin damage effect.Looking for compounds with improved MG function is an important research direction for the discovery of myelin protection and remyelination drugs.Salvia miltiorrhiza has the functions of promoting blood circulation,removing blood stasis,and relieving pain,calming the nerves.It is a common herbal medicine commonly used in the treatment of MS.Dihydrotanshinone I(DHT)is an effective ingredient of Salvia miltiorrhiza.There is no research on the treatment of MS by DHT.In this project,8-week-old male C57BL/6 mice were used as the target,and feed containing 0.2%Cuprizone was used to prepare a demyelinating animal model.DHT was administered by gavage.By comparing the CMC-Na normal group,CMC-Na model group and DHT The demyelination and MG polarization in the CNS of the intervention group.To explore the protective effect and mechanism of DHT on myelin sheath.Methods:(1)In vivo experiments,24 male C57BL/6 mice at 8 weeks of age were randomly divided into CMC-Na normal group,CMC-Na model group and DHT intervention group,8 mice in each group.Feed the diet containing 0.2%Cuprizone to prepare the demyelinating model;starting from the 15th day.Treatment:the normal group of CMC-Na will be given 0.5%aqueous solution of sodium carboxymethylcellulose(CMC-Na),and continue to feed without Cuprizone feed;CMC-Na model group was given0.5%CMC-Na solution and continued feeding with Cuprizone-containing feed;DHT intervention group,intragastric DHT(25 mg/kg/day,dissolved in 0.5%CMC-Na solution),continued feed with 0.2%Cuprizone.Until the 42nd day,the anesthesia was sacrificed,and brain tissue was separated to prepare frozen sections.Because the demyelination lesions induced by Cuprizone are mainly in the corpus callosum,therefore,all the experiments below only observe this area.The integrity of myelin sheath was detected by LFB histochemical staining,MBP and PLP immunofluorescence staining,TUNEL labeled apoptosis,and Iba-1,CD86 and CD163 immunofluorescence staining was used to detect MG polarization in vivo.(2)SIM-A9 MG was cultured in vitro,and the experiment was divided into CMC-Na control group,DHT control group,CMC-Na+LPS stimulation group and DHT combined LPS intervention group.After 1.5 h of adherence,the cells were stimulated with LPS(final concentration 25 ng/ml)for 4h,and then CMC-Na(0.005%)or DHT(100 nM)was added,and the culture was continued for 24 h.Set 3 complex holes in each group and repeat 3 times.Then,the collected cells were stained with CD14,CD16/32,TNF-?,iNOS,CD206,IL-10 and Arginase-1,and flow cytometry was used to detect the M1 and M2 polarization of SIM-A9 MG.Result:1.LFB histochemical staining,anti-MBP and PLP antibody immunofluorescence staining experiments marked the myelin sheath in the corpus callosum of mouse brain,showing similar results.Compared with the CMC-Na normal group,0.2%Cuprizone induced 80%of myelin sheath loss However,after DHT administration,only about 60%of the myelin sheath was lost,and DHT intervention significantly inhibited Cuprizone-induced demyelination.2.TUNEL can display apoptosis by combining with nucleic acid 3`-OH.The research group marked the apoptosis of the corpus callosum in the brain by TUNEL staining experiment.The results showed that compared with the CMC-Na normal group,0.2%Cuprizone induced A large amount of apoptosis(p<0.01),and after DHT administration,the number of apoptotic cells decreased significantly(p<0.01).3.Use anti-GFAP antibodies to label the astrocytes in the corpus callosum of the brain.The results showed that 0.2%Cuprizone induced significant astrocyte activation.After DHT administration,a large number of astrocytes still activated,and There was no significant difference between the CMC-Na model group.4.Two-color immunofluorescence staining with anti-MBP and Iba-1 antibodies was used to label the myelin sheath and MG of the corpus callosum,respectively.The results showed that 0.2%Cuprizone had no significant effect on the number of Iba~+MG compared with the normal CMC-Na group The cell body became larger and became amebic MG.The area of amebic MG in the CMC-Na model group increased significantly.After DHT administration,the area of amebic MG decreased significantly(p<0.05).5.Further using anti-CD86 and CD163 antibodies to detect the phenotype of MG in the corpus callosum,the results showed that compared with the CMC-Na normal group,the CMC-Na model group had more CD86~+MG and no CD163~+MG.Difference;in the DHT intervention group,compared with the CMC-Na normal group,the number of CD86~+MG and CD163~+MG increased significantly;but compared with the CMC-Na model group,the number of CD86~+MG was less,and the number of CD163~+MG increased significantly;Analyzing the ratio of the number of CD86~+MG to the number of CD163~+MG,the normal CMC-Na group was close to 1,the CMC-Na model group was significantly increased,and the DHT intervention group was significantly lower than 1,indicating that DHT changed the balance of MG to M2 type MG.6.Using SIM-A9 microglial cell line for verification,the results showed that LPS stimulation significantly induced an increase in the percentage of CD16/32~+,iNOS~+,TNF-?~+,CD206~+and IL-10~+cells(p<0.01),DHT intervention After LPS stimulation,the percentages of CD16/32~+,iNOS~+,TNF-?~+cells were significantly reduced(p<0.05),but the percentages of CD206~+,IL-10~+,and Arginase+cells were not significantly increased;for the number of M1 and M2 cells.The result showed that,compared with the CMC-Na control group and the DHT control group,the balance of MG in the LPS group was inclined to M1,and the balance of MG in the DHT and LPS intervention group was inclined to M2.Conclusion:The feeding of Cuprizone resulted in obvious demyelination of the CNS corpus callosum of the mice,which obviously induced apoptosis.The astrocytes and MG were obviously activated,and the balance of activated MG was inclined to M1;DHT intervention can obviously Reduced demyelination induced by Cuprizone significantly reduced apoptosis and had no effect on the activation of astrocytes.Both in vivo and in vitro experiments have shown that DHT can partially inhibit the activation of MG,tilting the balance of MG towards M2.It shows that DHT may reduce the inflammation of CNS by promoting the transformation of MG to M2,and play a protective role in myelin.