Study On The Effect Of Ethyl Pyruvate Targeting Glial Cells In The Regeneration Of Myelin Sheath

Objective:Ethyl pyruvate(EP)is a derivative of pyruvate,an endogenous energy substrate,with strong anti-inflammatory and antioxidant properties.Total flavonoids of Epimedium(TFE)is the extract of epimedium,but their role in the regeneration of myelin sheath has not been explored.In this study,EP effectively improved the behavior and histology of the demyelinating mice model induced by dicyclohexyldihydrazone(CPZ),and EP treatment enhanced the phagocytic function of meylindebris by microglia.In general,demyelination is accompanied by obvious activation of microglia and generation of neuroinflammation.EP can induce the polarization of inflammatory M1 microglia to anti-inflammatory m2 phenotype,which shows that demonstrate the decrease of i NOS / TNF-a and increase of Arg-1 / IL-10.In addition,EP also reduced the expression of TLR4/p-NF--k B/p65 and the secretion of IL-1?and IL-6,thus inhibiting microglial-mediated neuroinflammation in CPZ-induced demyelinating model.Astrocytes also play an important role in the process of demyelination and regeneration of myelin,which may be a potential target for the treatment of demyelination.On the basis of EP treatment for the improvement of the behavior abnormality and demyelination in CPZ-induced demyelinating mice,we further explore the effect of EP on the function of astrocytes,especially on the differentiation of astrocytes into oligodendrocytes.The results showed that EP increased the accumulation of astrocytes in the myelin sheath and promote the phagocytosis of myelin debris by astrocytes.At the same time,EP induced astrocytes to up-regulate the expression of CNTF and BDNF in corpus callosum and striatum,increased the expression of translation factors nestin,Sox2 and-catenin,and decreased the expression of Notch 1 in astrocytes.Because CNTF and BDNF from astrocytes can promote the mobilization and formation of oligodendrocyte precursor cells(OPCs),we also found that EP can effectively promote the generation of NG2+ and PDGF RA+ OPCs,and some of these OPCs can express mature astrocyte marker GFAP.By injecting CFSE-labeled primary astrocytes into the brain,we further confirmed that CFSE-labeled astrocytes expressing NG2 and Sox2 were observed in the corpus callosum after EP treatment,suggesting that EP might transform astrocytes into oligodendrocyte-like cells.Our results demonstrate that EP intervention can affect microglia and astrocytes during the pathological process of demyelination,form a microenvironment that can contribute to the regeneration of myelin sheath,and reveal the potential prospect for further clinical exploration and application.Clinically,the combination of Chinese and Western medicine is very common.The combination of traditional Chinese medicine and Western medicine will inevitably lead to some kind of drug interaction,which may have synergistic effect,and may also cause adverse reactions.The double-edged sword may contribute to the improvement of the disease,and may also cause tissue and organ damage,aggravating the process of the disease.Based on our previous research on the protection and regeneration of myelination by TFE,here we try to combine EP with TFE with different proportions.The combined dose of EP and TFE is the same as that of single EP and TFE(EP/TFE=50ug/ml,EP+TFE=50ug/ml).We compared antioxidant activity by microglia.At the same time,we compared the release of growth factors by astrocytes,providing the experimental basis for the choice of clinical combination for traditional Chinese medicine and Western medicine.The results show that that EP and TFE could effectively scavenge the oxidation products,inhibit the production of NO by microglia,and induce the expression of CNTF and bFGF by astrocytes.Method:In this experiment,C57BL/6 male mice(6 weeks old)were used to establish the demyelination model induced by CPZ feeding.The mice were divided into normal group,normal group + EP group,model group and EP treatment group.Mice of normal group were fed with normal diet for 6 weeks,mice of normal group + EP group were injected with EP alone,mice of model group and EP treatment group were fed with powder diet containing 0.2% CPZ for 6 weeks.At the end of fourth week(day 28)after feeding,EP or saline were injected intraperitoneally until the sixth week(day 42).Before the end of animal experiment,mice were performed by behavioral tests,including forced swimming,elevated cross maze and T maze,which were used to evaluate the depression,anxiety and cognitive impairment of mice.At the end of sixth week,mice were killed,the brain tissues were collected,frozen sections were made and the protein was extracted.The former was used for immunohistochemistry,and the latter was used for western blot and ELISA.Flow cytometry was used to detect the phagocytosis,phenotype and proliferation by BV2 microglia,primary microglia and astrocytes.The migration and differentiation of astrocytes were observed by stereotactic injection.The experimental data of each group were analyzed by graphpad prism 7.0.Result: 1.Consistent with previous reports,body weight of mice fed with CPZ decreased significantly in the first week compared with normal mice,and remained stable in the following three weeks.There was no significant difference in body weight between CPZ and CPZ + EP groups.Behavioral tests showed that mice fed CPZ for 6 weeks showed anxiety,depression and cognitive impairment compared with normal mice.However,EP treatment can effectively improve the above behavior abnormalities(P < 0.05).Pathologic staining showed that an obvious demyelination in corpus callosum of mice fed CPZ.Compared with the model mice fed with CPZ,EP treatment increased the intensity of Black Gold II staining and improved the demyelination(p<0.05).These results demonstrated that EP can effectively improve the demyelination,accompanied by behavioral improvement.2.Iba-1 immunofluorescence staining showed that Iba-1 + microglia were enriched in corpus callosum,striatum and cortex of mice fed with CPZ,and the expression of Iba-1 protein was also increased.EP treatment significantly reduced the concentration of Iba-1 microglia in the corpus callosum,striatum and cortex,and decreased the expression of Iba-1 protein in brain tissue(P < 0.05).3.The results of immunodouble staining showed that MBP staining was co-located in the cytoplasm of Iba-1microglia,suggesting that microglia could migrate to the myelinated tissue and phagocytize the myelin debris during demyelination.4.To further confirm that EP can promote the phagocytosis of myelin debris by microglia,we designed the experiments from primary microglia and BV2 cells in vitro.The results showed that EP increased the migration of microglia and the phagocytosis of myelin debris and induced the polarization of M2 phenotype,accompanied by cell proliferation and death.5.The results of immunodouble staining showed that EP reduced the expression of TLR4 and p-NF-k B/p65 in Iba-1microglia,decreased the production of IL-1b and IL-6 in the brain extract of mice fed with CPZ,and inhibited the microglia-mediated neuroinflammation.6.At the same time,immunodouble staining showed that EP decreased the expression of i NOS and TNF-a in Iba-1microglia;on the contrary,EP increased the expression of Arg-1 and IL-10 in Iba-1microglia(P < 0.05),suggesting that EP could induce the polarization of M1 to M2 microglia.7.Similarly,EP induced GFAP+astrocytes to accumulate and phagocytize myelin debris in myelin sheath,which was verified by cell experiments in vitro.8.EP can induce astrocytes to up-regulate the expression of CNTF and BDNF in corpus callosum and striatum,increase the expression of nestin,Sox2 and ?-catenin,and decrease the expression of Notch 1 by astrocytes.9.EP can also promote the formation of NG2+and PDGF RA+OPCs.Some cells can express Ki67 and MBP,suggesting that these OPCs are proliferative cells and may differentiate into mature oligodendrocytes.It was further confirmed that EP might induce astrocytes to differentiate into oligodendrocyte-like cells by injecting CFSE-labelled astrocytes in brain.10.EP and TFE were prepared in a specific proportion,which could more effectively remove oxidation products,in vitro BV2 microglia experiment,EP and TFE(5:5 ratio)could effectively inhibit the production of NO by BV2 cells;in vitro GL261 astrocytes experiment,EP and TFE(5:5 ratio)could effectively induce the expression of CNTF and bFGF by astrocytes.It was also found that p-STAT3 negatively regulated the formation of CNTF and bFGF by astrocytes.Conclusion: 1.EP treatment reduced the demyelination in corpus callosum of mice fed with CPZ,accompanied by the improvement of anxiety,depression and cognitive impairment.2.EP affected the migration and enrichment by microglia,enhanced the phagocytosis of myelin debris,and inhibited the neuroinflammation,which may be related to the polarization of M2 microglia.3.EP also affected the migration and enrichment by astrocytes,enhanced the phagocytosis of myelin debris,promoted the expression of CNTF and BDNF,and up-regulate the expression of nestin and Sox2.4.EP finally promoted the formation and proliferation of OPCs,and differentiated them into mature oligodendrocytes.At the same time,astrocytes were induced to differentiate into oligodendrocytes.5.The combination of EP and TFE(appropriate proportion)can more effectively eliminate the oxidation products,inhibit the production of NO by BV2 cells,and induce astrocytes to express CNTF and bFGF.