Jisuikang Improves Spinal Cord Injury By Regulating Gut Microbiota-induced Histone Acetylation In Microglia

BACKGROUND:Spinal cord injury causes heavy burden to society and family due to the high disability rate.In recent years,tissue-engineered nerve scaffolds have been proved effective on treating spinal cord injury,however,with some limitations.Traditional Chinese Medicine(TCM)has been more and more popular due to its advantages of multiple targets and low side effects as a good match for existing treatment methods.Jisuikang,Traditional Chinese Medicine compound,has been proved effective in treating human spinal cord injury.OBJECTIVE:To elucidate the mechanism of Jisuikang in treating spinal cord from the perspective of regulating gut microbiota and histone acetylation in microglia.METHODS:In vivo experiments involved 64 C57BL/6 mice,which were allocated into 4 groups using the stratified block randomization,namely,sham operation+normal saline group(SN group),sham operation+Jisuikang group(SJ group),spinal cord injury model+normal saline group(MN group),and spinal cord injury model+Jisuikang group(MJ group).A weight drop method was used to cause the spinal cord injury.After the operation,mice were raised in a single cage in a clean environment with constant temperature and humidity.The condition and weight of the mice were recorded after the operation.The motor function was evaluated by BMS score and inclined plate test.Mouse feces were collected 7 d,14 d,21 d and 28 d post operation.Blood plasma and spinal cord tissue were collected 28 d post operation.GC-MS was used to determine the concentration of short-chain fatty acids in feces and blood.16s rDNA sequencing was used to analyze the gut microbiota in mice feces.Hematoxylin eosin staining(HE staining)was used to observe the pathological changes of the spinal cord.Histone acetylation levels and microglial cell activation were detected by immunohistochemical staining(IHC).Function of various cells was analyzed by immunofluorescence multi-label staining(IF).In vitro experiments involved BV-2,which is a widely used immortal microglia.Various kinds of short-chain fatty acids with different concentrations were screened for effect on histone acetylation at different sites in BV-2 using Dot Blot,the result of which were further verified by Western Blot.Enzyme-linked immunoassay(ELISA)and real-time fluorescence quantitative polymerase chain reaction(qPCR)were used to determine the expression of inflammatory factors relevant with BV-2 activation.Immunofluorescence(IF)were used to detect Ibal expression in BV-2.CCK-8 was used to evaluate BV-2 proliferation.RESULTS:Spinal cord injury can significantly reduce the weight of mice,Basso Mouse Scale score and the angle in oblique plate test,lead to white matter loss,increase the expression of TREM2 and the acetylation level of H3K27 in the spinal cord,reduce the expression of Ibal,CD 13,GFAP and the acetylation level of H4K8,reduce the diversity of gut microbiota,reduce the concentration of propionate,butyrate and isobutyrate in the plasma,and change the concentrations of short chain fatty acids in feces.Jisuikang can improve Basso Mouse Scale score,reduce white matter loss,increase the expression of Ibal,NeuN,CD13,GFAP,TREM2 and the acetylation level of H3K27 in the spinal cord,reduce the expression of Ibal and the acetylation level of H4K8,increase the diversity of gut microbiota in mice with spinal cord injury,but reduce the diversity of gut microbiota in mice with sham operation.Jisuikang can regulate the concentration of short chain fatty acids in plasma and feces.In vitro experiments showed that different kinds and concentrations of short chain fatty acids and TSA could inhibit the proliferation of BV-2 cells,regulate the expression of TREM2 and acetylation of H3K27 and H4K8,improve the levels of IL-4 and IL-13,reduce the levels of IL-10,TNF-α,iNOS and Ibal,inhibit the expression of TREM2,promote the expression of TNF-α,IL-10 and Argl,and promote the expression of TNF-α,IL-10 and Arg1.The expression of iNOS and Mincle were changed depending on the time of intervention.CONCLUSION:Jisuikang may reduce white matter loss and improve motor function of hind limbs in mice by reduce secondary injury of spinal cord,which is related to M2a and Ml-type activation of microglia induced by short chain fatty acids from intestinal bacteria.