The Effect Of Oxymatrine On White Matter Injury And Gut Dysbiosis After Intracerebral Hemorrhage

BACKGROUND:Intracerebral hemorrhage(ICH)is a common subtype of stroke,with high mortality and morbidity due to lack of effective treatments.White matter injury(WMI)was a common pathological event in patients ICH(present in 77%patients)..Motor dysfunction after ICH is closely associated with WMI,especially the corticospinal tract(CST)injury,and secondary neuroinflammation plays a key role in this process.It is reported that gut microbiota participated in regulating the pathophysiology of the central nervous system,such as myelination,immune cell activation,neurogenesis and regeneration,via the gut-brain axis.Oxymatrine(OMT)is transformed in the gastrointestinal tract and proven with antiinflammation,anti-microbial activity,immunomodulation and neuroprotection.OBJECTIVE:The current study aimed to investigate the effect of OMT on post-ICH white matter injury,as well as distal CST injury and the neurological function recovery.Besides,the effects of OMT on gut dysbiosis and intestinal barrier dysfunction induced by ICH were also investigated.METHODS:ICH model was established by injecting type Ⅳ collagenase into the right striatum of C57BL/6J mice.Mice were randomly assigned to three groups,OMT was daily administrated via oral gavage after ICH onset in the OMT-treated group,while equivalent volume of sterile double-distilled water was orally administrated in the sham group and the ICH+Vehicle group.Neurological deficits were assessed by modified neurological severe score(mNSS)and corner turn test at days-1,1,3,7,and 14 after ICH.Hematoma volume and brain water content were studied at 3 days after ICH.On the 14 days after surgery,the WMI,glial scar formation and the structural integrity of CST were observed under microscopy.On days 3 and 14 after ICH,the mRNA expression levels of intestinal tight junction proteins and inflammatory indicators of both the brain and gut tissue were detected by RT-qPCR.Intestinal barrier permeability was detected by 4kD FITC-dextran(FD4)leakage assay and the systemic inflammatory reaction was also investigated by serum inflammatory factors level detection using ELISA on the 3 d and 14 d after ICH.Microbial 16S rRNA sequencing was performed to reveal the dynamic microbial profiles to explore the relative abundance,diversity and differential taxa at days 1,3,7 after ICH.Additionally,the effects of OMT on ICH-induced gut dysbiosis were investigated as well.RESULTS:Compared with the sham group,a larger hemorrhagic lesion in the right striatum and higher brain water content were detected in the ICH+Vehicle group.While OMT treatment significantly reduced hematoma volume and suppressed edema development after ICH.Compared with the ICH+Vehicle group,both the scores of mNSS and corner turn test were significantly decreased in the ICH+OMT group.Immunodetection showed that the expression of both MBP and NF200 in the perihematoma area were significantly increased in the ICH+OMT group on day 14 after ICH,but only NF200 expression was significantly increased in the distal CST of the cervical enlargement.Besides,the colocalization area of GFAP and CSPG were significantly reduced in both the brain white matter and the distal CST.On days 3 and 14 after ICH,RT-qPCR showed that the mRNA levels of the NLRP3 inflammasome(Nlrp3,Asc,Caspase-1),and other inflammatory indicators(I1-1β,Tnf-α,I1-6,and Nos2)in brain in the ICH+OMT group were significantly lower than those in the ICH+Vehicle group.Meanwhile,compared with the ICH+Vehicle group,the mRNA levels of intestinal tight junction proteins(ZO-1,Occludin and Claudin-4)were significantly increased,while those of the NLRP3 inflammasome did not reach statistical significance.The concentrations of serum FD4,LPS,IL-1β,IL-6 and TNFa were significantly decreased in the ICH+OMT group compared with the ICH+Vehicle group at 3 days and 14 days after ICH.Gut microbial profiles demonstrated that mice with ICH had lower microbial diversity and more opportunistic pathogens,while OMT favorably regulated ICH-induced gut dysbiosis by increasing the relative abundance of beneficial bacteria and restoring the diversity of gut microbiota.COCLUSIONS:OMT administration alleviated neurological deficits after ICH.Besides,OMT improved ICH-induced WMI and distal CST injury at cervical enlargement,and inhibited the formation of glial scar.OMT modulated the microbiota dysbiosis caused by ICH and ultimately improved the intestinal barrier dysfunction.This study provided the exprimental basis for the potential clinical transformation of OMT.