| ObjectiveTo explore the effect of SNX on the intestinal barrier and inflammatory microenvironment of rats after stroke and its possible mechanism of action using Shunaoxin dropping pills(SNX)intervention in transient middle cerebral artery occluded(tMCAO)rats.Methods1.The model of tMCAO in male SD rats was established by the intraluminal suture.The successfully modeled rats were randomly divided into the model group(tMCAO group)and the Xionggui group(tMCAO+SNX group).Rats in the sham-operated group(Sham group)underwent only neck incision and common carotid artery dissection.The tMCAO+SNX group was administered 24 h after ischemia reperfusion(SNX dissolved in saline,45 mg/kg/d for 7 days by gavage),and the Sham and tMCAO groups were given an equal volume of saline gavage.2.The neuroprotective effects of SNX on tMCAO rats were evaluated by neural defect score on days 1,3,7,14 and 21 after successful modeling in each group.3.HE staining,AB-PAS staining,and immunofluorescence staining(claudin-1,occludin,ZO-1)were used to observe the colon pathology,goblet cell expression,tight junction protein expression and distribution in each group of rats on the 7th and 21 st day after tMCAO.The relative expression of colon tight junction protein(claudin-1,ZO-1)in each group of rats on day 7 after TMCAO was measured by Western Blot.To evaluate the protective effect of SNX on gut barrier in tMCAO rats.4.Immunofluorescence staining and Western Blot was used to detect the expression and distribution of Toll-like receptor 4(TLR4)protein in the colon of rats on day 7 after tMCAO.The relative expression of TLR4/NF-κB signaling pathway-related proteins in the colon of rats on day7 after tMCAO was measured by Western Blot.The relative expressions of IL-6,TNF-α,IL-10,and TGF-β mRNA in the colon of rats in each group on day 7 and day 21 after tMCAO were measured using Real-time PCR.To evaluate the effect of SNX on gut inflammatory microenvironment and TLR4/NF-b in rats after successful modeling.Results1.SNX significantly improved the symptoms of neurological deficits on days 14 and 21 after ischemia-reperfusion in tMCAO rats(P<0.05).2.Colon HE staining showed that SNX improved colon pathological injury in tMCAO rats at days 7 and 21 after ischemia-reperfusion.Colon AB-PAS staining showed that compared with the tMCAO group,there was no statistically significant difference in the number of goblet cells in the tMCAO+SNX group at day 7(P>0.05),but at day 21,the number of goblet cell in the tMCAO+SNX group increased significantly(P<0.01).The colon immunofluorescence assay(claudin-1,occludin,ZO-1)showed that the distribution and expression of colon claudin-1,occludin,and ZO-1 proteins in the tMCAO+SNX group were consistent with those in the Sham group at days 7 and 21.Western Blot assay showed that: the relative expression of colon claudin-1and ZO-1 proteins was significantly higher in the tMCAO+SNX group compared with the tMCAO group at day 7(P<0.05,P<0.01).3.Colon immunofluorescence showed that colon TLR4 protein expression and distribution were reduced in the tMCAO+SNX group compared with the tMCAO group on day 7.Western Blot detection of colon TLR4/ NF-κB pathway-related proteins in each group at day 7 showed that the relative expression of colon NF-κB p65 and IκBα proteins were not statistically different between the groups(P > 0.05),and the expression of colon TLR4,p-NF-κB p65,and p-IκBα proteins was significantly lower in the tMCAO+SNX group(P<0.01).Real-time PCR results showed that at day 7,the expression levels of IL-10 and TNF-α mRNA were significantly lower in the tMCAO+SNX group compared with the tMCAO group(P<0.01),and the expression levels of IL-6 and TGF-β mRNA were not statistically significant in the tMCAO+SNX group compared with the tMCAO group(P>0.05).At day 21,the gene expression levels of IL-6 mRNA were significantly lower in the tMCAO+SNX group compared with the tMCAO group(P<0.05),and the differences in the gene expression levels of TNF-α、IL-10 and TGF-β mRNA in the tMCAO+SNX group were not statistically significant compared with the tMCAO group(P>0.05).Conclision1.SNX protects the intestinal barrier and improves neurological deficits in tMCAO rats.2.SNX may regulate intestinal inflammatory microenvironment by regulating the TLR4/ NF-κB signaling pathway. |
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