Study On The Effect And Mechanism Of The Matched Pair Of Scutellariae Radix And Sophora Japonica L.ameliorate Hypertension And Hypertensive Renal Injury

Objective:To construct a network about active components and targets for the treatment of hypertension of Scutellariae Radix(SR)and Sophora Japonica L.(SJ)and to analyze the material and mechanism.To further identify the active ingredients of SR and SJ and to study the effect and mechanism of SR and SJ on hypertension and hypertensive renal injury in vivoMethods:(1)All the chemical molecules and the relatived targets for SR and SJ were retrieved from Traditional Chinese Medicine Systems Pharmacology Database(TCMSP).Then intersected with the genes associated with hypertension that were obtained from TTD,OMIM,CTD and Genecards database.The corresponding active components and targets network was established by software Cytoscape.To analyze genes GO function and the KEGG pathway enrichment using the Metescape online analysis platform,and the molecular docking software Sybyl was used to analyze the binding of critical components with targets and further validated in vivo.(2)High performance liquid chromatography-mass spectrometry(HPLC-MS)was used to identify the active ingredients of SR and SJ.(3)Spontaneously hypertensive rats(SHR)were selected as the disease model of hypertensive renal injury,and to study the effects of SR and SJ,we observed blood pressure?renal structure and function of SHR.(4)16S rRNA high-throughput sequencing analysis was used to analyze intestinal flora of rat in each group to study the effect of SR and S J on the richness and diversity of intestinal flora,and we inspected the structure of intestinal barrier and detect the expression of tight junction protein in gut by the technology of immunofluorescence,immunohistochemistry and Western blot.(5)To clarify the effect of SR and SJ on indoxyl sulfate(IS),an intestinal toxin,and on oxidative stress response induced by IS,enzyme-linked immunosorbent assay(ELISA)was used to analyze the the content of IS in serum.To clarify the effect of SR and SJ on microbial metabolites and their receptors,we detected the content of short-chain fatty acids(SCFAs)in rat feces and used real-time quantitative PCR to detect the mRNA expressions of Olfr78 and GRP41,which were SCFAs receptors related to vasoconstriction and diastolic function in kidney.Results:(1)Network of compounds and targets related to hypertension in SR and SJ consisted of 26 compounds and 45 targets.Gene GO functional analysis about the antihypertension targets of SR and SJ suggested that molecular functions?biological processes and cell components were involved.Gene KEGG pathway enrichment analysis showed 17 signaling pathways including HIF-1 signaling pathway?calcium signaling pathway and Apelin signaling pathway were related to the improvement of hypertension.The docking results of Sybyl showed that isorhamnetin of SJ have good binding activity with endothelial nitric oxide synthase and moslosooflavone of SR have strong interaction with angiotensin converting enzyme.Isorhamnetin could increase eNOS in serum or endothelium and moslosooflavone could decrease ACE in serum of spontaneously hypertensive rats.(2)31 active ingredients of SR and SJ were identified by HPLC/MS.Combined with the analysis that active ingredients were less in the blood,it indicated that the most ingredients were not in blood may play a role through the intestinal flora.(3)The result in vivo showed that SR and SJ can reduce the blood pressure of SHR?decrease the levels of serum creatinine?blood urea nitrogen and urine microalbumin with improving kidney function,as well as reduce deposition of collagen and then inhibit renal fibrosis.(4)The result of 16S rRNA high-throughput sequencing analysis suggested that SR and SJ could improve the richness and diversity of intestinal flora,decrease the ratio of Firmicutes/Bacteroidetes(F/B),an index indecated intestinal flora disorder.SR and SJ increased the expression of intestinal tight junction protein and repaired the intestinal barrier structure.(5)SR and SJ can reduce the content of indoxyl sulfate in serum,and then reduce oxidative stress response and increase the level of antioxidant enzymes in kidney.SR and SJ can increase the content of SCFAs in rat feces and reduce inflammatory factors in serum,and inhibit the mRNA expression of Olfr78,a SCFAs receptor related to constriction of blood vessels,while increase the mRNA expression of GPR41,a SCFAs receptor related to dilation of blood vessels.Conclusion:SR and SJ have multi-component,multi-target and multi-pathway effects.SR and SJ can effectively improve hypertension and hypertensive nephropathy.The mechanism may be related to improvement of gut microbiota and intestinal barrier function,reduction of indoxyl sulfate and oxidative stress response,promotion of SCFAs,regulation the levels of SCFAs receptors and inhibition in inflammation.