基于血清药物化学解析拳参缓解腹泻型肠易激综合征的药效物质基础研究OA

Objective To observe the therapeutic effect of Bistortae Rhizoma on diarrhea-predominant irritable bowel syndrome(IBS-D),and to explore its pharmacodynamic material basis.Methods Forty-eight SPF-grade SD rats were randomly assigned,with eight rats in the normal group and forty rats in the modeling group.Except for the rats in normal group,others were administered senna leaf decoction at a dose of 10 mL/kg by gavage combined with tail-clamping stress to establish the IBS-D model for 21 consecutive days.After successful modeling,rats in the modeling group were randomly divided into a model group,a pinaverium bromide-positive group(10 mL/kg),and Bistortae Rhizoma high-,medium-,and low-dose groups(2,1.5,1 g/kg),with eight rats in each group.The normal and model groups received an equivalent volume of distilled water by gavage,while the other groups received the corresponding drug solutions.All treatments were performed once daily for 14 consecutive days.General condition of the rats was observed daily,and fecal water content and Bristol scores were recorded.Histopathological changes in colonic and brain tissues were examined by HE staining,and levels of 5-hydroxytryptamine(5-HT),tumor necrosis factor-alpha(TNF-α),and vasoactive intestinal peptide(VIP)in serum and colonic tissues were measured by ELISA to evaluate the therapeutic effect of Bistortae Rhizoma on IBS-D.Prototype components of Bistortae Rhizoma absorbed into blood were analyzed using quadrupole-Orbitrap mass spectrometry,and key pharmacodynamic compounds and core targets for IBS-D treatment were screened via network pharmacology,with molecular docking employed to validate the binding capacity between compounds and targets.Finally,an LPS-induced Caco-2 cell injury model was established,and the optimal survival concentration of pharmacodynamic compounds was determined by CCK-8 assay.Levels of 5-HT,TNF-α,and VIP in cells were measured by ELISA,and expression levels of tight junction proteins claudin-1,occludin,and ZO-1 were detected by Western blot to verify the in vitro cellular activity of the key pharmacodynamic compounds.Results Compared with the normal group,the model group exhibited a highly significant decrease in body weight(P<0.05)and highly significant increases in fecal water content,Bristol score,and levels of 5-HT,TNF-α and VIP in serum and colonic tissues(P<0.01);the colonic tissue architecture was abnormal,showing focal epithelial cell shedding,necrosis,and marked inflammatory cell infiltration;brain tissue also showed structural abnormalities,a sharp reduction in hippocampal neuron count,and numerous pyknotic and hyperchromatic neurons.Compared with the model group,the general condition,body weights,fecal water content,and Bristol scores of all treatment groups were improved to varying degrees;levels of 5-HT,TNF-α,and VIP were highly significantly decreased in the pinaverium bromide-positive group and the Bistortae Rhizoma high-and medium-dose group(P<0.01,P<0.05);colonic tissue architecture was essentially normal with only mild inflammatory cell infiltration,and brain tissue structure was largely normal with abundant and well-arranged hippocampal neurons and no evident neuronal pyknosis or hyperchromasia.Six prototype components absorbed into blood were identified from Bistortae Rhizoma,namely rutin,(+)-gallocatechin,kaempferide,gallic acid,chlorogenic acid,and 1-O-galloyl-β-D-glucose.Network pharmacology combined with mass spectrometry analysis suggested that all six above-mentioned components may be key pharmacodynamic compounds.They act on five core targets including SRC and HSP90AA1,with strong compound-target binding affinity.The CCK-8 assay revealed that 10 µmol/L was the optimal concentration for cell viability of the key pharmacodynamic compounds gallic acid,chlorogenic acid,and rutin.ELISA and Western blot results showed that each compound significantly ameliorated LPS-induced Caco-2 cell injury.Conclusion Bistortae Rhizoma effectively alleviates diarrheal symptoms of IBS-D,and its key pharmacodynamic compounds are gallic acid,chlorogenic acid,and rutin.The underlying mechanism is likely closely associated with the regulation of the PI3K-Akt,MAPK,and AGE-RAGE signaling pathways.