Effect Of Milk-made Physochlaina Physaloides On Nitroglycerin-induced Migraine In Rats Based On Metabolomics And Intestinal Flora

Objective:Metabolomics and intestinal flora sequencing techniques were used to elucidate the mechanism of milk-made Physochlaina physaloides anti-nitroglycerin-induced migraine in rats.Method:1.Randomly dividing SD rats into 6 groups-normal control,model control,and another-after 1 week of adaptive feeding,positive control group and milk-made Physochlaina physaloides low dose,milk-made Physochlaina physaloides medium dose,milk-made Physochlaina physaloides high dose and 6 groups,(12 rats in each group).The pharmacological effects of milk-made Physochlaina physaloides on nitroglycerin-induced migraine in rats were investigated using serum calcitonin gene-related peptide(CGRP),nitric oxide(NO),endothelin(ET-1),β-endorphin(β-EP),pentraxin(5-HT)and hippocampal histopathological changes as indicators.2.Metabolomics analysis: Serum samples from rats with migraine intervention by milk-made Physochlaina physaloides were analyzed by UHPLC-OE-MS technique to obtain metabolite profiles,Principal Component Analysis(PCA)and Orthogonal Partial Least Squares Discriminant Analysis(OPLS-DA)are both employed were used to explore the specific markers of milk-made hysochlaina for migraine and its pathway enrichment analysis for differential metabolites.3.Intestinal flora analysis:Based on Illumina Mi Seq sequencing method,the V3-V4 highly variable region of bacterial 16 s r RNA was sequenced.A further calculation of α-diversity & β-diversity,as well as a comparison of differences between groups,was carried out to explain the modulatory effect of milk-made Physochlaina physaloides on the structure and function of the intestinal flora of rats with migraine and the correlation analysis of metabolomics and intestinal flora,based on substance annotation.Results:1.The nitroglycerine-induced migraine model was successfully replicated in rats,Significantly higher serum levels of CGRP,NO,and ET-1 were observed(P<0.05),whereas brain tissue levels of β-EP and 5-HT were significantly lower(P<0.05)in the model control rats compared with the normal control group.Compared with the model control group,the three doses of milk-made Physochlaina physaloides low dose(0.13g/kg),milk-made Physochlaina physaloides medium dose(0.26g/kg)and milk-made Physochlaina physaloides high dose(0.52g/kg)The serum levels of CGRP,NO,and ET-1 were significantly reduced by Toutongning tablet(P<0.05),while the levels of β-EP and 5-HT in brain tissue were remarkably augmented(P<0.05).(P<0.05).Compared with the model control group,the milk-made Physochlaina physaloides group and the Toutongning tablet group improved the morphology of hippocampal nerve cells,with neural cells arranged more neatly and tightly,and the number of degenerated nerve cells reduced,indicating that milk-made Physochlaina physaloides can effectively prevent nitroglycerin-induced hippocampal tissue damage in rats.2.The results of the metabolomics experiment showed that 46 potential biomarkers of milk-made Physochlaina physaloides for migraine were screened according to VIP>1 and P value<0.05.Results of metabolic pathway annotation and enrichment analysis of the differential metabolites indicate that milk-made Physochlaina physaloides treats migraine mainly through the regulation of metabolic pathways such as Biosynthesis of secondary metabolites,Biosynthesis of cofactors,amino acid biosynthesis,Caebon metabolism,are all integral components.3.The results of 16 s RNA sequencing showed that,Beta Diversity analysis demonstrated that milk-made Physochlaina physaloides could modulate nitroglycerin-induced differences in flora.Further studies showed that,at the phylum level,milk-made Physochlaina physaloides increased the relative abundance of Firmicutes,Proteobacteria and Bacteroidetes,down-regulated Verrucomicrobia and promoted the growth of intestinal symbiotic bacteria;at the family level,the relative abundance of groups such as Lachnospiraceae,Peptostreptococcaceae,Prevotellaceae and Erysipelotrichaceae was elevated,while the relative abundance of Verrucomicrobiaceae,Ruminococcaceae and Lactobacillaceae decreased.Conclusion:Milk-made Physochlaina physaloides has a certain preventive effect on migraine induced by nitroglycerin in rats.Its mechanism may play a therapeutic role by reducing the activity of CGRP,NO and other enzymes in rat serum,improving amino acid metabolism,regulating metabolic disorders and intestinal flora structure.