| Objective:In this study,to clarify the antipyretic pharmacological effects of Pueraria lobata(P.lobata)and Pueraria lobata var.Thomsonii(P.thomsonii)on the rat model of wind-cold syndrome,and to investigate the mechanism of their antipyretic effects from the perspectives of intestinal flora and the mechanism of heat production in body temperature regulation.Method:1.Effect of P.lobata and P.thomsonii on intestinal flora in a rat model of wind-cold syndromeUsing the wind-cold body surface cooling and blowing method to establish the wind-cold syndrome rat model.Firstly,84 SD rats were randomly divided into control group(12 rats)and wind-cold syndrome modeling group(72 rats).After 4 days of continuous modeling,rats with fluctuating body temperature(ΔT<0.5°C)and single temperature below 38°C were excluded,and the remaining 60 rats in the wind-cold syndrome modeling group were randomly divided into 6 groups of 10 rats each:model group,paracetamol group,P.lobata 3.2 g/kg group,P.lobata 6.4 g/kg group,P.thomsonii 3.2 g/kg group and P.thomsonii 6.4 g/kg group.A preliminary study of the antipyretic pharmacodynamics of P.lobata and P.thomsonii by examining changes in body temperature,body weight and blood viscosity in rats.Based on DNA16S r RNA sequencing technology,the preliminary analysis of rat feces was performed to extract the abundance values of flora at different taxonomic levels,followed by the association analysis of body temperature with the taxonomic levels of intestinal flora species in rats by the Two-part model algorithm based on R language and the comparative 16S r RNA database to obtain P.lobata and P.thomsonii were correlated with body temperature.The data on the abundance values of the flora at the Species classification level were then combined to obtain the target flora associated with the antipyretic activity of P.lobata and P.thomsonii;The results of the intestinal flora were initially validated by detecting the changes of the inflammatory factors Interleukin-6(IL-6)and Interleukin-8(IL-8)in rat plasma using an enzyme-linked immunosorbent assay(ELISA).2.Effects of P.lobata and P.thomsonii on thermogenic pathways in a rat model of wind-cold syndromeUsing the wind-cold body surface cooling and blowing method to establish the wind-cold syndrome rat model.Firstly,110 SD rats were randomly divided into normal control group(15 rats)and wind-cold syndrome modeling group(95 rats).After screening,the remaining 84 rats were randomly divided into 6 groups:model group,paracetamol group,P.lobata 3.2 g/kg group,P.lobata 6.4 g/kg group,P.thomsonii 3.2 g/kg group,P.thomsonii 6.4 g/kg,14 rats in each group.Protein expression levels and m RNA expression levels of TRPM8 and TRPV1,which mediate cold and heat sensitive pathways,and uncoupling protein 1(UCP1)in brown adipose tissue(BAT)were measured by western blot and real-time quantitative fluorescence polymerase chain reaction(Real-time q RT-PCR).Observation of changes in BAT cells by Hematoxylin-eosin staining(HE)technique.Changes in skeletal muscle thermogenesis measured by ELISA assay of skeletal muscle ATP content and Na~+-K~+-ATPase activity.The blood flow velocity of rat neck skin was measured by XTL-30 multifunctional microcirculation color analyzer to measure changes of vascular diastolic or constrictive.To clarify the mechanism of antipyretic effect of P.lobata and P.thomsonii.Result:1.Effect of P.lobata and P.thomsoniion intestinal flora in a rat model of wind-cold syndromeThe experimental results showed that P.lobata and P.thomsonii could lower the body temperature,increase the body weight and lower the blood viscosity of rats,which tentatively verified that the antipyretic effect of P.lobata and P.thomsonii.A total of two strains of bacteria,uncultured＿bacterium＿g＿Prevotella and Lactobacillus＿johnsonii,were screened by intestinal flora analysis methods for their association with the antipyretic properties of P.lobata and P.thomsonii,which may affect body thermoregulation by regulating the plasma inflammatory factors IL-6 and IL-8.The ELISA results of IL-6 and IL-8 in rat plasma showed that P.lobata and P.thomsonii could reduce the levels of IL-6 and IL-8 in plasma.2.Effects of P.lobata and P.thomsonii on thermogenic pathways in a rat model of wind-cold syndromeThe results of western blot and Real-time q RT-PCR showed that P.lobata and P.thomsonii could reduce the protein expression level of TRPM8 and m RNA in the cold-sensitive pathway,increase the protein expression level of TRPV1 in the heat-sensitive pathway and m RNA expression levels in the heat-sensitive pathway,and reduce the protein expression level and m RNA expression level of UCP1 in BAT to reduce the body’s heat production.The results of HE staining showed that P.lobata and P.thomsonii could increase the diameter of BAT cells and reduce the heat production of BAT.The ELISA results of skeletal muscle ATP and Na~+-K~+-ATPase activity showed that P.lobata and P.thomsonii could reduce skeletal muscle thermogenesis thereby increasing skeletal muscle ATP content and Na~+-K~+-ATPase activity.The results of XTL-30 multi-functional microcirculation color analyzer showed that P.lobata and P.thomsonii could increase the vascular flow rate in the skin by causing vasodilation.The above results suggest that P.lobata and P.thomsonii could achieve the antipyretic effect on rats with wind-cold syndrome through cold and heat-sensitive pathways and thermogenic pathways.Conclusion:P.lobata and P.thomsonii have antipyretic effects by modulating the intestinal flora uncultured＿bacterium＿g＿Prevotella and Lactobacillus＿johnsonii to affect the release of inflammatory factors IL-6 and IL-8,which may affect the body’s thermoregulatory center to regulate cold and heat sensitive pathways and thus reduce the heat production of brown adipose tissue and skeletal muscle to achieve antipyretic effects. |
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