Study On The Effective Substances And Mechanism Of Buyang Huanwu Decoction In Treating Diabetic Peripheral Neuropathy

Diabetic peripheral neuropathy(DPN)is a complication caused by diabetes,involving peripheral nerves.The onset of DPN is hidden and slow,and the late stage of the disease can lead to serious nerve damage.Modern people due to lifestyle changes,increased number of obese DPN patients increased.Common hypoglycemic drugs to control blood glucose can not be a good intervention in the process of DPN.Clinically,opioid analgesics,tricyclic antidepressants,antiepileptic drugs are often used to alleviate neurological pain and other symptoms.However,these drugs do not aim at the pathogenesis of DPN,and the treatment is not radical,and the side effects are large.Therefore,the research and development of DPN specialized drugs has attracted worldwide attention.Unfortunately,most of the drugs in the research failed due to safety problems.Therefore,safe and effective specialized treatment drugs for DPN are urgently needed.DPN belongs to the category of pain syndrome and numbness in TCM theory.Buyang Huanwu Decoction recorded in Medical forest correction is a famous prescription for supplementing qi and activating blood circulation,which is used to cut DPN in TCM pathogenesis.Buyanghuanwu Decoction is effective in the treatment of DPN,but the effective substances and mechanism of Buyanghuanwu Decoction are not clear.In this study,Buyanghuanwu Decoction was taken as the research object.In view of the unclear effective substances and mechanism of action,the fingerprint construction,chemical composition characterization,blood component analysis,network pharmacology,active component screening and mechanism of action of Buyanghuanwu Decoction were studied.The specific research contents are as follows :1.Fingerprint of Buyang Huanwu DecoctionBased on the new core-shell chromatography and high resolution liquid chromatography-mass spectrometry,the quality evaluation method of Buyang Huanwu Decoction was established by combining chromatographic fingerprint and qualitative identification of common peak components.A new core-shell chromatographic column was used to establish the fingerprint of Buyang Huanwu Decoction within 38 min by optimizing the chromatographic conditions with the separation effect and number of chromatographic peaks as the evaluation indexes.Furthermore,21 common peaks were identified and assigned by high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry,covering all herbs in the prescription.Among them,10 peaks were from Astragalus membranaceus,3peaks were from Angelica sinensis,2 peaks were from Paeonia lactiflora,3peaks were from safflower,2 peaks were from Ligusticum chuanxiong,2peaks were from peach kernel,and 1 peak was from Dilong.2.Systematic Characterization of Chemical Constituents of Buyang Huanwu DecoctionIn order to further comprehensively and systematically characterize the chemical material basis of Buyang Huanwu Decoction,UPLC-Q-Orbitrap HRMS technology combined with the self-built chemical composition database of Buyang Huanwu Decoction was used to identify the chemical constituents in the water extract of Buyang Huanwu Decoction.Finally,101 components were identified,including 31 amino acids,29 flavonoids,17 organic acids,7 saponins,and 17 other compounds.The compound Chinese medicine flavor of these compounds was assigned,which laid the foundation for the subsequent study on the pharmacodynamic substances of Buyang Huanwu Decoction.3.Network Pharmacology Analysis of Buyang Huanwu Decoction on DPNOn the basis of systematic characterization of compound chemical constituents,serum pharmacochemistry method was used to identify the transitional compounds in blood of SD rats after intragastric administration of Buyang Huanwu Decoction,in order to clarify the pharmacodynamic material basis of Buyang Huanwu Decoction.Results 30 prototype blood components were found in rats,including 4 saponins,12 flavonoids,4 organic acids,4amino acids and 6 others.Furthermore,the action pathway of Buyang Huanwu Decoction in the intervention of DPN was predicted by combining serum pharmacochemistry with network pharmacology.Thirty blood components in the aforementioned Buyang Huanwu decoction were selected to study the intervention mechanism of Buyang Huanwu decoction on DPN by network pharmacology.A total of 30 core targets were obtained,including CASP3(Caspase-3)and TNF(tumor necrosis factor).GO function enrichment analysis showed 574 GO items,mainly involved in regulating metabolism,inflammation,apoptosis and other biological processes.KEGG enrichment results showed that AGEs-RAGE signaling pathway was the main signaling pathway of Buyang Huanwu Decoction in the intervention of DPN.4.Protective effect of blood components of Buyang Huanwu Decoction on Schwann cell damage induced by methylglyoxalFirstly,the RSC96 rat Schwann cell injury model induced by methylglyoxal was established.On this basis,the established cell pharmacology model was used to study the protective effect of 11 representative blood components in Buyang Huanwu Decoction on the rat Schwann cell injury induced by methylglyoxal.The results showed that kaempferol,calycosin isoflavone,ligustilide,astragaloside IV,gallic acid,and formononetin could all interfere with the damage of Schwann cells caused by methylglyoxal to varying degrees.At the same time,it was found that the representative components of calycosin,kaempferol and astragaloside IV decreased the level of reactive oxygen species in rat Schwann cells induced by methylglyoxal.Western blot results showed that the expression of Glo1 protein was up-regulated and the expression of RAGE protein was inhibited after the intervention of kaempferol(50μM).The above results suggest that kaempferol may play a protective role by inhibiting the MGO-AGEs-RAGE pathway.