Study On The Mechanism And Pharmacological Material Basis Of Buyang Huanwu Decoction In Treating Diabetic Kidney Disease

Objective:Buyang Huanwu Decoction(BHD)has a good effect on the clinical application of diabetic kidney disease(DKD).This article discusses the mechanism of BHD in treating DKD from the perspectives of mitochondrial protection,lipid metabolism,autophagy,and inflammation.The formula of BHD is disassembled to explore its compatibility mechanism.At the same time,the pharmacodynamic material basis of BHD for improving lipid metabolism is obtained through spectrum effect correlation.Methods:1.In vivo experiment: Randomly select 10 male C57BL/6 mice from 60 as the normal group.The other 50 mice were fed a high-fat diet for 6 weeks and were given STZ(110mg/kg)by intraperitoneal injection to induce the DKD model.Successful modeling mice were randomly divided into the model group,BHD group,Yiqi group(YQ),and Huoxue group(HX),with 12 mice in each group.On the 6th weekend of administration,collect 24-h urine,take blood,and euthanize the animals.Detect indicators related to 24-h UP,blood lipids,kidney function,inflammation,and antioxidant activity.Freeze slice kidney tissue and observe pathological changes in kidney tissue through HE,PAS,Masson,Oil Red O,and AFS staining.Using immunohistochemistry to detect changes in fibronectin FN levels in kidney tissue;Using the western blot to detect mitochondrial function-related proteins Drp1,MFF,MFN2,PINK1,Parkin,lipid metabolism-related proteins AMPK,p-AMPK,m TOR,p-m TOR,PPARα,autophagy-related proteins Beclin1 and P62,inflammation-related proteins NLRP3,NF-κB,GSDMD,and Caspase1 in kidney tissue.2.In vitro experiment:(1)High glucose stimulation of HUVEC cells in vitro,intervention with BHD and its YQ and HX containing serum,detection of NO levels in cell supernatant,observation of cell migration ability,and detection of cell mitochondrial membrane potential using JC-1 staining.Using the western blot to detect inflammation-related proteins NLRP3,NF-κB,GSDMD,and Caspase1.(2)High glucose stimulation of NRK-52 E cells in vitro,intervention with BHD and its YQ and HX containing serum.The morphology of the cytoskeleton was observed using TRITC and DAPI double staining.Autophagy levels were observed using MDC staining,and the mitochondrial membrane potential was detected using JC-1 staining.Using the western blot to detect mitochondrial function-related proteins Drp1,MFF,MFN2,PINK1,Parkin,energy metabolism-related proteins AMPK,p-AMPK,m TOR,p-m TOR,and autophagy-related proteins Beclin1 and P62 in NRK-52 E cells.(3)Oleic acid stimulation of NRK-52 E cells in vitro,intervention with BHD and its YQ and HX containing serum.Detect intracellular TG levels,and observe lipid deposition through Oil Red O staining.The morphology of the cytoskeleton was observed using TRITC and DAPI double staining.Autophagy levels were observed using MDC staining,and the mitochondrial membrane potential was detected using JC-1 staining.Using the western blot to detect mitochondrial function-related proteins Drp1,MFF,MFN2,PINK1,Parkin,lipid metabolism-related proteins AMPK,p-AMPK,m TOR,p-m TOR,PPAR,and autophagy-related proteins Beclin1 and P62 in NRK-52 E cells.3.Metabolomics and gut microbiota: Non-targeted metabolomics was performed on the kidneys of normal group,model group,and BHD group mice to detect differential metabolites in positive and negative ion modes in the kidneys of different groups of animals.KEGG pathway analysis was performed to obtain key metabolites and pathways that exert therapeutic effects.16 s r RNA sequencing was performed on the feces of mice in the normal group,model group,and BHD group to detect changes in gut microbiota at the phylum and genus levels.4.Spectrum-effect relationship: HPLC was used to establish fingerprint spectra of extracts from different polar parts of BHD.A lipid deposition model of L02 cells induced by oleic acid was used to treat with extracts from different polar parts of BHD.Oil red O staining,TG,and MDA detection were used to determine lipid deposition and antioxidant activity.Grey correlation analysis and PLSR analysis were used to establish a spectrum-effect relationship and obtain the pharmacological material basis.5.In vitro experimental verification of pharmacological substances: Oleic acid stimulates NRK-52 E cells in vitro,and calycosin-7-glucoside and ononin are used for intervention,respectively.Detect intracellular TG levels,and observe lipid deposition through Oil Red O staining.The morphology of the cytoskeleton was observed using TRITC and DAPI double staining.Autophagy levels were observed using MDC staining,and the mitochondrial membrane potential was detected using JC-1 staining.Using the western blot to detect mitochondrial function-related proteins,lipid metabolism-related proteins,and autophagy-related proteins in NRK-52 E cells.Results:1.In vivo experiment: By using the BHD,YQ,and HX to treat DKD mice,the results showed that the BHD group had the best effect,with good effects on improving lipid metabolism,protecting mitochondrial function,regulating autophagy,and reducing inflammatory reactions,thereby protecting the kidneys,reducing 24-h UP,BUN,and SCr,reducing glomerular enlargement,inflammatory infiltration,glomerular fibrosis,and proliferation of glomerular basement membrane,and it reduces the deposition of glycogen,collagen,and lipids in the kidneys.The effect of the YQ is similar to that of the BHD,but its effect on reducing inflammation is slightly weaker.Although the HX has a relatively small dosage,its effect on reducing inflammation is similar to that of the YQ.However,the HX has a weaker effect on improving lipid metabolism,with significant lipid deposition in the kidneys.These results indicate that the combination of the YQ and the HX may have a synergistic promoting effect,thereby enabling the total formula of BHD to play a better therapeutic effect.2.In vitro experiments:(1)BHD and its split formula group effectively alleviated high glucose-induced HUVEC cell damage,increased NO levels in cell culture medium,increased cell migration rate,and improved mitochondrial membrane potential.The mechanism of its action was related to improving high glucose-induced cellular inflammatory response.BHD and its split formula group significantly reduced the expression of high glucose-induced inflammatory proteins NF-κB and NLRP3 inflammasomes,thereby reducing the expression of cell pyroptosis proteins GSDMD and Caspase1.The total formula group of BHD has a better effect compared to the two groups of YQ and HX,while the effects of YQ and HX are similar.(2)Each group played a protective role against high glucose-induced damage to NRK-52 E cells,reducing changes in cell morphology,reducing cell death,improving autophagy levels,and protecting mitochondrial function.The medicated serum of BHD and its disassembled formula group regulated the AMPK/m TOR and PINK1/Parkin signaling pathways,enhanced autophagy and mitophagy,regulated the levels of mitochondrial fission and fusion-related proteins,and alleviated high glucose-induced damage to NRK-52 E cells.In terms of efficacy,BHD has a better effect on regulating the AMPK/m TOR and PINK1/Parkin signaling pathways,regulating mitochondrial fission and fusion-related proteins than the YQ and HX,while the YQ is better than the HX.(3)The medicinal serum of BHD and its disassembled formula has a protective effect on NRK-52 E cells damaged by oleic acid,reducing intracellular lipid deposition,alleviating changes in cell morphology,alleviating cell death,improving autophagy levels,and protecting mitochondrial function.In terms of the effectiveness of their effects,the total formula group of BHD and the YQ have better effects on regulating the AMPK/m TOR and PINK1/Parkin signaling pathways,regulating mitochondrial fission and fusion,mitophagy,autophagy,and lipid metabolism-related proteins than the HX,while the regulatory effect of the HX is weaker.3.Metabolomics and gut microbiota: Metabolomics analysis revealed that 25 differential metabolites may be the key to the therapeutic effect of BHD.Its KEGG pathway involves alanine,aspartate and glutamate metabolism,arginine biosynthesis,histidine metabolism,drug metabolism-cytochrome P450,insulin resistance,arachidonic acid metabolism.Through16 S r RNA sequencing analysis,it was found that the richness and diversity of gut microbiota in DKD model mice were significantly reduced compared to the normal group.The intervention of BHD increased the richness and diversity of gut microbiota,and the levels of Actinobacteriota and Proteobacteria in the feces of model group mice were significantly increased,while the levels of Bacteroidota were significantly reduced.At the genus level,compared to the normal group,20 genus level bacteria in the model group underwent significant changes.At the same time,the intervention of BHD led to a correction in the relative abundance of these microbiota groups.4.Spectrum-effect relationship: The results showed that the extracts from different polar parts of BHD could reduce the levels of TG and MDA.The grey relational analysis showed that the peaks that contributed greatly to the reduction of TG and MDA were peaks 3,16,14,10,1,15,2,and 11 respectively.Peaks 1,4,9,10,14,16,and 17 could reduce TG and MDA by PLSR analysis.According to the results of grey relational analysis and PLSR analysis,peaks 1,10,14,and 16 may have good lipid-lowering and antioxidant effects.5.In vitro experimental verification of pharmacological substances:calycosin-7-glucoside and ononin showed a dose-dependent protective effect on NRK-52 E cells damaged by oleic acid,reducing intracellular lipid deposition,alleviating changes in cell morphology,alleviating cell death,improving autophagy levels,and protecting mitochondrial function.In terms of the mechanism of effect,calycosin-7-glucoside and ononin regulate the AMPK/m TOR pathway and PINK1/Parkin pathway,regulating proteins related to mitochondrial fission and fusion,mitophagy,autophagy,and lipid metabolism.Conclusion:1.BHD and its disassembled formula can effectively improve the physiological indicators of DKD mice,improve lipid metabolism,protect mitochondrial function,improve autophagy,reduce inflammation,and protect the kidneys.Its good effect is closely related to the combination of YQ and HX.2.The serum containing BHD and its disassembled formula can effectively protect HUVEC from high glucose damage,improve cell function,and reduce inflammation.3.The serum containing BHD and its derivative can effectively protect NRK-52 E cells damaged by high glucose or oleic acid,improve lipid metabolism and autophagy levels,and protect mitochondrial function.4.BHD regulates multiple metabolic pathways in the kidneys and alleviates dysbiosis of gut microbiota,thereby improving glucose and lipid metabolism and inflammatory response in DKD mice,thereby protecting the kidneys.5.Spectrum-effect relationship screening identified 4 potential lipid-lowering and antioxidant components for BHD,providing the preliminary basis for quality control of BHD and lipid-lowering drug research.6.Calycosin-7-glucoside and ononin can effectively protect NRK-52 E cells from oleic acid damage,improve lipid metabolism and autophagy levels,and protect mitochondrial function.