Study On The Effects And Mechanism Of Moxibustion At CV4 Acupoint And Curculigo Orchioides Gaertn Tonifying Kidney Yang,moxibustion At ST36 Acupoint And Dried Ginger Tonifying Spleen Yang

Objective:From the perspective of energy metabolism,this study took medicine as the entry point to explore the effect of moxibustion at Guanyuan Point（CV4）and Xianmao（XM）on tonifying kidney yang,and moxibustion at Zusanli Point（ST36）and dried ginger（GJ）to tonifying spleen yang and its mechanism.studied the effect and mechanism of moxibustion at guanyuan acupoint（CV4）and curculigo orchioides gaertn（XM）to tonify kidney yang,moxibustion at zusanli acupoint（ST36）and dried ginger（GJ）to tonify spleen yang.Methods:In the study of moxibustion at CV4 and XM for the mechanism of invigorating kidney yang,40 SD rats were divided into 5 groups:blank control group,moxibustion at CV4 group（CV4）,high-dose of XM group（XMH）,middle dose of XM group（XMM）and low-dose of XM group（XML）,with 8 rats in each group.During the experiment,the rats’weight and body temperature were monitored.The activities of Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,SDH,ATP content,kidney mitochondria number and mitochondrial metabolic rate were measured.Biochemical methods were used to detect the contents of alanine aminotransferase（ALT）,asparagus amino acid aminotransferase（AST）,uric acid（UA）,urinary nitrogen（BUN）,creatinine（CR）,triglyceride（TG）,total cholesterol（TC）,total bile acid（TBA）,high density lipoprotein cholesterol（HDL-C）,low density lipoprotein cholesterol（LDL-C）in rats,and the pathological changes of liver and kidney to evaluate the liver and kidney functional of rats.Metabolomics technology was using to screen differential metabolites and analyze the metabolic pathways.RT-PCR and Western blot were used to detect the expressions of Sirt1,Pgc-1α,Ampk,Ppar-αm RNA and protein,and to explore the mitochondrial synthesis and function.In the study of moxibustion at ST36 and rhizoma zingiberis（GJ）for the mechanism of invigorating spleen Yang,40 SD rats were divided into 5 groups:blank control group,moxibustion at ST36 group（ST36）,high-dose group of GJ（GJH）,middle dose group of GJ（GJM）and low dose group of GJ（GJL）,with 8 rats in each group.During the experiment,the rats’weight and body temperature were monitored.The activities of Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,SDH,ATP content,gastric mitochondria number and mitochondrial metabolic rate were measured.Biochemical methods were used to detect the contents of ALT,AST,UA,BUN,Cr,TG,TC,TBA,HDL-C,LDL-C and the pathological changes of liver,stomach and kidney to evaluate the liver,stomach and kidney functional of rats.Metabolomics technology was using to screen differential metabolites and analyze the metabolic pathways.RT-PCR and Western blot were used to detect the expressions of Sirt1,Pgc-1α,Ampk,Ppar-αm RNA and protein,and to explore the mitochondrial synthesis and function.Results:1.Compared with the blank control group,the weight of rats in the CV4,XMH,XMM and XML groups were decreased（p<0.05,p<0.01）,but there was no significant change in body temperature（p>0.05）.Compared with the blank control group,the activity of ATPase and SDH were significantly enhanced in the CV4,XMH,XMM and XML groups（p<0.05,p<0.01）.The ATP content was increased in the CV4,XMH,XMM and XML groups（p<0.05,p<0.01）.The number of kidney mitochondria was increased in the CV4,XMH,XMM and XML groups（p<0.05,p<0.01）.CV4 group has no significant effect on the kidney mitochondrial metabolism rate（p>0.05）,and XMH significantly inhibits kidney mitochondrial metabolism rate（p<0.05）.XMM and XML groups can significantly increase the kidney mitochondrial metabolism rate（p<0.05,p<0.01）.Compared with the blank control group,there were no significant effects on liver and kidney function.For the CV4 groups,metabolomics results showed that six potential biomarkers were identified in the serum,namely xanthine,oleamide,2-arachidonylglycerophosphocholine,D-glutamine,（6b,7b,c13R）-6,7-diacetoxy-8,14-labdadiene-13-ol,Nutriacholic acid;Seven potential biomarkers were identified in feces,namely（1-（2-furylmethyl）-1H-pyrrole）,linoleic acid,2-phenylglycine,succinic acid,3-（3,5-dimethoxyphenyl）propionic acid,3-hydroxysebacic acid,and citric acid;A total of seven potential biomarkers were identified in the kidney tissues,namely 2-methylmalic acid,isoleucyl phenylalanine,oleamide,sphingosine,succinic acid,gluconic acid,tauroursodeoxycholic acid.For the XMH group,four potential biomarkers were identified in the serum,namely oleamide,retinyl ester,2-arachidonicglycerophosphocholine,and 9-oxoctadecanoic acid.A total of six potential biomarkers were identified in feces,namely 2-arachidonylglycerophosphocholine,capric acid,linoleic acid,11-cis-retinal,creatine,levulinic acid;Two potential biomarkers were identified in the tissues,namely succinic acid and gluconic acid.For the XMM group,five potential biomarkers were identified in the serum,namely oleamide,retinyl ester,N-lactate leucine,docosapentaenoic acid（22n-6）,2-arachidonic glycerol phosphate choline;A total of five potential biomarkers were identified in feces,namely oleamide,katonic acid,L-leucine,2-keto-3-deoxy-D-gluconic acid and 11-cis-retinal;A total of three potential biomarkers were identified in the kidney tissues,namely xanthine,succinic acid,and tauroursodeoxycholic acid.For the XML group,three potential biomarkers were identified in the serum,namely retinyl ester,2-arachidonicacidglycerophosphate choline,and oleamide.Five potential biomarkers were identified in feces,namely Nutriacholic acid,2-arachidonylglycerophosphocholine,11-cis-retinal,creatine and decanoic acid;A total of five potential biomarkers were identified in the kidney tissues,namely maleylacetic acid,isoleucine proline,Lyso PE（18:0/0:0）,succinic acid and adenosine monophosphate（AMP）.Metabolic pathway results show that the CV4 group involved citric acid cycle（TCA cycle）,alanine,aspartic acid and glutamate metabolism,D-glutamine and D-glutamate metabolism,butyrate metabolism,sphingolipid metabolism,pentose phosphate pathway metabolism,propionate metabolism,glyoxylic acid and dicarboxylic acid metabolism,purine metabolism.The XMH group involved TCA cycle,retinol metabolism,pentose phosphate metabolism,propionate metabolism,alanine,aspartate and glutamate metabolism.The XML group involved retinol metabolism,citric acid cycle metabolism（TCA）,biosynthesis and degradation of valine,leucine and isoleucine,butyrate metabolism,propionate metabolism,Alanine,aspartic acid and glutamate metabolism,aminoacyl biosynthesis,purine metabolism.The XML group involved retinol metabolism,butyrate metabolism,TCA cycle metabolism,propionate metabolism,alanine,aspartate and glutamate metabolism,and purine metabolism.RT-PCR and WB results showed that compared with the blank control group,CV4 group enhanced the expression of Ampk,Pgc-1α,and Ppar-αm RNA and protein,but had no significant effect on Sirt1.XM group enhanced Ampk,Pgc-1α,Ppar-αand Sirt1 m RNA and protein expression.2.Compared with the blank control group,the weight of rats in the ST36,GJM,and GJL groups were decreased（p<0.05,p<0.01）,but there were no significant change in body temperature（p>0.05）.The body weight and body temperature of the rats in GJH group did not change（p>0.05）.Compared with the blank control group,the activity of ATPase and SDH were enhanced in the ST36 group（p<0.05,p<0.01）,the GJH group can increase the activity of Ca²⁺-Mg²⁺-ATPase and SDH（p<0.05,p<0.01）,no significant effect on Na⁺-K⁺-ATPase activity（p>0.05）.Th GJM group enhanced the activity of Ca²⁺-Mg²⁺-ATPase and SDH（p<0.01）,but had no significant effect on the activity of Na⁺-K⁺-ATPase（p>0.05）.The GJL significantly enhanced the activity of SDH（p<0.01）,no significant effect on ATPase activity（p>0.05）.The ATP content was increased in the ST36,GJH,GJM and GJL groups（p<0.05,p<0.01）.The number of gastric mitochondria were increased in the ST36,GJH,GJM and GJL group（p<0.01）.ST36 and GJM groups had no significant change on gastric mitochondrial metabolism rate（p>0.05）.The GJH group inhibited the gastric mitochondrial metabolism rate（p<0.01）,and the GJL group could significantly increase the gastric mitochondrial metabolism rate（p<0.01）.Compared with the blank control group,there were no significant on liver,kidney and stomach function.For the ST36 group,metabolomics results showed that five potential biomarkers were identified in the serum,namely N-lactoleucine,2-arachidonicacidglycerophosphocholine,PC（20:2（11Z,14Z）/14:0）,isoleucine,D-glutamine;There were six potential biomarkers in feces,namely 4-Methylzymosterol intermediate,2b,3a,7a,12a-tetrahydroxy-5b-cholic acid,8-oxohexadecanoic acid,and diisophthalic acid Butyl ester,kynuric acid,arginine-asparagine;There were ten potential biomarkers in gastric tissue,namely oleamide,sphingosine,phytosphingosine,tetracosahexaenoic acid,Lyso PC（14:0）,Lyso PE（18:0/0:0）,oxidized glutathione Peptides,3-hydroxydecanoic acid,18-hydroxyarachidonic acid,PC（20:2（11Z,14Z）/14:0）.For the GJH,There were three potential biomarkers in the serum,namely PC（20:2（11Z,14Z）/14:0）,2-arachidonicacidglycerophosphocholine,Lyso PE（0:0/18:0）;There were eight potential biomarkers in feces,namely palmitic acid,linoleic acid,N-acetylleucine,ornithine,DG（18:2n6/0:0/18:2n6）,asparagine B,iso leucyl phenylalanine,sebacic acid;There were five potential biomarkers in the gastric tissue,namely 3-hydroxydecanoic acid,ursodeoxycholic acid,Lyso PG（16:0/0:0）,PC（20:2（11Z,14Z）/14:0）,Tauroursodeoxycholic acid.For the GJM group,the five potential biomarkers in the serum,namely xanthine,retinyl ester,2-arachidonicglycerophosphocholine,arachidonic acid,3-hydroxycholic acid;There were six potential biomarkers in feces,namely purine,ornithine,capric acid,palmitic acid,arginine-asparagine,diisobutyl phthalate;There were six potential biomarkers in the gastric tissue,namely cytosine,xanthine,oleamide,PC（20:2（11Z,14Z）/14:0）,tauroursodeoxycholic acid,D-leucine.For the GJL group,there were three potential biomarkers in the serum,namely 2-arachidonylglycerophosphocholine,PC（20:2（11Z,14Z）/14:0）,3-hydroxycholic acid;There were six potential biomarkers in the feces,namely palmitic acid,oleamide,Lyso PA（18:0e/0:0）,Sebiferic acid,3-（2-hydroxy-4-methoxyphenyl）propanoic acid,asparagine B.There were five potential biomarkers in gastric tissue,namely chenodeoxycholic acid glycineconjugate,PC（20:2（11Z,14Z）/14:0）,3-hydroxydecanoic acid,18-hydroxyarachidonic acid,tauroursodeoxycholic acid.Metabolic pathway analysis results showed that the metabolic pathways involved in ST36 group include sphingolipid metabolism,glycerophosphate metabolism,glutathione metabolism,linoleic acid metabolism,D-glutamine and D-glutamate metabolism,and valerian metabolism,the biosynthesis and degradation of amino acids,leucine and isoleucine,α-linolenic acid metabolism,arachidonic acid metabolism,aminoacyl t RNA biosynthesis.The metabolic pathways involved in the GJH group include linolenic acid metabolism,α-linolenic acid metabolism,arginine biosynthesis,glutathione metabolism,arachidonic acid metabolism,glycerol phosphate metabolism,arginine and proline metabolism.The metabolic pathways involved in the GJM group include arachidonic acid metabolism,linolenic acid metabolism,α-linolenic acid metabolism,arginine biosynthesis,retinol metabolism,glutathione metabolism,unsaturated fatty acid biosynthesis,glycerol phosphate,arginine and proline metabolism,purine metabolism.The metabolic pathways involved in the GJL group include linolenic acid metabolism,α-linolenic acid metabolism,arachidonic acid metabolism,glycerol phosphate metabolism,and primary bile acid biosynthesis.RT-PCR and WB results showed that compared with the blank control group,ST36 and GJ groups were enhanced Ampk,Pgc-1α,Ppar-α,Sirt1 m RNA and protein expression.Conclusion:1.Moxibustion at ST36 and GJ can promote the energy metabolism of the stomach tissue of rats,increase the energy storage of the stomach tissue,and have the effect of tonifying spleen yang.Moxibustion at CV4 and XM can promote kidney energy metabolism in rats,increase kidney energy storage,and have the effect of tonifying kidney yang.2.The CV4 and ST36 groups had no obvious effect on the mitochondrial metabolism rate,while the XMH and GJH groups inhibited the mitochondrial metabolism rate.They may promote energy metabolism by increasing the number of mitochondria.The medium-dose group and low-dose group may promote energy metabolism by increasing the rate of mitochondrial metabolism and the number of mitochondria.3.In terms of tonifying the spleen-yang,moxibustion at ST36 may mainly regulates the metabolism of glycerol phosphate,sphingolipid and glutathione to increase the body’s energy supply and storage.GJ may regulates energy metabolism mainly through arachidonic acid metabolism,retinol metabolism,glycerol phosphate metabolism and arginine biosynthesis.In terms of tonifying the kidney-yang,moxibustion at CV4 may mainly regulates the TCA cycle,sphingolipid metabolism,pentose phosphate metabolism and purine metabolism to increase the body’s energy supply and storage.XM may regulates energy metabolism mainly through retinol metabolism and TCA cycle metabolism.