| Object i ve:Herbal cake-separated moxibustion was used to intervene in hyperlipidemia rabbits.By observing the changes in blood lipid levels,intestinal flora structure,metabolites of colon tissue,and the correlation between microflora and metabolites to probe into the mechanism of herbal cake-separated moxibustion in regulating hyperlipidemia.Methods:32 New Zealand rabbits were randomly divided into normal group,model group,herbal cake-separated moxibustion group and atorvastatin calcium group with 8 rabbits in each group.The hyperlipidemia model was prepared by feeding a high cholesterol diet with 12 weeks.The rabbits of normal group and model group were fed with normal standard diet and high cholesterol diet respectively.The rabbits of atorvastatin calcium group were fed with 1 mg/(kg·d)atorvastatin calcium powder mixed with high cholesterol diet,the rabbits of herbal cake-separated moxibustion group were given high cholesterol diet and moxibustion every day for 12 weeks.After 12 weeks,the general behavior changes of rabbits and serum LDL-C,TG and TC contents were observed.The 16S rDNA technique was brought to observe the changes of intestinal flora structure of rabbits in each group,and to identify the different genera of bacteria after the intervention;1H NMR technique was brought to detect the changes of metabolites in colon tissues of rabbits in each group,and to find out the relevant metabolic pathways;The correlation analysis was conducted on the different bacteria and metabolites after intervention,to study the relationship between gut microflora and metabolites,and to explore the possible function mechanism.Results:1.The results of serum lipid showed that the LDL-C,TG and TC levels in the model group were significantly higher than those in the normal rabbits(P<0.01).Compared with atorvastatin calcium group,the level of TG,TC and LDL-C in the herbal cake-separated moxibustion group had no significant difference(P>0.06),and compared with the model group,the content of TG,TC and LDL-C decreased(P<0.05 or P<0.01).2.The results of intestinal flora structure showed the Shannon and Chao 1 indexes of rabbits in the model group decreased compared with the normal group,while the difference was not statistically significant(P>0.05);Compared with the model group,the Chao 1 index in the herbal cake-separated moxibustion group increased significantly(all P<0.05),and the Shannon index showed an upward trend.The Chao 1 and Shannon indexes in the atorvastatin calcium group showed a trend of retracement compared with the model group,while the difference was not statistically significant(P>0.05).Beta diversity analysis showed that there were some differences among the four groups of bacteria.At the phyla level,cyanobacteria in the model group were lower than those in the normal group(P<0.05);Compared with the model group,the cyanobacteria in the herbal cake-separated moxibustion group increased(P<0.05).Proteobacteria in atorvastatin calcium group was higher than that in model group(P<0.05).At the genus level,there were significant differences in 9 species among the top 20 genus with relative abundance.Compared with the normal group,un＿f＿Muribaculaceae,un＿o＿Gastranaerophilales,Ruminococcaceae＿UCG-014,Ruminococcaceae＿V9D2013 decreased(P<0.05 or P<0.01),Lachnospiraceae＿NK4A136,Ruminiclosterium＿6 and Bacteroides increased in the model group(P<0.05 or P<0.01);Compared with the model group,Ruminococcaceae＿UCG-014,un＿o＿Gastranaerophilales,un＿f＿Muribaculaceae,Akkermansia increased in the herbal cake-separated moxibustion group(P<0.05 or P<0.01),while Lachnospiraceae＿NK4A136,Ruminiclosterium＿6 and Bacteroides decreased(P<0.05 or P<0.01);Compared with the model group,Lachnospiraceae＿NK4A136,Ruminiclosterium＿6 reduced(P<0.05),while Ruminococcaceae＿UCG-014 increased(P<0.01)in the atorvastatin calcium group.3.The results of metabolites of colon tissue:OPLS-DA analysis results showed that there were differences of metabolites in each group.The metabolites betaine,alanine,lactate,VLDL,N-acetylglycoprotein and glutamate in the model group were higher than those in the normal group(P<0.05),while butyrate,glycerophosphatecholine,glycerol,glutamine,creatine,phosphatecholine,choline,sarcosine and glutamine were lower(P<0.05).Correlation analysis showed that many metabolites of the host were related to serum LDL-C,TG and TC.At the same time,D-glutamine/D-glutamate metabolism,glycerolipid metabolism,glycine/serine/threonine metabolism,alanine/aspartate/glutamate metabolism and glycerophospholipid metabolism changed.The intervention of herbal cake-separated moxibustion could adjust the changes of metabolites such as alanine,glutamine,glutamate,butyrate,glycerophosphatecholine,lactate,N-acetylglycoprotein in HLP rabbits.4.The results of flora-metabolism correlation analysis showed that the changes of metabolites in host were related to the changes of gut microbiota.57 correlation pairs(|r|>0.5)and 9 key strong correlation pairs(|r|≥0.8)were revealed from herbal cake-separated moxibustion group and the model group by the pearson correlation analysis between the difference metabolites and bacteria genus.un＿o＿Gastranaerophilales,Ruminococcaceae＿UCG-014,Akkermansia were the three bacteria genus that had the most remarkable correlation with metabolites,and their relative abundance were significantly correlated with the levels of choline,phosphocholine,butyrate,lactate and other metabolites(P<0.05 or P<0.01).7 correlation pairs(|r|>0.5)and 4 key strong correlation pairs(|r|≥0.8)were screened by pearson correlation analysis between the the difference metabolites and bacteria genus in the atorvastatin calcium group and the model group.Ruminococcaceae＿UCG-014 was associated with metabolites strongestly,related to choline,cholinephosphate,glutamine,alanine significantly(P<0.05 or P<0.01).Conclusion:1.The intervention of herbal cake-separated moxibustion can improve the general condition and dyslipidemia of HLP model rabbits induced by high cholesterol diet.2.Herbal cake-separated moxibustion can regulate the changes of intestinal flora structure effectively in HLP rabbits.3.The intervention of herbal cake-separated moxibustion can modulate the changes of metabolites in HLP model rabbits.HLP can be improved by regulating some metabolites to lower oxidative stress,inflammation and adjust energy metabolism.4.Intestinal flora was closely connected with metabolites of host.Herbal cake-separated moxibustion played the role of regulating energy metabolism and anti-inflammatory by adjustting the changes of intestinal flora structure and metabolites,un＿o＿Gastranaerophilales,Ruminococcaceae＿UCG-014 and Akkermansia may be the important target bacteria genus for the prevention and treatment of HLP by herbal cake-separated moxibustion... |
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