Molecular Mechanism Of "Salvia Miltiorrhiza And Siwu Decoction Shows Same Effects" Based On Weight Loss And Lipid Lowering

Objective:Based on weight reduction and lipid lowering,the new work of"one-flavor Salvia miltiorrhiza（Sal）is the same as the Siwu decoction（SWD）"is explored.In this study,we used network pharmacology to predict the active components and pathways of Sal and SWD,and used microbial diversity,metabolomics and transcriptomics methods to clarify the similarities and differences in the mechanisms of weight and lipid reduction by Sal and SWD,so as to provide experimental basis for the study of"one-flavor Sal,work is the same as SWD".Based on weight reduction and lipid lowering,the new work of"one-flavor Sal is the same as the SWD"is explored.In this study,we used network pharmacology to predict the active components and pathways of Sal and SWD,and used microbial diversity,metabolomics and transcriptomics methods to clarify the similarities and differences in the mechanisms of weight and lipid reduction by Sal and SWD,so as to provide experimental basis for the study of"one-flavor Sal,work is the same as SWD".Methods:1.Clarify the theory of"one-flavor Sal is the same as the SWD"based on the theory of traditional Chinese medicine and literature research.Through the Traditional Chinese medicine System Pharmacology Analysis Platform（TCMSP）and other databases and related literature,the ingredient databases of Sal and SWD were established with the screening criteria of"OB≥30%and DL≥0.18".The potential targets of active compounds were analyzed and predicted by target identification service platform.The DAVID platform was used to perform disease enrichment analysis of the targets of Sal and SWD to predict the potential diseases of Sal and SWD.2.Extensive targeted metabolomics was used to identify the components of Sal and SWD.Twelve male SD rats weighing 160±20 g were divided into blank control group（Ctrl）,Salvia miltiorrhiza treatment group（Sal）and Siwu decoction treatment group（SWD）,with 6 rats in each group.The rats in Sal group and SWD group were given 2.7 g/kg and 3.24 g/kg by gavage,respectively.The rats in blank group were given equal volume of normal saline as control.To analyze the blood components of Sal and SWD by serum non-targeted metabolomics.3.Male SD rats were treated with high-fat diet（HFD）for 7 weeks to set up a model of obesity.40 rats were divided into 5 groups:normal group（Control）,high-fat model group（HFD）,high-fat model plus Sal group(HFD+2.7g·kg^-1Sal),high-fat model plus SWD group(HFD+3.24g·kg^-1SWD)and high-fat model plus Orlistat group(HFD+0.03g·kg^-1Orlistat),with 8 rats per group.Weekly periodic monitoring of rat body weight,food intake and activity state and other general conditions;after 8weeks of continuous administration of the drug,rats were killed after weighted,blood collection from the abdominal aorta was used for serum lipid levels.Adipose tissue of abdomen,epididymis and perirenal was collected to calculate body fat index（BFI）.Adipose tissue and liver were taken for H&E staining.Determination of c AMP,PKA,HSL in hepatic and adipose tissues by ELISA.Rat feces were collected for microbial diversity and LC-MS lipidomics detection.Liver tissue was collected for transcriptomic detection.RT-PCR was used to detect the m RNA expression of PPAR and MAPK.The expression of basic proteins of PPAR and MAPK in the liver were detected by Western Bloting.Results:1.Network pharmacology predicts that Sal and SWD may be the same for weight loss and lipid loweringThe targets of Sal and SWD were constructed through Uniprot website,and the corresponding targets of Sal and SWD were 635 and 707,respectively,and contained453 identical targets,accounting for 51%of the total targets.The target enrichment analysis results showed that obesity had a high correlation with Sal and SWD.The"target-signaling pathway"network diagram showed that the potential"weight loss and lipid lowering"effects of Sal and SWD may be related to the MAPK signaling pathway.2.The active ingredients of Sal and SWD are the material basis for weight loss and lipid loweringThe compositions of Sal and SWD were identified by liquid mass spectrometry tandem mass spectrometry through widely targeted metabolomics.A total of 788metabolites were identified from Sal,including phenolic,organic,lipids,et al,among which phenol and lipids were the predominant.Salvianolic acid A,Danshensu,cryptotanshinone,salvian cycloheptatrienolone and methylsalvianquinone were among the top substances in terms of relative content.SWD screened 931 metabolites,which were classified as organic acids,phenolic acids,free fatty acids,etc.Similar to Sal,phenolic acids and lipids are dominant.The top substances were citric acid,gallic acid,ligustrolactone A,paeoniflorin side,L-piperidinic acid,etc.Most of the active components of Sal and SWD have the effect of regulating fat metabolism,indicating that the main active components of Sal and SWD are the material basis for weight loss and lipid lowering.3.Untargeted metabolomics showed that the blood components of Sal and SWD were closely related to lipid metabolismNon-targeted metabolomics studies of serum indicated that some of the metabolites were significantly different from each other such as5-O-p-Coumaroylquinic acid,Phenylalanine,12-Hydroxyoctadecanoic acid and PS（18:1（9Z）/0:0）in the rats after administration of Sal and SWD.Among them,compared with control rats without drug intervention,Sal changed the expression of25 differential metabolites in rats after intervention,among which 9 metabolites were significantly up-regulated and 14 were significantly down-regulated（P<0.01）;SWD changed the expression of 30 differential metabolite in rats after intervention,among which 12 metabolites were significantly up-regulated and 18 were down-regulated（P<0.01）.The KEGG classification indicated that the metabolites were more expressed in the metabolism of amino acids and biosynthesis,phenylalanine and2-oxycarboxylic acid metabolism.4.Sal and SWD can effectively reduce body weight and lipidThe rats of the control group had good psychological status.Their hair was thick and they did not shed much.While in the HFD group,the rats had poor mental state,sparse hair loss and decreased activity.After continuous instillation with Sal,SWD and Orlistat as a positive control,the mental state of rats was improved,the rate of weight growth slowed down,the BFI,waist circumference,TG,TC,LDL-C GLU and FFA were reduced,and HDL-C was elevated.Microscopic observation of hepatic tissue revealed that the hepatic lobules were intact,while the rats induced by HFD had obvious accumulation of fat droplets,depression necrosis infiltration with balloon like changes in hepatic tissue.Adipose tissue was observed under microscope,and adipose cell contour and volume increased in the HFD group.The intervention of Sal,SWD and Orlistat has an effective effect on the reduction of hepatic fat accumulation,steatosis and the proliferation of fat cells in obese rats with HFD.ELISA results showed that the expression levels of c AMP,PKA and HSL of liver and fat in HFD group were lower than that in control group（P<0.01）,and the reversal of the trend in the treatment group was significant（P<0.05 or P<0.01）,indicating that Sal,SWD and Orlistat can effectively play the role of reducing weight and fat.5.The effect of Sal and SWD on weight loss and lipid lowering are related to the regulation of intestinal flora in obese rats16S r DNA was used to determine the intestinal flora structure,and the results showed that the samples were completely separated among the groups with significant differences in components.Sal significantly reduced the diversity of intestinal flora in obese rats while SWD had no significant effect.At the phylum and genus levels,the relative abundance of Proteobacteria and Actinobacteriota has increased,and the relative abundance of Firmicutes and Patescibacteria has been reduced.Correlation analysis of environmental factors showed that BFI,body weight,GLU,FFA,TG,TC,LDL-C is positively correlated with Turicibacter,Quinella,Desulfovibrio,Monoglobus,and Blautia,and negatively correlated with NK4A214＿group,Dubosiella,Facklamia,Aerococcus and Jeotgalicoccus,while HDL-C showed opposite results.c AMP,PKA and HSL were negatively correlated with HFD group,and positively correlated with control group,Sal group,SWD group and Orlistat group.6.The effect of Sal and SWD on weight loss and lipid reduction are related to the regulation of lipid metabolites in obese ratsNon-targeted metabolomics of metabolites showed that Sal and SWD could change the expression level of endogenous metabolites in obese rats.For example,Sal improved the levels of metabolites such as TG,DG,CL,Hex1Cer and Cer.SWD improved the levels of TG,DG,OAHFA,PC,Cer and other metabolites.Sal and SWD improved the levels of DG（22:6/22:6）,TG（4:0/14:1/18:3）,TG（15:0/18:2/22:6）,TG（18:2/18:20:4）and Cer（d16:0/21:0）（P<0.05）.The results of KEGG enrichment indicated that Sal and SWD played an important role in the regulation of triglyceride metabolism,fat digestion and absorption,weight loss and lipid reduction.7.The effect of Sal and SWD on fat reduction in obese rats are associated with transcribed genes.The transcriptomic study of liver tissue showed that the expression levels of 587differential genes of obese rats were changed by Sal,among which 256 genes were significantly up-regulated and 331 genes were significantly down-regulated.SWD changed the expression levels of 699 differential genes in obese rats,which were significant up-regulation of 291 genes and significant down-regulation in 408;Orlistat changed the expression levels of 1099 differential genes in obese rats,of which 481genes were significantly up-regulated and 618 genes were significantly down-regulated.Enrichment analysis of KEGG indicated that Sal and SWD were mainly involved in weight loss and lipid reduction through lipid metabolism such as triglyceride metabolism,fatty acid degradation,unsaturated fatty acid biosynthesis and other pathways.8.The effect of Sal and SWD on weight loss and lipid lowering may be related to the regulation of PPAR and MAPK signaling pathwaysThe genes and metabolites detected in different groups were comprehensively analyzed to find potential biomolecules and explore their signaling pathways,and Western Blot and RT-PCR were performed for verification.Through joint analysis of differential metabolites and differential genes,the key genes PLC-γ1,LPL and Cyp7a1 were found,and the signal pathways PPAR and MAPK were selected for gene and protein verification,combined with the enrichment of differential metabolic set and differential gene set into pathway results and network pharmacology results.RT-PCR results showed that Sal,SWD and Orlistat significantly up-regulated m RNA expression of Raf,ERK1/2,PPARα,PPARγ,Adiponectin,CPT1,LPL,Cyp7a1;m RNA expression of p38MAPK,CEBP-α,FAS,SREBP-1c,a P2,PLC-γ1 were significantly down-regulated.Sal up-regulated m RNA expression of Ras and MEK,while SWD showed the opposite effect.The results of WB indicated that p38MAPK,P-p38MAPK,ERK1/2,PPARγ,CEBP-αand PLC-γ1 in liver tissues of Sal,SWD and Orlistat groups were significantly reduced,while the protein expression levels of PPARαand Adiponectin were significantly increased（P<0.05,P<0.01）.In conclusion,the effect of Sal and SWD on weight loss and lipid lowering may be related to the inhibition of MAPK and CEBP signaling pathways,and then intervention of PPAR pathway to reduce lipid production.9.Different mechanisms underlying the same effect of Sal and SWDFirst of all,Sal and SWD have their own unique components,which exert their weight-reducing and lipid-lowering effects through the same or different mechanisms.Secondly,Sal and SWD have different effects on the structure of different bacterial flora,lipid metabolites and transcription genes,so they may mediate different pathways to exert their related effects.In the present study,Sal and SWD showed different effects on the m RNA and protein expressions of genes related to MAPK and PPAR signaling pathways.Sal promoted the phosphorylation of ERK1/2,while SWD showed an inhibitory trend;Sal increased the m RNA expression of Ras/Raf/MEK/ERK pathway,while SWD decreased the m RNA expression of Ras and MEK,and increased the m RNA expression of Raf and ERK.Conclusion:The equivalent weight loss and lipid reduction mechanism of Sal and SWD may be realized by intervening and regulating lipid metabolism,intestinal flora and metabolites,liver transcription,and related genes and proteins in PPAR and MAPK signaling pathways in obese rats induced by high fat diet.However,the difference between the two is that their components are different,and they have different effects on the structure of some bacterial flora,lipid metabolites and transcription genes,so they may play the same or different effects through different mechanisms.In a word,there is a certain basis for"one-flavor salvia miltiorrhiza,the power is the same as the siwu decoction".