Study On The Mechanism Of Guanxinning Tablets On Tibet Minipigs With Atherosclerosis Based On The "Liver-gut" Axis

Objective To observe the effects of Guanxinning Tablets(GXN)on Tibetan minipigs with atherosclerosis(AS)induced by high-fat and high-cholesterol diet(HFC),and to explore the possible mechanism from the "liver-gut" axis,laying a foundation for the research of pharmacological mechanism of GXN.Methods(1)Eighteen male 3-4-month-old Tibetan minipigs were selected.After adaptive feeding,they were divided into three groups according to blood lipid,blood glucose and body weight,namely the normal control(NC)group,AS group and GXN group,6 pigs in each group.The NC group was always fed with regular diet.The AS and GXN groups were fed with HFC for 36 weeks.The GXN group was given GXN at 162 mg/kg daily from 24th week,and the intervention was continued for 12 weeks.After the end of the administration,blood was collected and a glucose tolerance test was performed,and then the animals were euthanized.The biochemical indexes of glucose and lipid metabolism and inflammatory indexes were measured,and AI and HOMA-IR indexes were calculated.The pathological structure and lipid deposition of the aorta was observed by HE and Sudan IV staining,and high-throughput sequencing analysis of the aortc transcriptome was performed.(2)TC,TG,SOD and MDA contents in liver were measured.HE staining was used to observe the pathological changes of liver and small intestine.The lipid deposition in the liver was evaluated by Oil red "O" staining.High-throughput sequencing analysis of the liver and small intestine transcriptome were performed,and 16S rDNA sequencing was used to analyze colon contents.Results(1)Compared with the NC group,the plasma TC,HDL-C,LDL-C,FFA,FMN,FINS,IL-6,IL-1?,TNF-?,CRP levels,and AI and HOMA-IR indices of Tibetan minipigs in the AS group were significantly increased(P<0.05,P<0.01);the minipigs in the AS group showed impaired glucose tolerance,increased aortic lipid deposition,and obvious AS plaques and inflammatory cell infiltration were observed in blood vessels,while IMT increased(P<0.01).Compared with the AS group,the GXN group significantly reduced the above-mentioned glucose and lipid metabolism and inflammation indexes and AI and HOMA-IR indexes(P<0.05,P<0.01),improved impaired glucose tolerance,suppressed aortic lipid deposition and AS plaque,and reduced IMT value(P<0.01).Analysis of aortic transcriptome:compared with NC group and AS group,2475 differential genes(DEG)were obtained(1792 up-regulated and 685 down-regulated);compared with AS group and GXN group,731 DEG were obtained(401 up-regulated and 330 down-regulated).Venn analysis found that 476 DEG showed a reverse regulation trend.GSEA analysis showed that these two data sets were enriched to 109 and 103 signal pathways,respectively,and 67 common pathways with opposite regulatory trends were obtained.It was also found that GXN significantly affects human unstable AS plaque genes set.In addition,GO-BP and KEGG analysis of 476 DEG showed that 35 GO-BP including immune response,inflammatory response,response to lipopolysaccharide(LPS),cell response to tumor necrosis factor,response to insulin,and cell adhesion were significantly enriched;At the same time,46 KEGG pathways including calcium signaling pathway,vascular smooth muscle contraction,steroid biosynthesis,TNF signaling pathway,NF-?B signaling pathway,cell adhesion molecules,and insulin secretion were significantly enriched.Furthermore,by constructing a PPI network of DEG in the aorta,six Hub genes(MMP9,IL6,CXCL10,ISG15,VCAM1 and LEP)and one core module using IRF1 as a transcription regulator were found.This module is related to the TNF signaling pathway,fluid shear stress and atherosclerosis,Toll-like receptor signaling pathway,NF-?B signal pathway and so on.(2)Compared with the NC group,the liver TC,TG,and MDA contents in the AS group were significantly increased(P<0.05,P<0.01).The minipigs in the AS group showed liver steatosis,a large number of inflammatory cell infiltration,and increased lipid deposition were observed.Compared with AS group,the liver of TC and MDA contents in the GXN group were significantly reduced(P<0.05,P<0.01),and the liver lipid deposition and inflammatory response were also reduced.Analysis of the liver transcriptome:compared with NC group and AS group,731 DEG were obtained(472 up-regulated and 259 down-regulated);compared with AS group and GXN group,216 DEG were obtained(106 up-regulated and 110 down-regulated).Venn analysis found that 71 DEG showed a reverse regulation trend.GSEA analysis showed that these two data sets were enriched to 72 and 60 signal pathways,respectively,and 46 common pathways with opposite regulatory trends were obtained.In addition,GO-BP and KEGG analysis of 71 DEG showed that 5 GO-BP including inflammatory response,immune response,monocyte chemotaxis,and response to LPS were significantly enriched;At the same time,5 KEGG pathways including Toll-like receptor signaling pathway,viral protein interaction with cytokine and cytokine receptor,apoptosis,and B cell receptor signaling pathway were significantly enriched.Furthermore,by constructing a PPI network of DEG in the liver,two Hub genes(CXCL10 and CCL4)and one core module were found.This module is related to the chemokine signaling pathway,cytokine-cytokine receptor interaction,and Toll-like receptor signaling pathway and so on.(3)The results of HE staining showed that the small intestine of Tibetan minipigs in the AS group can observe that intestinal villous edema,congestion of some capillaries,epithelial cell arrangement disorder,even cell necrosis,and the lamina propria of intestinal was infiltrated with a large number of monocytes and eosinophils.GXN can significantly improve the pathology.Analysis of small intestine transcriptome:compared with NC group and AS group,377 DEG were obtained(156 up-regulated and 221 down-regulated);compared with AS group and GXN group,1207 DEG were obtained(645 up-regulated and 562 down-regulated),and identified 205 DEG showed a reverse regulation trend.GSEA analysis showed that these two data sets were enriched to 41 and 70 signal pathways,respectively,and 26 common pathways with opposite regulatory trends were obtained.In addition,GO-BP and KEGG analysis of 205 DEG showed that 9 GO-BP such as inflammatory response,B cell receptor signaling pathway,myeloid dendritic cell chemotaxis,and response to LPS were significantly enriched;At the same time,18 KEGG pathways were enriched,which mainly involved the PPAR signaling pathway,cytokine-cytokine receptor interaction,glucagon signaling pathway,B cell receptor signaling pathway,fat digestion and absorption,and insulin secretion,cholesterol metabolism and so on.Furthermore,by constructing a PPI network of DEG in the small intestine,four Hub genes(CXCR5,CD 19,CD79B and CNR2)and one core module were found.This module is related to chemokine signaling pathway,cytokine-cytokine receptor Interaction,neuroactive ligand-receptor interaction,sphingolipid signaling pathways,etc.(4)Analysis of intestinal flora:There were 1853 OTUs in the intestinal contents of the three groups.Compared with NC group,diversity of intestinal flora of Tibetan minipigs in AS group(P<0.05).From the perspective of the phylum level,the abundance of Bacteroidete,Euryachaeota,and Spirochaetes in the AS group were significantly reduced(P<0.05),and the abundance of Proteobacteria,Verrucomicrobia and Synergistetes were significantly increased(P<0.05),while the ratio of Bacteroidetes/Firmicutes was significantly reduced(P<0.01);compared with the AS group,the abundance of Firmicutes was increased significantly(P<0.05),while the abundance of Proteobacteria decreased significantly(P<0.05).From the perspective of the genus level,compared with the NC group,the abundance of Escherichia and Gemmiger was significantly increased in the AS group(P<0.05),but significantly decreased in the GXN group(P<0.05);in contrast,compared with the NC group,the abundance of Paraprevotella was significantly decreased in the AS group(P<0.05),but significantly increased in the GXN group(P<0.05).Further analysis by LEfSe revealed that Escherichia,Gemmiger and Paraprevotella could be the target bacteria of GXN.Conclusion GXN can regulate glucose and lipid metabolism,improve insulin resistance and chronic low-grade inflammation,inhibit vascular lipid deposition and AS plaques formation in Tibet minipigs with AS,indicating that GXN has the effects of anti-AS;its mechanism may be related to affecting TNF signaling pathway,Toll-like receptor signaling pathway,NF-?B signaling pathway,cholesterol metabolism and insulin secretion pathways,and MMP9,IL-6,CXCL10,ISG15,VCAM-1 and LEP may be the key genes for the effects of GXN on the aorta,IRF1 may be the key transcription factor for GXN to exert anti-AS effect.On the one hand,GXN affect the PPAR signaling pathway and insulin secretion pathway in the intestine by regulating the flora of Paraprevotella and Gemmiger,thereby inhibiting the absorption of cholesterol and glucose by the small intestine,reducing lipid synthesis and insulin resistance in the liver,and delaying the occurrence of AS;On the other hand,GXN regulating the flora of Escherichia in the intestine,inhibiting the production of LPS,and maintains the intestinal barrier by regulating multiple signaling pathways,thereby reducing the entry of microbial molecular models such as LPS into the circulatory system and inhibits the activation of Toll-like receptor signaling pathway,NF-?B signaling pathway,TNF signaling pathway and other pro-inflammatory pathways in the liver and aorta,reduces the release of inflammatory factors,improves the chronic low-grade inflammatory response,and exerts anti-AS effect.It shows that the effects of GXN on AS in Tibet minipigs is closely related to the "liver-gut" axis pathway.