Effects Of Panax Notoginseng Saponins On Lipid Metabolism,Inflammation And Intestinal Microbiota In Hyperlipidemic Rabbits

This study aimed to explore the effects of panax notoginseng saponins（PNS）at different concentrations on intestinal microbiota,lipid metabolism and inflammation in hyperlipidemia rabbits.The experiment was divided into high fat diet feeding test and PNS prevention test.High-fat diet feeding experiment:12 healthy purebred male New Zealand rabbits of 30-days-old with uniform body weight（2.15 ± 0.05 kg）were selected.The rabbits were divided into normal diet group（CON）and high-fat diet group（HFD）on average.The normal diet group was fed with normal diet,and the high-fat diet group was fed with high-fat diet（the basic diet was mixed with 5%cholesterol,6%egg yolk powder and 8%lard for granulation）.The initial test period was 7 days and the positive test period was 80 days.PNS prevention test:25 healthy purebred male New Zealand rabbits（2.61 ± 0.10 kg）of 30-days-old were reselected.According to body weight and blood biochemical indexes,rabbits were divided into 5 groups:normal diet group（CON）,high-fat diet group（HFD）,high-fat diet prevention group Ⅰ（HFD+PNS1）,high-fat diet prevention group Ⅱ（HFD+PNS2）,high-fat diet prevention group III（HFD+PNS3）,with 5 replicates in each group.The CON group was fed a basal diet,and the other groups were fed a high-fat diet.At the end of the trial period,the high-fat diet group was given intragastric administration of panax notoginseng saponins,the HFD+PNS1,HFD+ PNS2 and HFD+ PNS3 groups were given intragastric administration of 100 mg/kg·d,200 mg/kg·d and 300 mg/kg·d PNS,respectively,and the CON and HFD groups were given the same amount of normal saline.The initial test period of the formal test was 7 days,and the positive test period was 60 days.Exp.1:Feeding rabbits with high-fat diet showed that:（1）the weight of HFD group was significantly higher than that of CON group on the 30th day of high-fat diet（P<0.01）;（2）The serum levels of total cholesterol（TC）（P<0.01）,triglycerides（TG）（P<0.05）,low-density lipoprotein cholesterol（LDL-C）（P<0.01）and high-density lipoprotein cholesterol（HDL-C）（P<0.01）in HFD group were significantly higher than those in CON group from 30th day to 80th day.（3）After the experiment,liver index and abdominal fat index in HFD group were significantly higher than those in CON group（P<0.05）;The average area of abdominal adipocytes and total area of liver lipid droplets in HFD group were significantly larger than those in CON group（P<0.05）.Exp.2:To investigate the effect of PNS on blood biochemical indexes and growth performance of hyperlipidemia.The results showed that:（1）The weight of rabbits in each group increased gradually,and the weight of rabbits in HFD group increased rapidly,but the difference was not obvious（P>0.05）;（2）On the 14th day,the serum TC,TG,LDL and HDL in the high-fat diet group were significantly higher than those in the normal group（P<0.001）,and the high-fat model was successfully established.（3）After the experiment,the liver indexes of HFD,HFD+PNS1,HFD+PNS2 and HFD+PNS3 groups were significantly higher than those of the CON group（P<0.05）.The liver index of HFD+PNS1,HFD+PNS2 and HFD+PNS3 groups decreased compared with HFD group,but the difference was not significant（P>0.05）.The abdominal fat index in HFD,HFD+PNS1,HFD+PNS2 and HFD+PNS3 groups was significantly higher than that in normal group（P<0.05）.The abdominal fat index of HFD+PNS2 and HFD+PNS3 groups was lower than that of HFD group,but the difference was not significant（P>0.05）.In the CON group,the size of hepatocytes was uniform,with clear cellular nucleus and uniform cytoplasm.In the HFD group,the cell area increased,the cells swelled in large areas and vacuoles were formed.HFD+PNS 1,HFD+PNS2 and HFD+PNS3 groups could significantly improve liver steatosis induced by high-fat diet.Compared with the high-fat group,the lipid droplets and vacuole areas in cells were significantly reduced.The abdominal aorta of rabbits in the high-fat group was thrombosis,with thick vascular wall and significantly smaller vascular diameter than those in the HFD+PNS2 group and the CON group（P<0.05）.In addition,the abdominal aorta of the HFD+ PNS2 and HFD+PNS3 groups was more flat than those in the HFD and HFD+PNS2 groups,with significantly reduced thrombosis and obvious improvement trend.Exp.3:At the end of the experiment,the morphological structure of small intestine,liver,abdominal fat and abdominal aorta in normal diet group,high-fat diet group and high-fat diet prevention group were observed,and the genes related to liver and ileum hyperlipidemia were detected and analyzed.The results showed that:（1）The morphological structure of rabbit intestinal tissue:After PNS prevention,the villus length and villus crypt ratio of duodenum,jejunum and ileum in hyperlipidemia rabbits were significantly higher than those in high-fat diet group（P<0.05）.The duodenal pH in HFD+PNS1 group and HFD+PNS2 group was significantly higher than that in HFD group（P<0.05）.The cecum pH of HFD+PNS2 group and HFD+PNS3 group in the CON group was significantly higher than that in the HFD group（P<0.05）.（2）Rabbit inflammatory genes:The mRNA expression levels of TNF-α,IL-6,TLR-2,TLR-4,MYD88 and NF-κB in liver and ileum of HFD group were significantly higher than those of CON group（P<0.05）,and the mRNA expression level of IL-10 was significantly lower than that of CON group（P<0.05）.After PNS prevention,the mRNA expression levels of liver inflammatory factors TNF-α,IL-6,TLR-2,TLR-4,MYD88 and NF-κB in HFD+PNS3 group were significantly lower than those in hyperlipidemia rabbits（P<0.05）.The expression of IL-10 mRNA in HFD+PNS3 group was higher than that in hyperlipidemia rabbits.The mRNA expressions of IL-6,NF-κB,TLR-2 and TLR-4 in ileum of HFD+PNS3 group were higher than those of HFD3 group.The expression of IL-10 mRNA in HFD+ PNS1,HFD+ PNS2 and HFD+PNS3 groups was higher than that in HFD group.（3）Rabbits lipid metabolism genes:The mRNA expression levels of genes AdipoR-1,AdipoR-2,LXR-α,LXR-β,CoA-α,PPAR-α and PPAR-γ related to lipid metabolism in liver and ileum were significantly decreased（P<0.05）,and the mRNA expression levels of genes SREBP-1 and SREBP-2 related to fat decomposition were significantly increased on average（P<0.05）.The mRNA expression levels of AdipoR-1,LXR-β,CoA-a and PPAR-α in liver were increased after PNS prevention,and the mRNA expression levels of SREBP-1 and SREBP-2 were decreased in HFD+PNS2 and HFD+PNS3 groups.The mRNA expression levels of AdipoR-1,AdipoR-2,LXR-β and PPAR-y in ileal promoting lipid degradation in HFD+PNS2 and HFD+PNS3 groups were higher than those in HFD group.The mRNA expression of SREBP-1 and SREBP-2 in ileum of HFD+PNS1,HFD+PNS2 and HFD+PNS3 groups decreased.（4）Rabbit ileum tight junction protein:the mRNA expression levels of Claudin-1（tight junction protein 1）,Occludin and ZO-1（atresia lactoprotein 1）in HFD group were lower than those in CON group.The mRNA expressions of Claudin-1,Occludin and ZO-1 in ileum of HFD+PNS2 and HFD+PNS3 groups were higher than those of HFD group.Exp.4:The 16S rRNA technology was used to study the relationship between PNS and ileal microbiota,metabolites of ileal microbiota and intestinal genes in hyperlipidemia rabbits.The contents of short chain fatty acids in ileum were detected and analyzed.The results showed that:（1）At the phylum level,it was found that the abundance of Bacteroidetes in HFD group was lower than that in other groups.With the increase of PNS concentration,the abundance of Firmicutes gradually increased,and the abundance of Firmicutes in HFD+PNS1 group was the highest in each group.At genus level,the abundances of Ruminococcaceae_NK4A214_group,Ruminococcus₁ and Ruminococcaceae_UCG-014 in CON,HFD+PNS2 and HFD+PNS3 groups were higher than those in HFD group;The abundance of Turicibacter in CON,HFD+PNS2 and HFD+PNS3 groups was lower than that in HFD group.The abundance of Planococcus in HFD+ PNS1 and HFD+PNS3 groups was significantly higher than that in HFD group（P<0.05）.（2）The functional analysis of PICRUSt2 showed that HDF was positively correlated with immune diseases,neurodegenerative diseases and cardiovascular diseases,while HFD,HFD+PNS1,HFD+PNS2 and HFD+PNS3 groups were negatively correlated.The analysis of functional differences at the genus level showed that the abundance of immune system functional pathway in the CON group was significantly higher than that in the HFD group（P<0.05）.（3）The analysis of ileal short-chain fatty acids showed that the contents of acetic acid,isovaleric acid and valeric acid in CON group were significantly higher than those in HFD group（P<0.05）,and the content of propionic acid in HFD+PNS1 group was significantly higher than that in HFD group（P<0.05）.n-butyric acid content in HFD+PNS3 group was significantly higher than that in HFD group（P<0.05）.（4）Correlation analysis of genus level microbes with ileum genes and short chain fatty acids showed that Turicibacter was negatively correlated with Acetic acid,Propionic acid,Isobutyric acid,Valeric acid concentrations and Claudin-1,ZO-1,AdipoR-2 and SREBF-2 mRNA expressions（P<0.05）.Ruminococcaceae_NK4A214_group was significantly positively correlated with LXR-β mRNA expression（P<0.05）.Ruminococcus₁ was significantly positively correlated with LXR-β and CoA-α mRNA expression（P<0.05）.Ruminococcaceae_UCG-014 was significantly positively correlated with Acetic acid,Isobutyric acid,N-butyric acid,Isovaleric acid concentration and Occludin,TLR-2,NF-κB,MyD88,PPAR-γ,SREBP-1 and SREBF-2 mRNA expression（P<0.05）,and extremely significantly positively correlated with TLR-4 and TNF-α mRNA expression（P<0.01）.（5）The pearson correlation analysis of ileum gene and short-chain fatty acid showed that acetic acid was significantly negatively correlated with the mRNA expression levels of TLR-2,TLR-4,NF-κB and SREBP-1（P<0.05）,and significantly positively correlated with the mRNA expression level of IL-10（P<0.05）.Propionic acid was significantly negatively correlated with TNF-α and IL-6 mRNA expression（P<0.05）.Isobutyric acid was significantly positively correlated with the mRNA expression levels of ZO-1,AdipoR-1 and LXR-α（P<0.05）.n-butyric acid was significantly negatively correlated with SREBP-1 mRNA expression（P<0.05）,and was significantly positively correlated with PPAR-α mRNA expression（P<0.05）.Isovaleric acid was significantly negatively correlated with TLR-2,NF-κB and IL-6（P<0.05）.In summary,PNS in the prevention of hyperlipidemia rabbits can improve the intestinal pH value and intestinal morphology of hyperlipidemia rabbits,improve the intestinal barrier’s inhibitory effect on harmful micromolecule substances,and further promote the proliferation of beneficial microbiota,so that the abundance and diversity of microbial microbiota in ileum content tend to normal levels,increase the production of short-chain fatty acids of beneficial bacteria in ileum,reduce the levels of pro-inflammatory genes and lipid metabolism-related genes in liver and ileum tissues,improve the levels of anti-inflammatory genes and lipid metabolism-related genes in liver and ileum,reduce blood cholesterol levels,improve the pathological changes of liver and artery,and improve the production and immune performance of hyperlipidemia rabbits.