| Objective:In this study,gastrodia elata extract and gastrodin were used as experimental drugs to observe the pathological changes of the thoracic aorta of model mice,the accumulation of lipids in the aortic root,the changes in blood lipid levels,the expression levels of LPS and inflammatory factors in the serum,and intestinal mucosal immunity.The changes in molecular SIg A,tight junction protein expression,intestinal flora and short-chain fatty acids,from the perspective of the relationship between intestinal flora and its metabolites and chronic inflammation throughout the body,a preliminary discussion on the role of gastrodia and gastrodin in alleviating the process of atherosclerosis The mechanism of action.Methods:1.8-week-old male C57BL/6J mice were randomly divided into a blank group,a model group,a gastrodia group and a gastrodin group.Except for the blank group,the other three groups were fed with a high-fat diet to replicate the AS model.2.Drug intervention: After 12 weeks,the model group continued to use high-fat feed,the gastrodia elata group and the gastrodin group continued to use the high-fat feed and the gastrodia extract and gastrodin were added for administration intervention,the blank group and the model group were given intragastric administration,etc.Measure normal saline once a day,and after feeding for a total of 20 weeks,the experiment was ended.3.Index detection:(1)HE staining method to detect the pathological changes of mouse thoracic aorta;BODIPY staining method to detect lipid accumulation in mouse aortic roots;(2)Blood lipid related indicators: single reagent direct method and double reagent method to detect small The expression of TC,LDL-C and HDL-C in mouse serum;(3)Inflammation related indicators: Detect TNF-α,IL-1β,ICAM-1,IL-6,IL-8in circulating blood of mice by Elisa method,IL-10 expression level;(4)Intestinal barrier and mucosal immune related indicators: the relative expression levels of ZO-1,OCLN,claudin-1 proteins in the colon tissue of each group of mice were detected by Wsetern blot;Changes in SIg A content in mouse colon tissue and expression levels of D-LA and LPS in serum(5)Intestinal flora and its metabolites related indicators:detection of intestinal flora by 16 Sr RNA gene high-throughput sequencing,GC-MS detection Short-chain fatty acids.Result:1.Pathological changes1.1 HE staining of mouse aorta in each group After 20 weeks,the HE staining results showed that the intima of the aorta in the blank group was smoother,the elastic fiber layer arranged concentrically was clear and complete,the subintimal space was smaller,the arrangement of smooth muscle cells was relatively regular,and there was no hyperplasia;the intima of the model group was rough,There is obvious thickening,a large number of smooth muscle cells proliferate,arrange disorderly,and have a tendency to migrate under the endothelium,the concentrically arranged elastic fiber layer is not clear,the subendothelial space is widened,and foam cells appear;the water extract group of gastrodia In the gastrodin group,the intima is thickened,smooth muscle cells in the media proliferate,and the arrangement is disordered,the concentrically arranged elastic fiber layer is unclear,and foam cells appear,but compared with the model group,there is a certain reduction.1.2 BODIPY staining of the aortic roots of mice in each group The results of BODIPY staining showed that the blank group had no obvious lipid droplets in the aortic valve area;compared with the blank group,the model group had obvious lipid droplet deposition;the gastrodia extract group and the gastrodin group had different degrees of lipid droplet infiltration,But significantly reduced compared with the model group.2.The influence of gastrodia and gastrodin on blood lipid levels Compared with the blank group,the serum TC and LDL-C content of the model group increased significantly(P < 0.001),while the HDL-C content decreased(P < 0.01).Compared with the model group,the serum TC and LDL-C content of the Gastrodia The content of TC was significantly reduced(P<0.001),the content of LDL-C was reduced(P<0.01),and the content of HDL-C was increased(P<0.05);the content of TC and LDL-C in the serum of the gastrodin group decreased(P<0.01)),the content of HDL-C increased(P<0.01).3.The influence of gastrodia and gastrodin on serum inflammatory factors TNF-α,ICAM-1,IL-1β,IL-6,IL-8,IL-10 Compared with the blank group,the serum levels of TNF-α,IL-1β,IL-6,and IL-8 in the model group were significantly increased(P<0.01),the content of ICAM-1 was extremely significantly increased(P<0.01),and IL-The content of 10 increased significantly(P<0.05).Compared with the model group,the serum TNF-α in the water extract group of Gastrodia elata decreased,the content of IL-10 increased(P<0.05),ICAM-1,IL-The contents of 1β and IL-8 were significantly reduced(P<0.01),and the content of IL-6 had a decreasing trend(P>0.05).The content of TNF-α,IL-1β and IL-6 in the gastrodin group was significantly reduced,and the content of IL-10 Significantly increased(P<0.01),and the content of ICAM-1 and IL-8 decreased(P<0.05).4.Comparison of the relative expression levels of tight junction protein in the colon tissue of mice in each group Compared with the blank group,the protein expression of ZO-1,OCLN,and claudin-1in the model group was significantly reduced(P < 0.01).Compared with the model group,the ZO-1,OCLN,The expression of claudin-1 protein increased significantly(P<0.01).5.Changes of s Ig A content in colon tissue of mice in each group Compared with the blank group,the immune molecule s Ig A content in the colon tissue of the model group decreased(P<0.01).Compared with the model group,the immune molecule s Ig A content of the gastrodin group increased(P <0.05),and the immune molecule s Ig A content of the gastrodia The content has an upward trend(P>0.05).6.Changes of serum D-LA content in mice of each group Compared with the blank group,the serum D-LA content of the model group was significantly increased(P<0.01).Compared with the model group,the serum D-LA content of the gastrodia elata group was significantly decreased(P<0.01).The serum D-LA content of the gastrodin The content decreased significantly(P<0.01).7.Effects of Gastrodia and Gastrodin on Serum LPS of Mice in Each Group Compared with the blank group,the serum LPS level of the model group was significantly increased(P<0.01).Compared with the model group,the serum LPS level of the gastrodiae group and the gastrodin group decreased.8.Changes in intestinal flora(1)Changes in abundance: From the perspective of the phylum level,the contents of the mouse intestine are mainly Firmicutes,Proteobacteria,Bacteroidetes and Actinobacteria.The relative abundance of Bacteroidetes and Actinobacteria decreased after modeling(P<0.05),and the relative abundance of Proteobacteria and Firmicutes increased(P>0.05).After administration intervention,this phenomenon changed.Compared with the model group,gastrodin increased the relative abundance of Bacteroidetes,Actinobacteria,and Firmicutes(P>0.05)and decreased the relative abundance of Proteobacteria(P> 0.05).Gastrodia extract significantly increased the relative abundance of Bacteroidetes(P<0.05)and Actinobacteria(P>0.05)after intragastric administration,and decreased the relative abundance of Proteobacteria and Firmicutes(P>0.05).At the genus level,compared with the blank group,the relative abundance of Romboutsia and Lactococcus(P<0.05)in the model group increased,and the relative abundance of Dubosiella(P<0.05),Alistipes(P<0.05),and unidentified-Enterobacteriaceae(P>0.05)The relative abundance of Faecalibaculum,Bifidobacterium,Bacteroides,Parabacteroides,Alloprevotella,Alipites,Odoribacter,Allobaculum decreased after the model was established,and the relative abundance increased after the administration intervention.There is an increase or an upward trend.(2)Diversity changes: Compared with the blank group,the intestinal flora diversity of rats in the model group decreased;compared with the model group,the intestinal flora diversity of rats in the drug intervention group increased.9.Changes in SCFAs content Compared with the blank group,the levels of acetic acid and propionic acid in the intestinal tract of the model group decreased(P<0.05),and butyric acid had a decreasing trend(P>0.05).Compared with the model group,the levels of acetic acid,propionic acid and butyric acid in the intestinal tract of gastrodia elata group and gastrodin group all had different degrees of increasing trend(P>0.05).Conclusion:1.Gastrodia extract and gastrodin can improve the pathological changes and lipid deposition of the aorta in AS mice,and regulate the expression of blood lipids.2.Gastrodia extract and gastrodin can reduce the expression level of pro-inflammatory factors,increase the expression level of anti-inflammatory factors,and regulate the inflammatory response in AS mice3.Gastrodia extract and gastrodin can enhance the immune function of the intestinal mucosa of AS mice,repair the intestinal mucosal barrier,and improve the circulation of endotoxin through the intestinal barrier.4.The water extract of gastrodia and gastrodin can regulate the intestinal flora of AS mice,increase the abundance of probiotics,reduce the abundance of pathogenic bacteria,increase the level of short-chain fatty acids,and reshape the structure of the intestinal flora.Is one of the mechanisms of preventing AS5.After quadruple antibiotics were given to clear the intestinal flora,the aortic lesions and lipid deposition in mice were more serious,the expression levels of blood lipids and inflammatory factors were more obvious,and the intestinal mucosal immune function decreased more severely than before the uncleared bacteria.,The effect of drug treatment is poor.The results indicate that the intestinal flora is indeed involved in the process of atherosclerosis in mice,and may play a role in it. |
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