Effects Of Vitamin D On Liver Function And Intestinal Microflora In Rats With Alcoholic Liver Disease

Objective Objectives was to investigate the effect of vitamin D(VD)on liver function and intestinal microflora in rats with alcoholic liver disease(ALD).Methods 1.Animal grouping: 75 8-week-old SPF male SD rats.After 3 weeks of adaptive feeding,they were randomly divided into 5 groups,each with 15 rats,namely the control group(C),alcohol model group(M),Diamine glycyrrhizinate intervention group(DG),low-dose VD group(LD)and high-dose VD group(HD).The group M(56% liquor intragastric administration): the dose is 8 m L/(kg·d)for the 1st to 4th week,and the dose is 10 m L/(kg·d)for the 5th to 16 th week;the group DG(200 mg/(kg·d)diamine glycyrrhizinate and 56° liquor gavage);the group LD(1.5 μg/(kg·d)and 56% liquor gavage),the HD group(3.0 μg /(Kg·d)VD and 56% liquor gavage),the liquor dosage was the same as that of the group M;the group C(intragastric administration of the same amount of normal saline),and the experiment lasted for 16 weeks.After the last gavage,3-5 pellets of rat feces from each group were collected in a sterile squirrel cage,and frozen at-80°C for later use.At the end of the 16 th week,the rats were fasted with water for 12 hours.The rats were anesthetized with 3% sodium pentobarbital,and blood was taken from the abdominal aorta.Leave it at room temperature for more than 30 minutes,centrifuge at 3000r/min for 10 minutes at 4°C,and save the serum for biochemical index detection.After harvesting liver tissues,keeping HE specimens and electron microscope specimens,the remaining liver tissues were quick-frozen with liquid nitrogen and stored in a refrigerator at-80°C for subsequent experiments.2.During the experiment,the food intake and body weight of rats were measured every week.3.Hematoxylin eosin(H & E)staining was used to observe the histopathological changes of rat liver,and transmission electron microscope was used to observe the ultrastructural changes of hepatocytes.4.Enzyme linked immunosorbent assay(ELISA)detects serum 25(OH)D,tumor necrosis factor(TNF-α),interleukin-6(IL-6)and lipopolysaccharide(LPS)level.5.A fully automated analyzer detects serum Glutamic-pyruvic transaminase(ALT),glutamic oxalacetic transaminase(AST),Gamma-glutamyl transpeptidase(GGT)and choline ester Enzyme(CHE)content.6.Using 16 S r DNA gene sequencing technology to analyze the differences in the intestinal microbiotaof rats.Results 1.During the experiment,the body weight and food intake of rats in each group were generally lower than those in the C group.2.The serum 25(OH)D level in the M group was significantly decreased by 66.9% compared with that in the C group(18.22±1.02)ng/ m L.After diamine glycyrrhizinate intervention,the serum 25(OH)D level of rats reached(15.69±0.82),significantly increased by 1.6 times compared with the M group.Compared with the M group,serum 25(OH)D levels in the HD group and the LD group were significantly increased by 96.84% and 36.32%,respectively.Statistical analysis showed that there were significant differences in the results(P<0.05).3.The results of H & E staining showed that the hepatocytes in the C group were complete,the nuclei were clear,and the hepatic cords were arranged radially;In the M group,the hepatocytes were swollen and deformed;Compared with the M group,the number of fat vesicles decreased in the LD group;Int he DG group and the HD group,the hepatic cords were arranged orderly and the histological structure tended to be normal.4.The results of transmission electron microscope showed that the nuclear membrane of hepatocytes in the C group was intact,the nucleolus was clear,and the morphology of mitochondria was normal.In the M group,the mitochondria and rough endoplasmic reticulum were swollen,deformed and cristae disordered.Compared with the M group,the structure of hepatic lobules in the LD group was not clear,but the degree was lower than that in the M group;In the HD group and the DG group,the above lesions were significantly improved.5.The serum levels of alt,AST and GGT in the M group were significantly increased(P< 0.05),which were 74.30%,43.17% and 53.16% higher than those in the C group respectively,and the serum level of CHE was decreased by 29.96%(P<0.05).Compared with the M group,the DG group and the VD group were significantly improved after intervention.6.Compared with the C group,the levels of IL-6 and TNF-α in the M group were significantly increased by 161.64% and 87.72%,respectively(P<0.05).Compared with the M group,the IL-6 level was significantly decreased by 19.62%(P<0.05)after high dose VD intervention,but the serum TNF-α level was not significantly changed(P>0.05).7.The serum LPS level of rats in the M group reached(35.85±5.24)Eu/L,which was significantly increased by 100.0 % compared with the group C(17.88±4.95)Eu/L(P< 0.05).Compared with the M group,serum LPS level in the HD group and the DG group was significantly decreased by 26.44% and 19.83%,respectively(P<0.05).8.The 16 S r DNA sequencing results showed that the chao1 index and Shannon index showed no significant difference in species richness and diversity among the three groups.The results of Beta diversity analysis suggest that there are significant differences in species structure and composition among the three groups.Among them,the HD group is more similar to the C group,while the M group is quite different from the other two groups.At the phylum level,Firmicutes,Bacteroidetes and Candidatus Saccharibacteria were the main phyla in the three groups,and the relative abundance of the other phyla was low.At the genus level,the most dominant genus of the C group was Saccharibacteria_genera_incertae_sedis,while the most dominant genus of the model group and the HD group were Prevotella and Lactobacillus,respectively.Compared with the C group and the HD group,the relative abundance of Lactobacillus in the M group was significantly reduced(P<0.05),while the abundance of Prevotella and Alitipes was significantly increased(P<0.05).The analysis results also found that Bifidobacterium was significantly enriched in the HD group(P<0.05).Conclusion 1.Appropriate supplementation of VD can significantly improve the histomathological changes of liver in ALD rats,and alleviate the inflammatory response mediated by enteric endotoxin.2.High-dose VD significantly reduced the abundance of pathogenic bacteria Prevotella and Alistipes in the intestinal tract of ALD rats,and significantly increased the abundance of probiotics Bifidobacterium and Lactobacillus,and regulated the intestinal microecological disturbance of ALD rats,thereby alleviating liver damage.