Effect Of Combined Intervention Of Trehalose And Lactobacillus Acidophilus GDMCC1.208 On The Inhibition Of Atherosclerosis

ObjectiveThis study aims to investigate the role of trehalose in the proliferation and stress resistance of Lactobacillus acidophilus GDMCC1.208,explore the effect and mechanism of trehalose and Lactobacillus acidophilus GDMCC1.208 combined gavage intervention on the development of atherosclerosis in apolipoprotein E gene knockout(Apo E^-/-)mice.The findings might provide new research ideas for the treatment of atherosclerotic diseases and lay a theoretical foundation for the development of Lactobacillus acidophilus related products.Methods1.Lactobacillus acidophilus GDMCC1.208 was identified by plate marking,Gram staining and 16S r RNA sequencing2.The effects of trehalose on the proliferation and acid and bile salt resistance of Lactobacillus acidophilus GDMCC1.208 were investigated by microbial metagenomic sequencing and other methods.3.The effect of Lactobacillus acidophilus GDMCC1.208 on the proliferation of human colorectal adenocarcinoma（Caco-2）cells was detected by CCK-8.4.Apo E^-/-mice in the experimental group were given trehalose or Lactobacillus acidophilus GDMCC1.208 or a combination of both by gavage,while mice in the control group were only given PBS.All groups were given gavage every morning for 12 weeks.During the gavage experiment,wild type（WT）mice were fed ordinary diet and Apo E^-/-mice were fed high-fat and high-cholesterol diet.They were weighed every 2 weeks.After gavage,the mice were dissected and the heart weight and liver weight were recorded.5.Oil red O staining was used to detect atherosclerotic lesions on the intima surface of the main aorta and the transverse section of the root.6.Paraffin sections and hematoxylin-eosin（HE）staining were used to investigate the structures of small intestine and colon of mice.7.Serum levels of total cholesterol（TC）,triglyceride（TG）,high density lipoprotein cholesterol（HDL-C）and low density lipoprotein cholesterol（LDL-C）were determined by the cholesterol test kit.8.Real-time quantitative PCR（RT-q PCR）was used to detect the expression levels of cholesterol metabolism related genes such as cholesterol 7α-hydroxylase（CYP7A1）,HMG-Co A reductase（HMGCR）,ATP-binding cassette transporter A1（ABCA1）and ATP-binding cassette transporter G1（ABCG1）in liver.9.RT-q PCR was used to detect the expression levels of inflammatory genes such as tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）in aorta.Results1.The morphology of plate colony and Gram staining microscopy of the strain were consistent with the characteristics of Lactobacillus acidophilus.16S r RNA sequencing showed that the strain was Lactobacillus acidophilus GDMCC1.208.2.Trehalose with a concentrations of 0.5%had no significant effect on the proliferation of Lactobacillus acidophilus GDMCC1.208,while trehalose with concentrations of 1.0%,2.0%,3.0%and 5.0%could promote the proliferation of Lactobacillus acidophilus GDMCC1.208 and improve its acid and bile salt tolerance.Among all concentrations,2.0%had the best effect.3.Lactobacillus acidophilus GDMCC1.208 at 1×10⁷CFU/m L and 1×10⁸ CFU/m L could significantly promote the proliferation of Caco-2 cells,with 1×10⁸ CFU/m L having a better effect.4.There were no significant differences in body weight,cardio-body ratio and hepato-body ratio of Apo E^-/-mice in each group.5.Compared with WT mice,the aorta of Apo E^-/-mice in the experimental control group showed obvious atherosclerotic plaques.The combined intervention of trehalose and Lactobacillus acidophilus GDMCC1.208 could significantly reduce the area of atherosclerotic lesions,and the effect was better than that of Lactobacillus acidophilus GDMCC1.208 alone.6.There was no significant difference in the structural state of small intestine and colon among all groups.7.After the combined intervention of trehalose and Lactobacillus acidophilus GDMCC1.208,the levels of TC and LDL-C in Apo E^-/-mice were significantly reduced,while the levels of TC and HDL-C were not significantly changed.8.After the combined intervention of trehalose and Lactobacillus acidophilus GDMCC1.208,the expression levels of CYP7A1,ABCA1 and ABCG1 genes in the liver of Apo E^-/-mice were significantly up-regulated,the expression levels of HMGCR gene were significantly down-regulated.9.After the combined intervention of trehalose and Lactobacillus acidophilus GDMCC1.208,the expression levels of TNF-αand IL-6 genes in the aorta of Apo E^-/-mice were significantly down-regulated.Conclusions1.Trehalose with a concentration of 2.0%can promote the proliferation of Lactobacillus acidophilus GDMCC1.208 and improve its acid and bile salt tolerance.2.Lactobacillus acidophilus GDMCC1.208 can inhibit the atherosclerotic lesions of Apo E^-/-mice aorta.The combined intervention of trehalose and Lactobacillus acidophilus GDMCC1.208 has a better inhibitory effect.3.Combined intervention of trehalose and Lactobacillus acidophilus GDMCC1.208may inhibit atherosclerotic lesions in Apo E^-/-mice by reducing serum cholesterol levels and participating in inflammatory response.