Effect of LBP on body weight in experiment mice
As shown in table 1, body weight of the control group moderately increased throughout the pregnancy, from a mean of 17.9 g before feeding trial to 20.8g at end of HFD, 23.1g at GD7 and 29.3g at GD16. In contrast, the HFD group rapidly increased in body weight from a mean of 17.8 g before feeding trial to 22.4g at end of HFD, 25.1g at GD7 and 30.9g at GD16.The HFD-treated mice weighed significantly more than the controls at end of HFD and GD7. Body weight of mice in the HFD+LBP group was not significantly different as compared with that of HFD group.
Effect of LBP on glucose intolerance in experiment mice
At the end of HFD and GD16, glucose tolerance was examined by oral glucose tolerance test. HFD feeding tended to increase glucose AUC at the end of HFD, but this effect failed to reach statistical significance (Fig.1A and B). At GD16, the HFD group showed significantly higher fasting blood glucose level than control group (10.24±0.29 vs 8.98±0.15). HFD dams exhibited prominent glucose intolerance compared with the control group, as indicated by significantly elevated glucose levels at 30, 60, 90 and 120 min and greater AUC (Fig. 1C and D). LBP treatment was able to largely alleviate glucose levels at 0, 90 and 120 min and glucose AUC in comparison with the HFD group (Fig. 1C and D).
Effect of LBP on plasma lipid levels in experiment mice
Table 2 showed significant increases in the levels of plasma TG, TC and LDL-C and a decrease in the level of plasma HDL-C of HFD group in comparison with the control group. The administration of LBP prevented that increase of TC, TG and LDL-C and decrease of HDL-C in HFD mice. The results indicated that LBP could attenuate the abnormal changes of lipid profiles in GDM.
Histopathological analysis
As shown in Figure 2, liver sections of mice in the control group had a normal structure in hepatic cell with distinct nucleus, preserved cytoplasm and central vein. Livers of HFD treated mice displayed lymphocytic infiltration, massive fatty degeneration and loss of cellular boundaries. Mice in the HFD+ LBP group exhibited marked improvements in fat deposition and inflammatory cell infiltration, suggesting that LBP can prevent the pathomorphological changes of liver histopathology.
Exosomal small RNA transcriptome profiling
Exosomes have been demonstrated to contain several categories of small RNAs, including miRNA, tRNA, rRNA, snRNA, snoRNA, piRNA, Y_RNA, and unannotated RNAs. The percentages of miRNA in the total small RNA isolated from control group, HFD group and HFD+LBP group corresponded to 16.76, 17.83 and 15.77%, respectively (Fig. 3). The clean miRNA reads of each sample were mapped to miRBase 21.0. The results of sequence statistics among the samples are listed in Table 3.
Differentially expressed miRNAs between samples
Using P < 0.05 as threshold cutoff, the differentially expressed miRNAs between groups were analyzed. Compared with the control group, 18 miRNAs were found to be significantly differentially expressed in the HFD group (7 down-regulated, 11 up-regulated). Compared with the HFD group, 16 miRNAs were found to be significantly differentially expressed in the HFD+LBP group (6 down-regulated, 10 up-regulated). As the table 4 shown, 6 common differentially expressed miRNAs (miR-93-3p, miR-188-5p, miR-466k, miR-1188-5p, miR-7001-3p and miR-7115-5p) were identified in the two comparison and LBP treatment can recover the expression levels of the 6 miRNAs to normal level.
Enriched GO and KEGG Pathways analysis
Candidate target genes for the 6 common differentially expressed miRNAs in the two comparison were predicted bioinformatically. GO analysis classified genes by biological process, molecular function and cellular component. In biological process, genes were mainly enriched in protein modification by small protein conjugation or removal, cellular protein catabolic process and ubiquitin-dependent protein catabolic process. In cellular component, genes were mainly enriched in synapse part, postsynapse and synapse. In molecular function, genes were mainly enriched in ubiquitin-like protein transferase activity, ubiquitin-protein transferase activity and GABA receptor activity (Fig. 4).
KEGG pathway analysis suggested that the genes were evidently enriched in phospholipase D signaling pathway, MAPK signaling pathway, foxO signaling pathway, dorso-ventral axis formation, insulin resistance, choline metabolism in cancer, renal cell carcinoma, insulin signaling pathway and cAMP signaling pathway (Fig. 5).
Effect of LBP on proteins associated with insulin resistance in livers
We used western blot to determine the effect of LBP on proteins expression level of the candidate target genes involved in insulin resistance in mice livers. In HFD and HFD+LBP groups, the protein expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) which is the target gene of miR-93-3p was similar to the those of control group (Fig.6A). In HFD group, protein expression of miR-188-5p target gene Carnitine O-palmitoyltransferase 1 (CPT1A) was significantly reduced, compared with that of control group (Fig. 6A). The decrease of protein expression of CPT1A in HFD mice was notably reversed by LBP treatment (Fig. 6B).