Effect of PLRS on biochemical indicators
Effects of PLRS on body weight, visceral fat accumulation, blood glucose and lipid levels, and histopathology in mice were presented as supplementary information.
Effect of PLRS on hepatic impairment
ALT and AST were the two main enzymes in the human liver, which used to evaluate the liver function. TBA is a class of water-soluble compound produced by the catabolism of cholesterol in liver. To explore the effect of PLRS on the lipid metabolism, the levels of ALT, AST, and TBA were measured in HFD-induced mice. As shown in Fig.1. HFD mice had high level of ALT, AST, and TBA compared with the NFD group (P<0.01). Instead, the levels of various indicators decreased after treatment with Orlistat and PLRS, and the down-regulation effect of ALT and AST in the HFD+HP group is better than that of the HFD+Orli group. The above description showed that PLRS could improve HFD cause liver damage by down-regulating the levels of ALT, AST, and TBA.
PLRS determination of intestinal contents by 16S rDNA
The changes of gut microbiota structural was studied by high-throughput Illumina HiSeq sequencing of the V3-V4 region of the 16S rRNA gene. The PCA score plot showed that the microbiota composition of cecum content samples differed significantly between the NFD and HFD groups (Fig.2a). The results of the α-diversity analysis suggested that both HFD+LP and HFD+HP groups significantly increased the diversity index of Simpson and Chao1, however, the HFD+Orli decreased the richness of the Chao1 index (Fig.2b-c). The results suggested that PLRS enhances the diversity on HFD mice. At the phylum level, a total of 10 phyla were identified. Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, unidentified_Bacteria, Actinobacteria, Chloroflexi, Deferribacteres, Acidobacteria, and Cyanobacteria were the major gut microbiota in all groups. In the NFD group, the main gut microbiota was dominated by Bacteroidetes (40.56%), Firmicutes (37.99%), and Proteobacteria (17.11%), however, the proportion in the HFD group changed to Firmicutes (56.18%), Bacteroidetes (33.35%), and Proteobacteria (22.07%), respectively (Fig.2d). After the Orlistat and PLRS treatment, the number of Firmicutes decreased compared with the HFD group. More importantly, the Firmicutes to Bacteroidetes (F/B) ratio as the two main bacterial phyla, which is widely used as a biomarker for predicting obesity (Fig.2e). Compared with the HFD group, the ratio of HFD+Orli, HFD+LP, and HFD+HP were reduced but without statistically significant. In the second stage, the abundance of gut bacteria was analyzed and compared separately at the genus level (Fig.2f). Compare with the NFD group, The HFD reduced the abundance of Ruminiclostridium, Helicobacter, and Alloprevotella in the intestine, while in the HFD group, the content of Staphylococcus was significantly increased relative to the other groups. In PLRS groups, we observed a significant increase in the relative abundance of Akkermansia, Lactobacillus, Blautia, and Dubosiella and a significant decrease in the abundance of Staphylococcus. Interestingly, at the genus level, Orlistat did not increase the abundance of intestinal flora but only decreased the abundance of Alloprevotella and Staphylococcus.
PLRS consumption regulated hepatic lipid metabolism genes and proteins.
HFD leads to lipid deposition in serum and liver, possibly due to differences in the expression levels of genes and proteins involved in the regulation of hepatic lipid metabolism. Based on this, we investigated mRNA and protein expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), sterol regulatory element-binding protein-1c (SREBP-1c), cholesterol 7α-hydroxylase (CYP7A1), and fatty acid synthase (FAS) in the fatty acid metabolic pathway and the PPAR metabolic pathway.
As shown in Fig.3a, the mRNA expression levels of HMGCR, SREBP-1c, and FAS were increased in HFD group as compared with the NFD group, and the increased level of SREBP-1c and FAS were significant (P<0.01). After Orlistat and PLRS gavage treatment, the mRNA expression was down-regulated. The mRNA expression level of CYP7A1 was down-regulated significantly in HFD mice compared with the NFD group (P<0.05). Nevertheless, PLRS supplementation significantly increased the levels of these genes compared with the HFD group. The up-regulation level of the CYP7A1 gene was higher in the HFD+HP group compared with the HFD+Orli group.
As shown in Fig.3b-c, compared with the NFD group, HFD mice showed significantly increased protein expression levels of HMGCR, SREBP-1c, and FAS (P < 0.01), and significantly decreased protein expression of CYP7A1 (P < 0.01); Compared with the HFD group, PLRS groups showed significant differences in regulating the expression levels of other proteins, CYP7A1 especially, which was even better than the NFD group, suggesting that PLRS may have the ability to target CYP7A1 to reduce obesity symptoms.
Correlation analysis of gut microbiota and lipid metabolism
To investigate the underlying interaction of PLRS on HFD-induced intestinal flora and lipid metabolism, the pearson algorithm was further used to assess the association among intestinal flora and serum biochemical indicators and key genes and proteins in the liver, as shown in Fig.4. Staphylococcus is positively correlated with hepatic TBA, levels of AST in the liver were positively correlated with the abundance of Bacteroides and unidentified_Clostridiales, while serum levels of TG were negatively correlated with the abundance of both Staphylococcus and Alloprevotella, the abundance of Alloprevotella and Akkermansia increased with increasing levels of ALT. In addition, the abundance of Akkermansia was highly correlated with both of the gene and protein expression level of SREBP-1c, and that elevated levels of FAS expression were linked with increased abundance of Alloprevotella, Staphylococcus, and Dubosiella. By contrast, CYP7A1 expression was in a negative correlation with the abundance of Akkermansia, unidentified_Lachnospiraceae, and Dubosiella. These results suggest that PLRS can inhibit the disruption of lipid metabolism caused by HFD, possibly due to involvement in pathways related to microbiota that can regulate lipid metabolism and improve intestinal damage by increasing the abundance of these beneficial bacteria in the gut.