Reglementary-dose CHI-induced MetS in mice
We treated ICR and C57BL/6 mice with 0.2 mg/L CHI for 12 weeks (Fig. 1A and Fig. S2A) and used liquid chromatography tandem mass spectrometry (LC-MS/MS) to measure the concentrations of CHI in serum of mice. The results of LC-MS/MS analysis showed that the concentration of CHI in serum of ICR and C57BL/6 mice were 0.0011 ± 0.0002 mg/L and 0.0012 ± 0.0003 mg/L, respectively (Fig. S1B). After 12 weeks of exposure to CHI, the body weights of the 18 week-old ICR mice increased significantly as compared to the Ctrl group. Eventually, the bodyweight of ICR mice also increased significantly from 6 weeks to 18 weeks (Fig. 1B). In addition, the weights of liver tissues and epididymal white adipose tissue (epiWATs)also significantly increased after CHI exposure (Fig. 1C and D). Meanwhile, the ratios of liver weight and epiWAT weight to body weight were significantly changed in the CHI groups as compared to the Ctrl group (Fig. 1C and D). In particular, there were no significant differences in food and water intake between the two treatment groups (Fig. S3A and B), indicating that the effects of CHI on mice, increasing their body weight, were not due to the increased food consumption. Furthermore, CHI significantly increased the contents of triglyceride (TC) and low-density lipoprotein-cholesterol (LDL-C) and significantly decreased that of total cholesterol (TG) in the serum of ICR mice, while that of high-density lipoprotein-cholesterol (HDL-C) was not observed to change in the CHI group (Fig. 1E and F). Similarly, the content of glucose and insulin in the serum of ICR mice increased significantly with the CHI exposure. Importantly, exposure to CHI caused a significant increase in insulin resistance index (HOMA-IR) of ICR mice (Fig. 1G). Subsequently, the intrinsic effects of CHI on the liver tissue and epiWAT of ICR mice were further evaluated. The histopathological analysis showed that the CHI promoted lipids accumulation in liver tissues and caused liver damage (Fig. 1H). Meanwhile, CHI also induced significant increase in the aspartate aminotransferase (ALT) activity in serum and content of TC contents in mice liver (Fig. 1I and 1J). In addition, the CHI also promoted lipids accumulation in the epiWAT tissue of ICR mice (Fig. 1H). The average size of the epiWAT cells markedly increased in the CHI group as compared to Ctrl group (Fig. 1K). Meanwhile, the mRNA expression levels of IL22, TLR4, TNF-α, IL6, MCP1, IL-1β, and IFN-γ, as inflammatory marker genes in liver tissue and epiWAT, were quantified. The results showed that CHI significantly increased the mRNA expression levels of IL22, TLR4, TNF-α, IL6, and MCP1 genes in the liver tissue and epiWAT (Fig. S4). These results also indicated that CHI could cause obesity, hepatic steatosis, dyslipidemia, and insulin resistance in ICR mice. Furthermore, obesity, hepatic steatosis, dyslipidemia, and insulin resistance in C57BL/6 mice after CHI exposure were also observed (Fig. S2, Fig. S3 and Fig. S4). In short, these results implied that the reglementary-dose exposure to CHI could induce MetS in mice.
Reglementary-dose CHI disrupted gut microbiota of mice
The emerging exploration of the association between pesticides and the host’s gut microbiota has completely changed the traditional understanding of pesticide-induced health effects of the host [41, 42]. Several studies have proved the close association between gut microbiota and MetS [45-47]. The imbalance of gut microbiota has been considered an important factor, inducing MetS in the host. Therefore, in this study, the effects of CHI on the gut microbiota of mice were evaluated using 16S rRNA gene sequencing. The principal component analysis (PCA), based on OTUs, showed that the gut microbiota of ICR mice altered with CHI exposure. PC1 and PC2 showed 23% and 19.5% variations, respectively. Differences in the gut microbiota between CHI and Ctrl groups Ewere observed (Fig. 2A). This indicated that CHI exposure induced remarkable changes in the overall structure and composition of gut microbiota in ICR mice. Furthermore, as α-diversity indicators of gut microbiota, the Simpson, Shannon, Chao1, and Observed_species indices did not change significantly in the ICR mice after CHI exposure (Fig. 2B). Subsequently, changes in the compositions of gut microbiota between the CHI and Ctrl groups were analyzed. The results showed that there were significant differences in the relative abundances of gut microbiota in ICR mice between the CHI and Ctrl groups (Fig. 2C and F). At the phylum level, exposure to CHI resulted in a significant increase and decrease in the relative abundances of Firmicutes and Bacteroidetes, respectively (Fig. 2D). Meanwhile, the ratio of Firmicutes to Bacteroidetes in the CHI group also increased significantly (Fig. 2E). At the genus level, the relative abundance of Clostridiales significantly increased, while that of S24-7, Lachnospiraceae, and Bacteroides significantly decreased in the CHI group as compared to the Ctrl group (Fig. 2G). In addition, the OTUs that significantly altered between the CHI and Ctrl groups also showed imbalances in the gut microbiota of ICR mice (Fig.2H). Similarly, CHI also caused significant changes in the gut microbiota of C57BL/6 mice (Fig. S5). The analysis of gut microbiota in C57BL/6 mice showed that CHI altered the relative abundances of Firmicutes and Bacteroidetes and resulted in a significant change in the ratio of Firmicutes to Bacteroidetes at the phylum level (Fig. S5C-E). Moreover, at the genus level, the relative abundances of Clostridiales and S24-7 also changed significantly after CHI exposure (Fig. S5F and G). Collectively, these results demonstrated substantial alterations in the gut microbiota of mice exposed to a low dose of CHI.
Reglementary-dose CHI altered BAs metabolic profiles of mice
The effects of gut microbiota on a host’s health are usually closely related to the metabolic axis of host gut microbiota [32, 48]. The above results showed that, at the genus level, the reglementary-dose CHI exposure caused significant changes in the relative abundances of Clostridiales, S24-7, and Bacteroides. Previous studies have also shown that Clostridiales, S24-7, and Bacteroides were closely related to the host’s BAs metabolism [33, 34]. In order to further explore the role of gut microbiota in reglementary-dose CHI-induced MetS in mice, the contents of 41 BAs in serum were determined using UHPLC-MS/MS (Table S3). The typical chromatograms of 41 BAs are provided in Fig. S6. The lowest limit of detection (LLOD) was 0.24 - 1.95 nmol/L and the lowest limit of quantification (LLOQs) was 0.49 - 3.91 nmol/L. The linear regression coefficients were all above 0.9733. The quantitative results of 41 BAs in the serum samples of each treatment group are listed in Tables S4 and S5. For ICR mice, the PCA analysis showed a significant difference between the CHI and Ctrl groups (Fig. 3A). In addition, as compared to the control group, the total BA contents in the serum of the CHI treatment group significantly increased (Fig. 3B). Additionally, the content of primary and conjugated BAs increased significantly (Fig. 3C and F). In the Ctrl and CHI groups, a total of 18 BAs were detected (Fig. 3G). Among them, exposure to CHI caused significant changes in the content of 11 BAs (Fig. 3H). Specifically, the contents of 5 BAs, including CDCA, taurodeoxycholic acid (TDCA), cholic acid (CA), lithocholic acid (LCA), and deoxycholic acid (DCA), in the serum of CHI group significantly decreased (Fig. 3I). In addition, the contents of 6 BAs, including taurine-α-murocholic acid (T-α-MCA), taurine-β-murocholic acid (T-β-MCA), taurolithocholic acid (TLCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), and ursodeoxycholic acid (UDCA), in the serum of the CHI group significantly increased (Fig. 3I). Consistently, the reglementary-dose CHI exposure also caused significant changes to the BAs metabolic profiles in the serum of C57BL/6 mice (Fig. S7). Similar to the ICR mice, the contents of 3 BAs, including T-α-MCA, T-β-MCA, and UDCA, increased significantly, while that of 5 BAs, including CDCA, TDCA, CA, LCA, and DCA, decreased significantly in the serum of C57BL/6 mice after CHI exposure (Fig. S7I). In short, the reglementary-dose CHI disrupts the BAs metabolism in mice.
Reglementary-dose CHI induced MetS in mice in a microbiota-dependent manner
In order to further explore whether the reglementary-dose CHI exposure-induced MetS in mice was dependent on the presence of gut microbiota, the ICR mice were treated with CHI and a mixture of antibiotics, containing vancomycin, neomycin, metronidazole, gentamicin, and ampicillin (Fig. 4A). Mixed antibiotics could significantly reduce the α-diversity of gut microbiota and the mRNA expression level of 16S in cecal contents of mice (Fig. S9B and C). This indicated that mixed antibiotics could significantly inhibit the gut microbiota of mice. Consistent with the above findings, the CHI exposure induced obesity, hepatic steatosis, dyslipidemia, and insulin resistance in the ICR mice as compared to Ctrl group. However, when the gut microbiota of mice was inhibited by antibiotics, the symptoms of MetS in the CHI-induced ICR mice showed significant improvement. Specifically, the body weight and body weight gain of ICR mice in the CHI+ABX treatment group decreased significantly as compared to the CHI group (Fig. 4B). Consistently, the liver weight, epiWAT weight, and tissue coefficients were significantly reduced after CHI+ABX exposure (Fig. 4C and D). In addition, the contents of TG, TC, and LDL-C in the serum of ICR mice after CHI+ABX exposure also decreased significantly (Fig. 4E and F). As compared to the CHI group, the serum glucose level, insulin level, and HOMA-IR index of the mice in CHI+ABX group were significantly reduced. In addition, the histopathological analysis showed that the ABX treatment also improved the deposition of lipids in the liver tissue and epiWAT of mice (Fig. S8D). Meanwhile, the activities of ALT in serum and the contents of TC in liver were significantly down-regulated after exposure to CHI+ABX (Fig. S8A and B). Similarly, the average size of epiWAT cells in the mice treated with CHI+ABX was significantly reduced (Fig. S8C). Subsequently, analysis of the gut microbiota composition of mice in each groups showed that mixed antibiotics could significantly affect the relative abundances of Firmicutes and Bacteroidetes at the phylum level (Fig. S9D and E), and could significantly improve the CHI-induced elevation of the relative abundance ratio of Firmicutes to Bacteroidetes (Fig. S9F). Furthermore, mixed antibiotics resulted in significant increases in the relative abundances of Bacteroides, Sutterella, Shigella and Eubacterium at the genus level (Fig. S9G and H). Meanwhile, some bacteria such as S24-7, Clostridiales and Lactobacillus were not detected after mixed antibiotics treatment (Fig. S9G and H). In summary, these results indicated that the reglementary-dose CHI induced MetS in mice in a gut microbiota-dependent manner.
Reglementary-dose CHI-induced MetS in mice were transferable through gut microbiota
In order to further illustrate that gut microbiota mediated the occurrence of reglementary-dose CHI-induced MetS in mice, the cecal contents of reglementary-dose CHI-treated mice were transferred to the recipient mice, followed by the examination of MetS-related indicators (Fig. 5A). In order to verify the effectiveness of gut microbiota transplantation, 16S rRNA gene sequencing analysis was performed on the cecal contents of T-Ctrl and T-CHI groups. The PCA analysis showed significant differences between the T-CHI and T-Ctrl groups (Fig. 5B). Consistently, as compared to the T-Ctrl group, the relative abundance of Firmicutes in the T-CHI group significantly increased, while that of Bacteroidetes decreased significantly (Fig. 5C). Meanwhile, the relative abundance ratio of Firmicutes to Bacteroidetes in the T-CHI group also increased significantly (Fig. 5D). This changes of gut microbiota in the T-Ctrl and T-CHI groups were similar to that in the Ctrl and CHI groups, respectively (Fig. S10B andC). This indicated that the gut microbiota transplantation could effectively transplant the microbiota-associated phenotypes from the mice in Ctrl and CHI groups into the recipient mice. Subsequently, the MetS-related indicators in the recipient mice were analyzed. As compared to the T-Ctrl group, the bodyweight and body weight gain of mice in the T-CHI group increased significantly (Fig. 5E and Fig. S10D). Similarly, the weights of liver and epiWAT and its tissue coefficients also increased significantly in the T-CHI group (Fig. 5E and Fig. S10E and F). In addition, the serum-related biochemical indicators in the T-CHI group also were altered significantly. Specifically, the contents of TG and HDL-C significantly decreased, while those of LDL-C significantly increased in the T-CHI group (Fig. 5F). Meanwhile, the serum glucose levels, insulin levels, and HOMA-IR index of the mice in T-CHI group also increased significantly (Fig. 5G). The histopathological analysis showed that, as compared to the T-Ctrl group, the liver tissue and epiWAT of the mice in T-CHI group exhibited obvious lipid deposition (Fig. 5K). In comparison with the T-Ctrl group, the activities of ALT in serum and the contents TG of in liver in the T-CHI group were altered significantly (Fig. 5H and 5I). The size and area of epiWAT cells in the T-CHI group also increased significantly (Fig. 5J and Fig. S10K).
In addition, the metabolic profiles of serum BAs also altered significantly in the T-Ctrl and T-CHI groups (Tables S6 and Fig. S11A). In comparison with the T-Ctrl group, the contents total BAs in T-CHI group did not change significantly (Fig. S11B). However, the contents of secondary and unconjugated BAs were significantly down-regulated, while that of conjugated BAs were significantly up-regulated in the serum of T-CHI group mice (Fig. S11D, E and F). A total of 26 BAs were detected in the T-Ctrl and T-CHI groups (Fig. S11G). As compared to the T-Ctrl group, the contents of 13 BAs in the serum of T-CHI group changed significantly (Fig. S11H). Among them, the contents of TLCA, T-β-MCA, and TCDCA increased significantly (Fig.S11I). Moreover, the contents of 10 BAs, including α-murocholic acid (α-MCA), allocholic acid (ACA), CA, CDCA, 3-dehydrocholic acid (3-DHCA), LCA, DCA, taurodeoxycholic acid (TDCA), 23-deoxycholic acid medeoxycholic acid (23norDCA), and hyodeoxycholic acid (HDCA), decreased significantly (Fig. S11I). Together, these results demonstrated that the recipient mice recapitulated metabolic phenotypes as observed in their respective donor mice. The gut microbiota-mediated MetS and BAs metabolism disorders in mice were induced by reglementary-dose CHI exposure.
Reglementary-dose CHI affected BAs receptors FXR signaling pathway
After exposure to reglementary-dose CHI, the metabolic profile of serum BAs metabolism in mice changed significantly. In particular, it was found that the major BAs, which could regulate the signaling pathway of an important nuclear BAs receptor FXR and included CDCA, CA, DCA, LCA, UDCA, TαMCA, and TβMCA, were all changed significantly [34]. Therefore, we first analyzed changes in the FXR signaling pathway in the intestinal tissue. The results showed that CHI could significantly inhibited the mRNA expression of Fxr and its downstream regulator Fgf15 in the intestine of ICR mice (Fig. S12A). And the content of FGF15 in serum of ICR mice was significantly reduced in CHI treatment group (Fig. S12C). However, CHI exposure did not result in altered mRNA expression of major BAs synthetic enzymes (Cyp7a1, Cyp8a1, Cyp27a1 and Cyp7b1) in the liver tissue of ICR mice (Fig. S12B). For C57BL/6 mice, CHI only resulted in a significant reduction in mRNA expression level of Fxr in the intestinal tissue (Fig. S12D). Similarly, the mRNA expression level of Fxr in the intestinal tissue of mice in the T-CHI treatment group compared with the T-Ctrl group (Fig. S12F). On the other hand, We also explore the mRNA expression levels of Fxr and its main downstream genes, such as Shp, Srebp-1c, and Chrebp, in the liver and epiWAT tissues of ICR mice. The CHI exposure significantly inhibited the relative RNA expression of Fxr (Fig. 6A). In addition, the relative mRNA expression levels of Srebp-1c and Chrebp significantly increased. In particular, the CHI exposure also significantly reduced the relative mRNA expression levels of Shp in liver tissues of ICR mice (Fig. 6A). Similarly, the CHI exposure also significantly inhibited the relative mRNA expression of Fxr gene in liver tissue and epiWAT of C57BL/6 mice. Furthermore, the relative mRNA expression levels of Shp, Srebp-1c, and Chrebp were also significantly changed after CHI exposure in liver tissue and epiWAT of C57BL/6 mice (Fig. 6B). Importantly, the recipient mice after receiving the transplantation of gut microbiota also demonstrated significant changes in the mRNA expression levels of Fxr and other related genes. As compared to the T-Ctrl group, the mRNA relative expression of Fxr gene in the liver tissue and epiWAT of the T-CHI group was significantly down-regulated, while that of the Chrebp gene was significantly up-regulated (Fig. S12H). In addition, in the T-CHI group, the relative mRNA expression of Shp gene in the liver tissue was significantly reduced, while that of the Srebp-1c gene in the epiWAT was significantly increased (Fig. S12H). These results demonstrated that the reglementary-dose CHI exposure could inhibit the Mrna expression level of the BAs receptor Fxr and have a pronounced effects on FXR signaling pathway in liver and epiWAT tissues.
Reglementary-dose CHI induced glucose and lipid metabolism disorders in mice
As an important nuclear BAs receptor, Fxr regulates the expression of various genes, which are related to glucose and lipid metabolism metabolism. Therefore, the mRNA expression levels of genes related to glucose and lipid metabolism metabolism in the liver tissue and epiWAT after CHI exposure were analyzed, which included fatty acid synthesis genes (Fasn, Acaca, Gpat1, Agpat1, Pparγ, Mogat1, Dgat1 and Dgat2), fatty acid uptake and transport genes, (Cd36, Fabp1, Fabp2 and Fatp5), fatty acid oxidation genes (Cpt1α, Acot1, Acox1 and Pparα), lipoprotein secretion genes (Mttp and Apod), glycogen synthesis (Gys2), gluconeogenesis genes (Pepck and G6pase), glycolysis genes (Gck and Pklr), and glucose transport gene (Glut2) (Fig. 6C). For the liver tissue of ICR mice, CHI exposure significantly increased the relative mRNA expression levels of Dgat1 and Fatp5 (Fig. 6D and E). As compared to the control group, the relative mRNA expression levels of Cpt1α and Pparα in the CHI group were significantly down-regulated (Fig. 6F). In addition, CHI exposure also significantly increased the relative mRNA expression levels of G6pase, Gck, and Glut2 (Fig. 6H). For the epiWAT of ICR mice, exposure to CHI significantly increased the relative mRNA expression levels of 6 fatty acid synthesis genes, including Fasn, Acaca, Agpat1, Pparγ, Mogat1, and Dgat1 (Fig. 6 D). Similarly, the relative mRNA expression levels of Cd36 and Fatp5 in the CHI group also significantly increased (Fig. 6E). At the same time, exposure to CHI significantly reduced the relative mRNA expression levels of Cpt1α, Acox1, and Pparα (Fig. 6F). In addition, CHI exposure also significantly increased the relative mRNA expression level of G6pase (Fig. 6H). In particular, CHI exposure could also promote the mRNA expression levels of fatty acid synthesis and uptake-related genes, inhibit the expression of fatty acid oxidation-related genes, and activate the expression of gluconeogenesis -related genes in the liver tissue and epiWAT of C57BL/6 mice (Fig. S13). These results implied that CHI exposure could cause the disorders of glucose and lipid metabolism metabolism in the liver tissue and epiWAT of ICR and C57BL/6 mice. In conclusion, these results confirmed that the reglementary-dose CHI exposure could induce glucose and lipid metabolism metabolism disorders in mice.
FXR activators ameliorated the MetS of mice induced by reglementary-dose CHI
In order to further determine the key role of BAs receptor FXR in the reglementary-dose CHI-induced MetS, the ICR mice were treated with CHI and FXR activators, which included GW4064 and CDCA (Fig. 7A). The analysis of MetS-related indicators in each treatment group showed that both the FXR activators, GW4064 and CDCA, effectively ameliorated the reglementary-dose CHI-induced MetS in mice. In particular, as compared to the CHI group, the body weight and body weight gain of mice in the CHI+GW4064 and CHI+CDCA groups were significantly reduced (Fig. 7B and Fig. S14A). Similarly, in the CHI+GW4064 and CHI+CDCA groups, the weight of liver tissue and epiWAT and its tissue coefficients were also significantly reduced (Fig.7B and Fig. S14A). In addition, the GW4064 and CDCA activators also significantly reduced the serum glucose level, insulin level, and HOMA-IR index in mice (Fig. 7D). Meanwhile, the serum LDL-C contents in the CHI+GW4064 group were significantly reduced as compared to the CHI group (Fig. 7C). Furthermore, the histopathological analysis showed no significant lipid deposition in the liver tissue and epiWAT of the mice in both the CHI+GW4064 and CHI+CDCA treatment groups as compared to the CHI group (Fig. 7G). As compared to the CHI group, the liver ALT activity and TC and TG contents in the CHI+GW4064 and CHI+CDCA groups were significantly reduced (Fig. 7E and Fig. S14E). Similarly, the size and area of epiWAT tissue cells in the CHI+GW4064 and CHI+CDCA groups were also significantly reduced (Fig. 7F and and Fig. S14F). These results indicated that both the FXR activators GW4064 and CDCA could effectively ameliorate the reglementary-dose CHI-induced MetS in mice, such as obesity, hepatic steatosis, dyslipidemia, and insulin resistance.