Nod-like receptor protein 3 inflammasome-mediated pyroptosis contributes to chronic NaAsO2 exposure-induced fibrotic changes and dysfunction in the liver of SD rats.

The metalloid arsenic, known for its toxic properties, is widespread presence in the environment. Our previous research has confirmed that prolonged exposure to arsenic can lead to liver fibrosis injury in rats, while the precise pathogenic mechanism still requires further investigation. In the past few years, the Nod-like receptor protein 3 (NLRP3) inflammasome has been found to play a pivotal role in the occurrence and development of liver injury. In this study, we administered varying doses of sodium arsenite (NaAsO2) and 10 mg/kg.bw MCC950 (a particular tiny molecular inhibitor targeting NLRP3) to Sprague-Dawley (SD) rats for 36 weeks to explore the involvement of NLRP3 inflammasome in NaAsO2-induced liver injury. The findings suggested that prolonged exposure to NaAsO2 resulted in pyroptosis in liver tissue of SD rats, accompanied by the fibrotic injury, extracellular matrix (ECM) deposition and liver dysfunction. Moreover, long-term NaAsO2 exposure activated NLRP3 inflammasome, leading to the release of pro-inflammatory cytokines in liver tissue. After treatment with MCC950, the induction of NLRP3-mediated pyroptosis and release of pro-inflammatory cytokines were significantly attenuated, leading to a decrease in the severity of liver fibrosis and an improvement in liver function. To summarize, those results clearly indicate that hepatic fibrosis and liver dysfunction induced by NaAsO2 occur through the activation of NLRP3 inflammasome-mediated pyroptosis, shedding new light on the potential mechanisms underlying arsenic-induced liver damage.

Calcitriol Inhibits NaAsO2 Triggered Hepatic Stellate Cells Activation and Extracellular Matrix Oversecretion by Activating Nrf2 Signaling Pathway Through Vitamin D Receptor.

Previous studies, including our own, have demonstrated that arsenic exposure can induce liver fibrosis, while the underlying mechanism remains unclear and there is currently no effective pharmacological intervention available. Recent research has demonstrated that vitamin D supplementation can ameliorate liver fibrosis caused by various etiologies, potentially through modulation of the Nrf2 signaling pathways. However, it remains unclear whether vitamin D intervention can mitigate arsenic-caused liver fibrosis. As is known hepatic stellate cells (HSCs) activation and extracellular matrix (ECM) deposition are pivotal in the pathogenesis of liver fibrosis. In this study, we investigated the intervention effect of calcitriol (a form of active vitamin D) on arsenite-triggered Lx-2 cells (a human hepatic stellate cell line) activation and ECM oversecretion. Additionally, we also elucidated the role and mechanism of Nrf2 antioxidant signaling pathway. Our results demonstrated that calcitriol intervention significantly inhibits Lx-2 cell activation and ECM oversecretion induced by arsenite exposure. Additionally, calcitriol activates Nrf2 and its downstream antioxidant enzyme expression in Lx-2 cells, thereby reducing ROS overproduction caused by arsenite exposure. Further investigation reveals that calcitriol activates the Nrf2 signaling pathway and inhibits arsenite-triggered Lx-2 cell activation and ECM oversecretion by targeting vitamin D receptor (VDR). In conclusion, this study has demonstrated that vitamin D intervention can effectively inhibit HSC activation and ECM oversecretion triggered by arsenite exposure through its antioxidant activity. This provides a novel strategy for targeted nutritional intervention in the treatment of arsenic-induced liver fibrosis.

Hyperphosphorylation of EGFR/ERK signaling facilitates long-term arsenite-induced hepatocytes epithelial-mesenchymal transition and liver fibrosis in sprague-dawley rats.

Arsenic is a well known environmental hazardous material, chronic arsenic exposure results in different types of liver damage. Among them, liver fibrosis has become a research hotspot because of its reversibility, while the underlying mechanism is still unclear. Previous studies revealed that EGFR/ERK signaling appears to play an important role in fibrosis diseases. In this study, sprague-dawley rats were exposed to different doses of arsenite for 36 weeks to investigate the roles of EGFR/ERK signaling on arsenite-induced liver fibrogenesis. Our results showed that long-term arsenite exposure induced liver fibrosis, accompanied by hepatic stellate cells (HSCs) activation, excessive serum secretion of extracellular matrix (ECM), and hepatocytes epithelial-mesenchymal transformation (EMT). In addition, arsenite exposure caused hyperphosphorylation of EGFR/ERK signaling in liver tissue of rats, indicating that EGFR/ERK signaling may be involved in arsenite-induced liver fibrosis. Indeed, erlotinib (a specific phosphorylation inhibitor of EGFR) intervention significantly decreased arsenite induced hyperphosphorylation of EGFR/ERK signaling, thereby suppressed hepatocytes EMT process and alleviated liver fibrogenesis in arsenite exposed rats. In summary, the present study provides evidences showing that hyperphosphorylation of EGFR/ERK signaling facilitates long-term arsenite-induced hepatocytes EMT and liver fibrosis in rats, which brings new insights into the pathogenesis of arsenic-induced liver injury.

[Protective effect of active vitamin D on liver fibrosis induced by sodium arsenite in SD rats].

OBJECTIVE: To explore the protective effect of active vitamin D(VD) on liver fibrosis injury induced by sodium arsenite(NaAsO_2) in SD rats. METHODS: Eighteen healthy newly weaned SD rats, half male and half female, were randomly divided into Control group(gavaged with 10 mL/kg normal saline), NaAsO_2-treated group(gavaged with 10 mg/kg NaAsO_2), Active VD(calcitriol) intervention group(gavaged with 10 mg/kg NaAsO_2 and 1.0 µg/kg calcitriol was given by gavage along with NaAsO_2 administration after 12 weeks), all rats were administered 6 days a week for 36 weeks and weighed every week. Enzyme-linked immunosorbent(ELISA) was used to detect the secretion levels of 25(OH)D_3 and hyaluronic acid(HA), laminin(LN), type Ⅲ pre-collagen amino-terminal peptide(PⅢNP), type Ⅳ collagen(COL-Ⅳ) in the serum of rats in each group; HE staining was used to observe the basic pathological changes of liver tissues in each group, Masson and Sirius Red staining were used to observe the fibrosis and collagen deposition of liver tissues in each group; Western Blot was used to detected the protein levels of fibrosis-related markers α-smooth actin(α-SMA), transforming growth factor-ß1(TGF-ß1) and Vimentin in each group. RESULTS: After 36 weeks of NaAsO_2 exposure, the weight of rats was significantly decreased compared with the control group, and the weight of female rats after calcitriol intervention was significantly increased compared with NaAsO_2-treated group(P<0.05). The result of liver coefficient showed increasing in NaAsO_2-treated group compared with the control group, while decreasing in calcitriol intervention group compared with NaAsO_2-treated group, and the difference was statistically significant in female rats. ELISA assay showed that compared with the control group((550.21±29.16) ng/L), the serum level of 25(OH)D_3 in NaAsO_2-treated group((436.82±74.37) ng/L) was significantly decreased(P<0.05), while the serum level of 25(OH)D_3 was significantly higher in calcitriol intervention group than that of NaAsO_2-treated group(P<0.05). HE staining found that, compared with the control group, the liver tissue of rats in NaAsO_2-treated group showed abnormal morphology, the liver tissue was structurally disordered, false lobules and fat vacuoles were also increased. Masson and Sirius Red staining also revealed abnormal hepatic lobule structure, enlarged and deformed portal area and abundant collagen fiber deposition in NaAsO_2-treated group. Further analysis showed that the positive staining area of collagen deposition in liver tissue of rats exposed to NaAsO_2 increased significantly compared with the control group(P<0.05). Those above changes in calcitriol intervention group were significantly alleviated compared with NaAsO_2-treated group(P<0.05). Western Blot analysis showed that the protein levels of α-SMA, TGF-ß1 and Vimentin were obviously higher in NaAsO_2-treated group(1.12±0.21, 1.12±0.26, 1.31±0.15) than that in the control group(0.57±0.10, 0.64±0.13, 0.72±0.16)(P<0.05). In addition, the serum levels of HA, LN, PⅢNP and COL-Ⅳ in rats exposed to NaAsO_(2 )((87.92±9.67), (89.04±11.91), (12.09±2.97) and(19.86±3.40)ng/mL) were also higher than those in control group. After calcitriol intervention, the protein levels of α-SMA, TGF-ß1 and Vimentin(0.68±0.16, 0.85±0.21, 0.84±0.09) in liver tissue and the serum levels of HA, LN, PⅢNP and COL-Ⅳ((54.29±7.23), (55.56±9.43), (6.49±1.08), (10.15±1.99) ng/mL) were significantly lower than those of NaAsO_2-treated group(P<0.05). CONCLUSION: Calcitriol can effectively alleviate liver fibrosis injury caused by long-term NaAsO_2 exposure in SD rats.

Hypermethylation of Mig-6 gene promoter region inactivates its function, leading to EGFR/ERK signaling hyperphosphorylation, and is involved in arsenite-induced hepatic stellate cells activation and extracellular matrix deposition.

Arsenic is a widespread naturally contaminant. Previous studies have highlighted the issue of liver fibrosis induced by arsenic exposure, while the exact mechanisms are not yet fully understood. Recent studies suggest that Mig-6/EGFR/ERK signaling appear to play important roles in fibrosis caused by various factors. In this study, we focused on the epigenetic modification combined with the signaling dysregulation to validate the role of Mig-6 in regulating EGFR/ERK signaling in arsenite-induced human hepatic stellate cells (HSCs) activation. Our results revealed that arsenite exposure induced HSCs activation and extracellular matrix (ECM) deposition. The EGFR/ERK signaling was significantly hyperphosphorylated in arsenite-exposed HSCs, and Mig-6 inactivation was involved in arsenite induced hyperphosphorylation of EGFR and activation of HSCs. Additionally, we further illustrated that hypermethylation of Mig-6 gene promoter region was responsible for the downregulation of Mig-6 induced by arsenite exposure. Moreover, 5-Aza-dC (a DNA methyltransferase inhibitor) can efficiently rescue hypermethylation of Mig-6 gene, decrease the hyperphosphorylation of EGFR/ERK signaling, then reverse arsenite induced HSCs activation. Taken together, the present study strongly suggests that inactivating of Mig-6 function by hypermethylation of its promoter region leading to hyperphosphorylation of EGFR/ERK signaling, and is involved in arsenite-induced HSCs activation and ECM deposition.