Fluoride is widely present in the natural environment . And long-term fluorine exposure can have certain toxic effects on the organism and cause significant hepatic pathological damage . Currently, a study demonstrated that fluoride exposure could cause liver damage by the mitochondrial apoptosis pathway . AST and ALT are the earliest and most sensitive indicators of the appearance of liver injury . When liver tissue is necrotic or damaged, ALT and AST escape from hepatocytes and enter the bloodstream, significantly increasing serum ALT and AST activity . Therefore, the increase of ALT and AST activity in serum reflects the degree of hepatocellular injury to a certain extent. In our study, we further proved that NaF treatment could induce necrosis and nuclear sequestration in hepatocytes and reduce intracellular organelles and swollen mitochondria in liver tissues of rats. Meanwhile, NaF also could elevate the levels of liver injury-related indicators (ALT and AST) and inflammatory indicators (IL-18, TNF-a) in rat serum. These results suggested that fluorine could induce liver injury in a dose-dependent manner.
Hepatic fibrosis (HF) is a repair response of the liver in response to chronic injury . HF is also an intermediate stage in the progression of chronic liver disease to cirrhosis, which is a key stage in reversing the disease . Late-stage HF may progress to irreversible cirrhosis . And cirrhosis may further cause ascites, splenomegaly, formation of collateral circulation, upper gastrointestinal bleeding, and even death . Our data further verified that fluorine exposure also could accelerate liver fibrosis in rats. Therefore, fluorine exposure can enhance liver injury and induce liver fibrosis.
Fluorosis is mainly associated with oxidative stress, hormonal regulation, and apoptosis . Research showed that signaling pathways and related factors are relevant to fluorosis [42, 43]. To further explore the underlying mechanisms of fluorosis-induced liver injury, we investigated the effects of fluorine on hepatocyte ERS and apoptotic pathways. Apoptosis, a form of programmed cell death, can be induced by different toxic stimuli . The literature reported that excess NaF could cause apoptosis in different cell types, including osteoblasts and human embryonic stem cells [45, 46]. And ERS is one of the key pathways of fluorine-induced apoptosis . ERS acts as a cellular self-protection mechanism and normally has a role in protecting cells from damage. Adverse environments, such as oxidative stress and toxic stimuli, can accumulate unfolded and misfolded proteins in the ER, which can activate the unfolded protein response (UPR) . UPR can maintain the balance of ER quantity and normal function in the body during ERS. Under stress, GRP78 can activate ERS through PERK, ATF6, and IRE1 . While excessive ERS instead can activate ERS-associated apoptotic proteins such as CHOP, it can eventually trigger apoptosis . In vivo study also showed that high fluorine concentrations can induce ERS and apoptosis in osteoblasts . Our study further verified that fluoride exposure could upregulate ERS-and apoptosis-related proteins in liver tissues and AML12 cells. Thus, fluoride exposure could induce ERS and apoptosis in hepatic cells. Meanwhile, we discovered that ERS alleviator (4-PBA) could induce proliferation and inhibit ERS and apoptosis in fluorine-exposed AML12 cells, suggesting that fluorine exposure to hepatocyte ERS is critical.
More importantly, our data showed that fluoride exposure could prominently downregulate Sirt-1 in liver tissues and AML12 cells. Sirt-1 is a deacetylase that can regulate biological metabolism through deacetylation . Besides, Sirt-1 has been reported to play key regulatory roles in physiological processes such as apoptosis, differentiation, oxidative stress, senescence, signaling, transcriptional regulation, and metabolic regulation through the regulation of histones, NF-kB, FOXO, and p53 [53, 54]. In recent years, studies confirmed that Sirt-1 is essential in liver-related diseases, such as liver transplantation , liver ischemia/reperfusion injury , liver fibrosis , fatty liver , alcoholic liver injury, and fibrosis . At the same time, the role and mechanism of Sirt-1 in liver injury induced by fluoride exposure are unclear. Our results further indicated that Sirt-1 knockdown could further enhance the induction of ERS and apoptosis mediated by fluorine exposure in AML12 cells.
In our study, we first constructed fluorosis rat and cell models using NaF and clarified the influences of fluorine exposure on liver injury and fibrosis in rats. Besides, we explored the impacts of fluorine exposure on ERS- and apoptosis-related proteins in fluorosis rats and cells. Moreover, we further verified the action of Sirt-1 silencing and ERS alleviator (4-PBA) in fluorine-exposed rat liver tissues in vivo and AML12 cells in vitro. Therefore, the investigation of the protective mechanism of SIRT-1 against fluoride exposure-induced liver injury may provide a laboratory basis for the future clinical mitigation of fluorosis.
We demonstrated that fluorine exposure could induce hepatocyte injury through modulation of ERS and apoptotic pathways. Besides, Sirt-1 knockdown could further enhance the ERS and apoptotic processes in hepatocytes induced by fluoride exposure and enhance the toxic effects of NaF (Figure 6).
The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.
Conflicts of Interest
The authors declare no competing interests.
Yanlong Yu and Ling Li contributed equally to this work.
This work was supported by the Special Funds for the Central Government to Guide Local Science and Technology Development (grant no. QKZYD(2019)4008).
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