3-MCPD Induces Renal Cell Pyroptosis and Inflammation by Inhibiting ESCRT-III-Mediated Cell Repair and Mitophagy.

3-Monochloropropane-1,2-diol (3-MCPD) is a chloropropyl alcohol contaminant mainly from the thermal processing of food and could affect kidneys. Pyroptosis is programmed cell death mediated by inflammasomes and gasdermins, and excessive cellular pyroptosis and inflammation can lead to tissue injury. In the present study, we found that 3-MCPD increased lactate dehydrogenase (LDH) levels in vitro and in vivo, increased the protein expression of NOD-like receptor family pyrin domain containing 3 (NLRP3), N-terminal domain of GSDMD (GSDMD-N), and cleaved caspase-1 and promoted the release of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18), which induced renal cell pyroptosis and inflammation. Mechanistic studies indicated that the addition of N-acetylcysteine (NAC), a ROS scavenger, inhibited NLRP3 activation and attenuated pyroptosis. Furthermore, we revealed that 3-MCPD induced ROS accumulation by inhibiting ESCRT-III-mediated mitophagy. These results were further validated by the overexpression of charged multivesicular body protein 4B (CHMP4B), a key subunit of ESCRT-III, and the addition of the mitophagy activator carbonyl cyanide m-chlorophenylhydrazone (CCCP) and rapamycin (Rapa). Thus, our results showed that 3-MCPD could induce mitochondrial damage and produce ROS. 3-MCPD suppressed mitophagy, leading to the accumulation of damaged mitochondria and ROS, thereby activating NLRP3 and pyroptosis. Meanwhile, 3-MCPD-mediated suppression of ESCRT-III hindered the repair of GSDMD-induced cell membrane rupture, which further caused the occurrence of pyroptosis. Our findings provide new perspectives for studying the mechanisms underlying 3-MCPD-induced renal injury.

Ginsenoside Rb1 alleviates 3-MCPD-induced renal cell pyroptosis by activating mitophagy.

Ginsenoside Rb1 (Gs-Rb1) is among the most significant effective pharmacological components in ginseng. 3-monochloropropane-1,2-diol (3-MCPD), a chloropropanol-like contaminant, is produced in the production of refined oils and thermal processing of food. Pyroptosis is a type of programmed cell death triggered by inflammasomes. Excessive pyroptosis causes kidney injury and inflammation. Previous studies have revealed that 3-MCPD induced pyroptosis in mice and NRK-52E cells. In the present study, we find that Gs-Rb1 attenuates 3-MCPD-induced renal cell pyroptosis by assaying GSDMD-N, caspase-1, IL-18, and IL-1ß in mice and NRK-52E cells. In further mechanistic studies, we show that Gs-Rb1 removes damaged mitochondria via mitophagy and reduces intracellular reactive oxygen species (ROS) generation, therefore alleviating 3-MCPD-induced NOD-like receptor family pyrin domain containing 3 (NLRP3) activation and pyroptosis. The above results are further validated by the addition of autophagy inhibitor Chloroquine (CQ) and mitophagy inhibitor Cyclosporin A (CsA). Afterward, we explore how Gs-Rb1 activated mitophagy in vitro. We determine that Gs-Rb1 enhances the protein expression and nuclear translocation of Transcription factor EB (TFEB). However, silencing of the TFEB gene by small interfering RNA technology reverses the role of Gs-Rb1 in activating mitophagy. Therefore, we conclude that 3-MCPD damages mitochondria and leads to ROS accumulation, which causes NLRP3 activation and pyroptosis in ICR mice and NRK-52E cells, while Gs-Rb1 mitigates this phenomenon via the TFEB-mitophagy pathway. Our findings may provide new insights for understanding the molecular mechanisms by which Gs-Rb1 mitigates renal injury.

Apigenin Alleviated High-Fat-Diet-Induced Hepatic Pyroptosis by Mitophagy-ROS-CTSB-NLRP3 Pathway in Mice and AML12 Cells.

Apigenin is considered the most-known natural flavonoid and is abundant in a wide variety of fruits and vegetables. A high fat diet (HFD) can induce liver injury and hepatocyte death in multiple ways. Pyroptosis is an innovative type of programmed cell death. Moreover, excessive pyroptosis of hepatocytes leads to liver injury. We used HFD to induce liver cell pyroptosis in C57BL/6J mice in this work. After gavage of apigenin, apigenin can significantly reduce the level of lactate dehydrogenase (LDH) in liver tissue ignited by HFD and reduce the levels of NLRP3 (NOD-like receptor family pyrin domain containing 3), the N-terminal domain of GSDMD (GSDMD-N), cleaved-caspase 1, cathepsin B (CTSB), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) protein expression and the colocalization of NLRP3 and CTSB and increase the level of lysosomal associated membrane protein-1 (LAMP-1) protein expression, thus alleviating cell pyroptosis. In a further in vitro mechanism study, we find that palmitic acid (PA) can induce pyroptosis in AML12 cells. After adding apigenin, apigenin can clear the damaged mitochondria through mitophagy and reduce the generation of intracellular reactive oxygen species (ROS), thus alleviating CTSB release caused by lysosomal membrane permeabilization (LMP), reducing the LDH release caused by PA and reducing the levels of NLRP3, GSDMD-N, cleaved-caspase 1, CTSB, IL-1ß, and IL-18 protein expression. By adding the mitophagy inhibitor cyclosporin A (CsA), LC3-siRNA, the CTSB inhibitor CA-074 methyl ester (CA-074 Me), and the NLRP3 inhibitor MCC950, the aforementioned results were further confirmed. Therefore, our results show that HFD-fed and PA can damage mitochondria, promote the production of intracellular ROS, enhance the lysosomal membrane permeabilization (LMP), and cause the leakage of CTSB, thus activating the NLRP3 inflammatory body and inducing pyroptosis in C57BL/6J mice and AML12 cells, while apigenin alleviates this phenomenon through the mitophagy-ROS-CTSB-NLRP3 pathway.

Ginsenoside Rb1 induces autophagic lipid degradation via miR-128 targeting TFEB.

In recent years, the effect of lipid metabolism on health has attracted more and more attention. Ginseng is a traditional Chinese herbal medicine in China and is widely used as food in Asia. Ginsenoside Rb1 (Gs-Rb1) is the most abundant ingredient in ginsenoside, which has a variety of biological activities. In this study, we found that Gs-Rb1 can reduce lipid accumulation in mice and HepG2 cells induced by a high-fat diet (HFD) and palmitic acid (PA). At the same time, we also found that Gs-Rb1 could stimulate the autophagic flux of HFD-fed mice and PA-treated HepG2 cells, and it is further verified by adding the autophagy activator rapamycin (Rapa) and autophagy inhibitor chloroquine (CQ). Furthermore, we found that Gs-Rb1 promoted the nucleus translocation of the transcription factor EB (TFEB) and the target role of miR-128, thus stimulating autophagic flux. Therefore, our results showed that Gs-Rb1 enhanced the transcription of TFEB and its downstream lysosome-related genes by inhibiting miR-128, improved the degradation ability of lysosomes to autophagosomes, and then promoted autophagic lipid degradation.

1,3-Dichloro-2-propanol induced ferroptosis through Nrf2/ARE signaling pathway in hepatocytes.

1,3-Dichloro-2-propanol (1,3-DCP) is a representative chloropropane environmental contaminant with multiple toxicities. Ferroptosis is a novel iron-dependent form of regulated cell death that is closely associated with the accumulation of lipid peroxides, Fe2+ and reactive oxygen species (ROS). In this study, we found that 1,3-DCP could induce mouse liver injury via ferroptosis. Administrating of C57BL/6J mice with 12.5, 25, and 50 mg/kg 1,3-DCP for 4 weeks via oral gavage, the data showed that 1,3-DCP exposure led to the pathological changes in mouse livers, remarkably induced accumulation of malondialdehyde (MDA) and Iron, reduction of glutathione (GSH), and changed in the expression of ferroptosis marker proteins glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase-4 (ACSL4). Then, we also proved the results with HepG2 cells in vitro. The data showed that treatment 1,3-DCP significantly triggered the ferroptosis in vitro. Furthermore, we found that the ferroptosis-related signal pathways were significantly activated in mice livers and HepG2 cells in response to 1,3-DCP exposure. The data showed that 1,3-DCP induced ferroptosis by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into nuclear and thereby suppressing the expression of its downstream target proteins including GPX4, ferritin heavy chain (FTH), ferroportin (FPN), cystine/glutamate transporter xCT (SLC7A11), and heme oxygenase 1 (HO-1). Taken together, our findings confirmed that 1,3-DCP induced ferroptosis via the Nrf2/ARE signaling pathway in hepatocytes. Our works provide new toxicity mechanisms of 1,3-DCP with ferroptosis on hepatocytes injury.

Apigenin alleviated PA-induced pyroptosis by activating autophagy in hepatocytes.

Apigenin is a kind of natural flavonoid that abundantly exists in fruits and vegetables. Pyroptosis is a new, pro-inflammatory type of programmed necrosis cell death, and the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is the key molecule to induce pyroptosis. Excessive hepatic pyroptosis results in liver injury. In the study, we found for the first time that apigenin could alleviate palmitic acid (PA)-induced NLRP3 inflammasome activation and pyroptosis in HepG2 cells and primary mouse hepatic cells. Meanwhile, apigenin could promote the autophagy of hepatocytes. When the autophagy inhibitor chloroquine (CQ) was added, the data showed that the recovery effect of apigenin on PA-induced pyroptosis was weakened, indicating that apigenin could alleviate PA-induced pyroptosis by activating autophagy. Further mechanistic studies showed that apigenin regulated the NLRP3 inflammasome through two ways, so as to alleviate PA-induced pyroptosis. On the one hand, apigenin eliminated damaged mitochondria by activating autophagy, thereby clearing reactive oxygen species (ROS) production and inhibiting the activation of the NLRP3 inflammasome, and on the other hand, activation of autophagy could directly degrade the NLRP3 inflammasome. Our study provides a new idea and target for the use of functional factors in food to alleviate liver injury.

Apigenin induced autophagy and stimulated autophagic lipid degradation.

Apigenin, as a natural flavonoid, has been proved to have many biological effects. Our previous research has found the antiadipogenic effects of apigenin on HepG2 cells. Autophagy is intimately associated with the metabolism of lipid droplets (LDs) and is considered to be one of the lipid breakdown pathways. However, there is no study to elucidate the lipid-lowering mechanism of apigenin from the perspective of autophagy. Here, we investigated the possible role of apigenin in autophagy and lipid accumulation in palmitic acid (PA)-induced HepG2 cells. Our results showed that apigenin increased autophagosome formation and the LC3-II/I ratio, but decreased the p-mTOR/mTOR ratio and P62 protein expression. The effects of apigenin were blocked by chloroquine (CQ). Likewise, apigenin significantly stimulated autophagic flux in the cytoplasm. This effect also could be blocked by CQ. Moreover, apigenin decreased the lipid content and co-localization of LDs with LC3, and CQ could block these effects. Thus, we proposed that apigenin induced autophagy and stimulated autophagic lipid degradation in PA-treated HepG2 cells.

Apigenin reduces the excessive accumulation of lipids induced by palmitic acid via the AMPK signaling pathway in HepG2 cells.

In recent years, increasing attention has been paid to diseases caused by excessive accumulation of lipids in the liver with therapeutic agents derived from natural products offering an alternative treatment to conventional therapies. Among these therapeutic agents, apigenin, a natural flavonoid, has been proven to exert various beneficial biological effects. In the present study, the antiadipogenic effects of apigenin in HepG2 cells was investigated. It was demonstrated that the treatment of cells with different concentrations of apigenin for 24 h significantly decreased the palmitic acid-induced increases in total cholesterol (TC) and triglyceride (TG) levels as well as intracellular lipid accumulation. In addition, apigenin increased the phosphorylated-AMP-activated protein kinase (AMPK) levels but decreased the expression levels of 3-hydroxy-3-methylglutaryl CoA reductase, sterol regulatory element-binding protein (SREBP)-1, fatty acid synthase, and SREBP-2 in a concentration-dependent manner. The present findings suggested that apigenin might improve lipid metabolism by activating the AMPK/SREBP pathway to reduce lipid accumulation in the liver.

1,3-Dichloro-2-Propanol inhibits autophagy via P53/AMPK/mTOR pathway in HepG2 cells.

As a common food processing pollutant, 1,3-Dichloro-2-propanol can be found in foodstuffs, especially in soup spices and instant soups. Mounting researchers have unfolded the relation between 1,3-DCP and various diseases. Autophagy is a process of self-regulation and defects in autophagy have been bound up with multifarious human pathologies and metabolic diseases. Here, we explored the effects of 1,3-DCP on autophagy and traced the molecular mechanism. Our results demonstrated that 1,3-DCP dose-dependently inhibited autophagy. Meanwhile, inhibition of autophagy was accompanied by reduced P53 and p-AMPK/AMPK expressions and stimulated p-mTOR/mTOR expression. Use of a specific mTOR inhibitor (rapamycin), a reversible AMPK activator(A-769662) and a selective P53 activator (Nutlin-3a) abolished the ability of 1,3-DCP to inhibit the induction of autophagy. These evidences suggested that P53/AMPK/mTOR signalling pathway played an important role in the regulation of 1,3-DCP-inhibited autophagy. Together, our results revealed new insights into the toxicity mechanism of 1,3-DCP, supplying theoretical and scientific basis for food safety.

Alliin attenuates 1, 3-dichloro-2-propanol-induced lipogenesis in HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway.

Accumulating evidence reveals the association of 1, 3-dichloro-2-propanol (1, 3-DCP) exposure and lipogenesis. Alliin, the most abundant sulphur compound in garlic, has been demonstrated to exhibit hypoglycemic, antioxidant and anti-inflammatory activities. Here, we showed that alliin attenuated lipogenesis induced by 1,3-DCP and that the reduction was due to activation of the AMPK pathway. HepG2 cells exposed to 1,3-DCP exhibited significant increase of triglyceride(TG) and total cholesterol(TC), and alliin reduced the accumulation. Most importantly, alliin could up-regulate the phosphorylation of AMPK and down-regulate protein and gene expressions of SREBP-1; FAS; SREBP-2;HMGCR in 1,3-DCP-induced HepG2 cells. The results demonstrated that alliin was effective on attenuating 1,3-DCP-induced lipogenesis via activation of the AMPK-SREBPs signaling pathway in HepG2 cells.

Protective effects of allicin on 1,3-DCP-induced lipid metabolism disorder in HepG2 cells.

Allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester), with quite a good range of hepatoprotective and antineoplastic properties, is a functional substance from garlic (Allium sativum L.) The purpose of this study was to provide evidence that allicin could protect 1,3-DCP-induced lipid metabolism disorder in HepG2 cells. Allicin reduced the accumulation of triglycerides (TG) and total cholesterol (TC) in 1,3-DCP-induced HepG2 cells. Allicin significantly increased the phosphorylation of AMP-activated protein kinase (AMPK) and down-regulated the levels of sterol regulatory element binding protein-1 (SREBP-1) and sterol regulatory element binding protein-2 (SREBP-2) in 1,3-DCP-induced HepG2 cells. Additionally, allicin had obvious recovery influence on the phosphorylation level of PKA and CREB in 1,3-DCP-induced HepG2 cells. These observations indicated that allicin alleviated lipid metabolism disorder induced by 1,3-DCP in HepG2 cells by regulating AMPK-SREBPs and PKA-CREB signaling pathways.