TNF-α derived from arsenite-induced microglia activation mediated neuronal necroptosis.

Arsenic, an identified environmental toxicant, poses threats to the health of human beings through contaminated water and food. Recently, increasing reports focused on arsenic-induced nerve damage, however, the underlying mechanism remains elusive. Microglia are important immune cells in the nervous system, which produce a large number of inflammatory factors including TNF-α when activated. Recent reports indicated that TNF-α is involved in the process of necroptosis, a new type of programmed cell death discovered recently. Although there were evidences suggested that arsenic could induce both microglia activation and TNF-α production in the nervous system, the mechanism of arsenic-induced neurotoxicity due to microglia activation is rarely studied. In addition, the role of microglia-derived TNF-α in response to arsenic exposure in necroptosis has not been documented before. In this study, we found that arsenite induced microglial activation through p38 MAPK signaling pathway, leading to the production of TNF-α. Microglia-derived TNF-α further induced necroptosis in the neuronal cells. Our findings suggested that necroptosis induced by microglia-derived TNF-α upon arsenite exposure partially played a role in arsenic-induced cell death which underlie the fundamental event of arsenic-related neurotoxicity.

Resveratrol Exerts Anti-Osteoarthritic Effect by Inhibiting TLR4/NF-κB Signaling Pathway via the TLR4/Akt/FoxO1 Axis in IL-1ß-Stimulated SW1353 Cells.

PURPOSE: Osteoarthritis (OA) is associated with chronic low-grade inflammation. Resveratrol exerts protective effects on OA through its anti-inflammatory property; however, the mechanism of resveratrol on anti-inflammatory signaling pathways has not been fully elucidated yet. The aim of the present study was to investigate whether resveratrol-mediated PI3K/Akt expression is linked to TLR4/NF-κB pathway and the role of TLR4/Akt/FoxO1 axis in the anti-osteoarthritic effect of resveratrol. METHODS: SW1353 cells stimulated by IL-1ß (10 ng/mL) were cultured in the presence or absence of resveratrol (50 µM) and then treated with TLR4 siRNA, PI3K inhibitor LY294002 or FoxO1 siRNA, respectively. The associated proteins of TLR4 signaling pathways and TLR4/Akt/FoxO1 axis were evaluated by Western blot. The level of IL-6 in the supernatant was detected by ELISA. RESULTS: IL-1ß treatment increased the expression of TLR4/NF-κB and phosphorylation of PI3K/Akt and FoxO1, while additional resveratrol further upregulated the expression of PI3K/Akt and FoxO1 phosphorylation but downregulated TLR4 signals in SW1353 cells. Further analyses by the inhibition of TLR4, PI3K/Akt and FoxO1 signaling pathways, respectively, showed that the activation of TLR4 can induce PI3K/Akt phosphorylation, which increases the phosphorylation of FoxO1 and inactivates it. Next, inactivated-FoxO1 can reduce the expression of TLR4, which forms a self-limiting mechanism of inflammation. Resveratrol treatment can upregulate PI3K/Akt phosphorylation and inactivate FoxO1, thereby reducing TLR4 and inflammation. CONCLUSION: This study reveals that TLR4/Akt/FoxO1 inflammatory self-limiting mechanism may exist in IL-1ß-stimulated SW1353 cells. This study reveals a novel cross-talk mechanism which is between integrated PI3K/Akt/FoxO1 signaling network and TLR4-driven innate responses in IL-1ß-stimulated SW1353 cells. Resveratrol may exert anti-OA effect by enhancing the self-limiting mechanism of inflammation through TLR4/Akt/FoxO1 axis.

Calreticulin regulated intrinsic apoptosis through mitochondria-dependent and independent pathways mediated by ER stress in arsenite exposed HT-22 cells.

Arsenic is a naturally occurring environmental toxicant. Chronic exposure to arsenic is linked with neurological damage. Although the mechanisms remain to be elucidated, it is currently believed that neural cell apoptosis is one of the underlying mechanisms of arsenic-induced neurotoxicity. Calreticulin (CRT) is a quality control chaperone located in the lumen of the endoplasmic reticulum (ER), which participates in many signaling pathways including apoptosis. However, the role of CRT in apoptosis is controversial. Whether CRT plays a role in arsenite-induced apoptosis and the relationship between CRT and ER stress-mediated apoptosis have not been mentioned before. In this study, we found that CRT expression as well as the cell apoptosis levels increased in a dose dependent manner upon arsenite exposure in HT-22 cells, a mouse hippocampal neural cell line. In addition, arsenite exposure resulted in the up-regulation of ER stress indicator GRP78 and ER stress-related proteins including p-PERK, ATF4, CHOP, calpain2 and cleaved caspases-12, accompanied by the down-regulation of Bcl-2 and up-regulation of Bax and cleaved caspase-3. Silence of CRT remarkably alleviated arsenite-induced apoptosis and reversed the expression of the proteins above. Our findings confirmed the role of CRT in the induction of apoptosis upon arsenite exposure and suggested that CRT mediated the intrinsic apoptotic cell death including both mitochondria-dependent (PERK/ATF4/CHOP/Bcl-2) and independent (calpain2/caspases-12) pathways initiated by ER stress, which we believed to be a previously undocumented property of arsenite-induced apoptosis.

Akt Regulated Phosphorylation of GSK-3ß/Cyclin D1, p21 and p27 Contributes to Cell Proliferation Through Cell Cycle Progression From G1 to S/G2M Phase in Low-Dose Arsenite Exposed HaCat Cells.

Arsenic is a toxic environmental contaminant. Long-term exposure to arsenic through drinking water induces human cancers. However, it is as yet uncertain about the mechanisms of arsenic induced carcinogenesis. Although the effects of low-dose arsenicals on proliferation and cell cycle have been revealed by short time exposure, the evidences for long-term exposure were seldom reported. The detailed mechanism has been unclear and supplemented constantly. In the present study, we used normal human keratinocytes (HaCat) to study the effects of long-term, low-dose sodium arsenite (NaAsO2) exposure on cell proliferation with emphasis on the Akt regulated cell cycle signaling pathways. Treatment of NaAsO2 resulted in increased cell proliferation and promotion of cell cycle progression from G1 to S/G2M phase, both of which could be attenuated by MK2206, a highly selective inhibitor of Akt. Along with the increased expression of phospho-Akt (p-Akt, Ser 473), increased expression of p-GSK-3ß (Ser 9), p-p21 (Thr 145), p-p27 (Thr 157) and total cyclin D1, and decreased expression of p-cyclin D1 (Thr 286), p21 and p27 were also found in the NaAsO2 exposed cells. Treatment of MK2206 markedly reversed the expression of all of the above proteins. Our findings indicated that the phosphorylated activation of Akt played a role in the proliferation of HaCat cells upon long-term, low-dose NaAsO2 exposure through the phosphorylative regulation of its downstream cell cycle regulating factors of GSK-3ß/cyclin D1, p21 and p27, which could induce the promotion of cell cycle progression from G1 to S/G2M phase.

Resveratrol inhibits the development of obesity-related osteoarthritis via the TLR4 and PI3K/Akt signaling pathways.

Aim of the study: Obesity leads to mild, chronic inflammation which is a primary risk factor for osteoarthritis (OA). Resveratrol exerts a protective effect on OA through its anti-inflammatory properties, but the precise mechanism remains unknown. The present study aimed to investigate the mechanism by which resveratrol alleviates obesity-related OA, and whether it is linked to the TLR4 and PI3K/Akt signaling pathways. Materials and methods: C57BL/6J male mice were fed a high-fat diet (HFD) with or without resveratrol treatment and knee joints were collected for analysis. In addition, IL-1ß-induced SW1353 cells were used to study in vitro the reciprocal effects of TLR4 and PI3K/Akt pathways. Results: Resveratrol inhibited the development of OA in mice fed a HFD. TLR4 and PI3K/Akt signaling pathways were both activated in the articular cartilage; resveratrol treatment down-regulated TLR4 but up-regulated PI3K/Akt signaling. Further in vitro results showed that the effect of resveratrol alone on activation of PI3K/Akt was attenuated but not abolished by the TLR4 inhibitor CLI-095, and resveratrol failed to reduce TLR4 protein expression in IL-1ß stimulated cells pretreated with the PI3K inhibitor LY294002. Conclusions: Resveratrol may exert an anti-osteoarthritic effect by inhibiting TLR4 via the activation of PI3K/Akt signaling pathways. Resveratrol has potential as a drug for OA prevention.

Oral Administration of Resveratrol Alleviates Osteoarthritis Pathology in C57BL/6J Mice Model Induced by a High-Fat Diet.

Obesity has been associated with osteoarthritis (OA) due to increased mass and metabolic factors which are independent of the biomechanical contribution to joint load. Resveratrol, a natural polyphenolic compound, exerts protective effects on OA through its anti-inflammatory property. However, the mechanism of resveratrol on obesity-related OA is unclear. To investigate the effect and possible mechanism of oral resveratrol on obesity-related OA, we fed C57BL/6J mice with a high-fat diet (HFD) for 16 weeks to establish obesity-related OA model; then two doses (22.5 mg/kg and 45 mg/kg) of resveratrol were given by gavage for additional 12 weeks. Mice with HFD significantly increased body weights compared to the control mice, while resveratrol treatment did not cause obvious weight loss. Histological assessments showed that resveratrol at 45 mg/kg significantly improved OA symptoms. Levels of serum IL-1ß and leptin were decreased by resveratrol treatment and positively correlated with Mankin scores. Moreover, resveratrol significantly inhibited the expression of TLR4 and TRAF6 in cartilage. These results suggest that HFD induced obesity can lead to the occurrence of OA, and resveratrol may alleviate OA pathology by decreasing the levels of systematic inflammation and/or inhibiting TLR4 signaling pathway in cartilage. Thus, resveratrol might be a promising therapeutic treatment for obesity-related OA.