Blocking reverse electron transfer-mediated mitochondrial DNA oxidation rescues cells from PANoptosis.

PANoptosis is a new type of cell death featured with pyroptosis, apoptosis and necroptosis, and is implicated in organ injury and mortality in various inflammatory diseases, such as sepsis and hemophagocytic lymphohistiocytosis (HLH). Reverse electron transport (RET)-mediated mitochondrial reactive oxygen species (mtROS) has been shown to contribute to pyroptosis and necroptosis. In this study we investigated the roles of mtROS and RET in PANoptosis induced by TGF-ß-activated kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (Oxo) plus lipopolysaccharide (LPS) as well as the effects of anti-RET reagents on PANoptosis. We showed that pretreatment with anti-RET reagents 1-methoxy PMS (MPMS) or dimethyl fumarate (DMF) dose-dependently inhibited PANoptosis in macrophages BMDMs and J774A.1 cells induced by Oxo/LPS treatment assayed by propidium iodide (PI) staining. The three arms of the PANoptosis signaling pathway, namely pyroptosis, apoptosis and necroptosis signaling, as well as the formation of PANoptosomes were all inhibited by MPMS or DMF. We demonstrated that Oxo/LPS treatment induced RET and mtROS in BMDMs, which were reversed by MPMS or DMF pretreatment. Interestingly, the PANoptosome was co-located with mitochondria, in which the mitochondrial DNA was oxidized. MPMS and DMF fully blocked the mtROS production and the formation of PANoptosome induced by Oxo plus LPS treatment. An HLH mouse model was established by poly(I:C)/LPS challenge. Pretreatment with DMF (50 mg·kg-1·d-1, i.g. for 3 days) or MPMS (10 mg·kg-1·d-1, i.p. for 2 days) (DMF i.g. MPMS i.p.) effectively alleviated HLH lesions accompanied by decreased hallmarks of PANoptosis in the liver and kidney. Collectively, RET and mtDNA play crucial roles in PANoptosis induction and anti-RET reagents represent a novel class of PANoptosis inhibitors by blocking oxidation of mtDNA, highlighting their potential application in treating PANoptosis-related inflammatory diseases. PANoptotic stimulation induces reverse electron transport (RET) and reactive oxygen species (ROS) in mitochondia, while 1-methoxy PMS and dimethyl fumarate can inhibit PANoptosis by suppressing RETmediated oxidation of mitochondrial DNA.

Anti-Necroptotic Effects of Itaconate and its Derivatives.

Itaconate is an unsaturated dicarboxylic acid that is derived from the decarboxylation of the Krebs cycle intermediate cis-aconitate and has been shown to exhibit anti-inflammatory and anti-bacterial/viral properties. But the mechanisms underlying itaconate's anti-inflammatory activities are not fully understood. Necroptosis, a lytic form of regulated cell death (RCD), is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) signaling. It has been involved in the pathogenesis of organ injury in many inflammatory diseases. In this study, we aimed to explore whether itaconate and its derivatives can inhibit necroptosis in murine macrophages, a mouse MPC-5 cell line and a human HT-29 cell line in response to different necroptotic activators. Our results showed that itaconate and its derivatives dose-dependently inhibited necroptosis, among which dimethyl itaconate (DMI) was the most effective one. Mechanistically, itaconate and its derivatives inhibited necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling and the oligomerization of MLKL. Furthermore, DMI promoted the nuclear translocation of Nrf2 that is a critical regulator of intracellular redox homeostasis, and reduced the levels of intracellular reactive oxygen species (ROS) and mitochondrial superoxide (mtROS) that were induced by necroptotic activators. Consistently, DMI prevented the loss of mitochondrial membrane potential induced by the necroptotic activators. In addition, DMI mitigated caerulein-induced acute pancreatitis in mice accompanied by reduced activation of the necroptotic signaling in vivo. Collectively, our study demonstrates that itaconate and its derivatives can inhibit necroptosis by suppressing the RIPK1/RIPK3/MLKL signaling, highlighting their potential applications for treating necroptosis-associated diseases.

Dimethyl fumarate inhibits necroptosis and alleviates systemic inflammatory response syndrome by blocking the RIPK1-RIPK3-MLKL axis.

Necroptosis has been implicated in various inflammatory diseases including tumor-necrosis factor-α (TNF-α)-induced systemic inflammatory response syndrome (SIRS). Dimethyl fumarate (DMF), a first-line drug for treating relapsing-remitting multiple sclerosis (RRMS), has been shown to be effective against various inflammatory diseases. However, it is still unclear whether DMF can inhibit necroptosis and confer protection against SIRS. In this study, we found that DMF significantly inhibited necroptotic cell death in macrophages induced by different necroptotic stimulations. Both the autophosphorylation of receptor-interacting serine/threonine kinase 1 (RIPK1) and RIPK3 and the downstream phosphorylation and oligomerization of MLKL were robustly suppressed by DMF. Accompanying the suppression of necroptotic signaling, DMF blocked the mitochondrial reverse electron transport (RET) induced by necroptotic stimulation, which was associated with its electrophilic property. Several well-known anti-RET reagents also markedly inhibited the activation of the RIPK1-RIPK3-MLKL axis accompanied by decreased necrotic cell death, indicating a critical role of RET in necroptotic signaling. DMF and other anti-RET reagents suppressed the ubiquitination of RIPK1 and RIPK3, and they attenuated the formation of necrosome. Moreover, oral administration of DMF significantly alleviated the severity of TNF-α-induced SIRS in mice. Consistent with this, DMF mitigated TNF-α-induced cecal, uterine, and lung damage accompanied by diminished RIPK3-MLKL signaling. Collectively, DMF represents a new necroptosis inhibitor that suppresses the RIPK1-RIPK3-MLKL axis through blocking mitochondrial RET. Our study highlights DMF's potential therapeutic applications for treating SIRS-associated diseases.

Baicalin inhibits necroptosis by decreasing oligomerization of phosphorylated MLKL and mitigates caerulein-induced acute pancreatitis in mice.

Necroptosis is a form of regulated necrosis mainly controlled by receptor-interacting protein kinases 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Necroptosis has important roles in defensing against pathogenic infections, but it is also implicated in various inflammatory diseases including pancreatitis. Baicalin, a flavonoid from Scutellaria baicalensis Georgi, has been shown to possess anti-inflammatory and anti-pyroptosis properties, yet it is unclear whether baicalin can inhibit necroptosis and confer protection against necroptosis-related diseases. Here we reported that baicalin significantly inhibited necroptosis in macrophages induced by lipopolysaccharide plus pan-caspase inhibitor (IDN-6556), or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). Mechanistically, baicalin did not inhibit the phosphorylation of RIPK1, RIPK3 and MLKL, nor membrane translocation of p-MLKL, during necroptotic induction, but instead inhibited p-MLKL oligomerization that is required for executing necroptosis. As intracellular reactive oxygen species (ROS) has been reported to be involved in p-MLKL oligomerization, we assessed the effects of N-acetyl-L-cysteine (NAC), an ROS scavenger, on necroptosis and found that NAC significantly attenuated TSI-induced necroptosis and intracellular ROS production concomitantly with reduced levels of oligomerized p-MLKL, mirroring the effect of baicalin. Indeed, inhibitory effect of baicalin was associated with reduced TSI-induced superoxide (indicating mitochondrial ROS) production and increased mitochondrial membrane potential within cells during necroptosis. Besides, oral administration of baicalin significantly reduced the severity of caerulein-induced acute pancreatitis in mice, an animal model of necroptosis-related disease. Collectively, baicalin can inhibit necroptosis through attenuating p-MLKL oligomerization and confers protection against caerulein-induced pancreatitis in mice.

Dimethyl fumarate ameliorates autoimmune hepatitis in mice by blocking NLRP3 inflammasome activation.

Dimethyl fumarate (DMF) is a fumaric acid derivative clinically approved for the treatment of some inflammatory diseases, but the underlying mechanism for its therapeutic effects remains incompletely understood. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation has critical roles in innate immune responses to various infections and sterile inflammations. In this study, we aimed to explore whether DMF affects auto-immune hepatitis (AIH) in mice induced by concanavalin A (Con A) by modulating NLRP3 inflammasome activation. The results showed that DMF suppressed the activation of NLRP3 inflammasome activation in lipopolysaccharide-primed murine bone marrow-derived macrophages upon ATP or nigericin treatment, as evidenced by reduced cleavage of pro-caspase-1, release of mature interleukin-1ß (IL-1ß) and generation of gasdermin D N-terminal fragment (GSDMD-NT). DMF also greatly reduced ASC speck formation upon the stimulation of nigericin or ATP, indicating its inhibitory effect on NLRP3 inflammasome assembly. Consistent with reduced generation of GSDMD-NT, ATP or nigericin-induced pyroptosis was markedly suppressed by DMF. Moreover, DMF treatment alleviated mitochondrial damage induced by ATP or nigericin. Interestingly, all these effects were reversed by the protein kinase A (PKA) pathway inhibitors (H89 and MDL-12330A). Mechanistically, DMF enhanced PKA signaling and thus increased NLRP3 phosphorylation at PKA-specific sites to attenuate its activation. Importantly, DMF decreased serum levels of inflammatory cytokines and ameliorated liver injury in Con A-induced AIH of mice, concomitant with reduced the generation of caspase-1p10 and GSDMD-NT and alleviating mitochondrial aggregation in the liver. Collectively, DMF displayed anti-inflammatory effects by inhibiting NLRP3 inflammasome activation likely through regulating PKA signaling, highlighting its potential application in treating AIH.

Taraxasterol mitigates Con A-induced hepatitis in mice by suppressing interleukin-2 expression and its signaling in T lymphocytes.

Discovery of anti-inflammatory drugs that can suppress T lymphocyte activation and proliferation by inhibiting TCR/CD3 and IL-2/IL-2R signaling is still needed in clinic, though rapamycin and other related reagents have made great success. Taraxasterol (TAS) is an active ingredient of dandelion, an anti-inflammatory medicinal herb with low in vivo toxicity that has long been used in China. Yet the action mechanism of TAS on lymphocytes remains elusive. The anti-inflammatory effects of TAS were evaluated in C57BL/6 mouse primary lymphocytes stimulated with concanavalin A (Con A) in vitro and in mouse model of Con A-induced acute hepatitis in vivo. Our results showed that TAS significantly suppressed Con A-induced acute hepatitis in a mouse model, reducing the hepatic necrosis areas, the release of aminotransferases, and the production of IL-2 and other inflammatory cytokines. Supporting this, in vitro study also showed that TAS reduced the production of IL-2 and the expression of IL-2 receptor subunit α (CD25) upon the stimulation of Con A, which was likely mediated by suppressing NF-κB activation. The downstream pathways of IL-2/IL-2R signaling, including the activation of PI3K/PDK1/mTOR, STAT3 and STAT5, were also suppressed by TAS. Consistently, Con A-induced T cell proliferation was also inhibited by TAS in vitro. Our data indicate that TAS can suppress both T lymphocyte activation and cell proliferation by down-regulating IL-2 expression and its signaling pathway thereby ameliorating Con A-induced acute hepatitis, highlighting TAS as a potential drug candidate for treating inflammatory diseases including autoimmune hepatitis.

[Health care status of female workers exposed to occupational hazards in Haidian district of Beijing].

OBJECTIVE: To investigate the health care status of female workers exposed to occupational hazards in Haidian district of Beijing and improve the labor protection of female workers. METHODS: A questionnaire provided by National Center for Women and Children's Health of Chinese CDC was used in the survey conducted to collect information about health care status of female workers in 141 factories with occupational hazards including chemical poisons and physical factors (noise, libration, microwave, high frequency and low temperature). RESULTS: 141 factories were investigated, including 53 state-owned enterprises, 21 collective enterprises, 46 joint-stock enterprises, and 21 non-public enterprises. 12 251 female workers were surveyed, 10.19% (1249/12 251) of whom were exposed to occupational hazards. Of 141 factories studied, 16.31% (23/141) had no labor protection management organization.27.66% (39/141) did not provide pre-employment physical examination service to female workers.48.94% (69/141) didn't establish labor protection system for female workers in menstrual period. While, 21.28% (30/141) of the studied institutes deducted some salaries in the pregnancy, and 32.62% (46/141) deducted their wages during the puerperal period. 2.13% (3/141) arranged female workers in the posts which are forbidden by law (continuous heavy work load operation).9.93% (14/141) arranged pregnant female workers on the post forbidden by law.31.91% (45/141) and 33.33% (47/141) would deduct the time of prenatal medical examination and lactation from their working hours, respectively.39.01% (55/141) didn't afford the cost of fertility. 68.09% (96/141) had annual gynecological examination.45 factories were collected occupational examination reports, accounted for 31.91% (45/141). No female workers were found suffering from occupational disease. Of the 1865 occupational hazard factor monitoring points in 34 factories, there were 155 monitoring points, which were all noise monitoring points, did not meet the standard. CONCLUSION: The current health-care status of female workers is not optimistic. It is necessary to consistently improve health care legislations, establish coordinated management mechanism and strengthen the publicity of policy to protect female workers.