Lactate dehydrogenase A mediated histone lactylation induced the pyroptosis through targeting HMGB1.

Cerebral ischemia (CI), as the cerebrovascular disease with the highest incidence rate, is treated by limited intravenous thrombolysis and intravascular therapy to recanalize the embolized vessels. Recently, the discovery of histone lactylation proposes a potential molecular mechanism for the role of lactate in physiological and pathological processes. This study aimed to analyze the lactate dehydrogenase A (LDHA) mediated histone lactylation in CI reperfusion (CI/R) injury. Oxygen-glucose deprivation/reoxygenation (OGD/R) treated N2a cells and middle cerebral artery occlusion (MCAO) treated rats was used as the CI/R model in vivo and in vitro. Cell viability and pyroptosis was assessed using CCK-8 and flow cytometry. RT-qPCR was performed to detect the relative expression. The relationship between histone lactylation and HMGB1 was verified by CHIP assay. LDHA, HMGB1, lactate and histone lactylation was up-regulated in the OGD/R treated N2a cells. Additionally, LDHA knockdown decreased HMGB1 levels in vitro, and relieved CI/R injury in vivo. Besides, LDHA silencing declined the histone lactylation mark enrichment on HMGB1 promoter, and lactate supplement rescued it. What?s more, LDHA knockdown decreased the IL-18 and IL-1β contents, and the cleaved-caspase-1 and GSDMD-N protein levels in the OGD/R treated N2a cells, which was reversed by HMGB1 overexpression. Knockdown of LDHA suppressed the pyroptosis in the N2a cells induced by OGD/R, which was reversed by HMGB1 overexpression. Mechanistically, LDHA mediated the histone lactylation induced pyroptosis through targeting HMGB1 in the CI/R injury.

Yao X ，Li C 《-》

DDX3X Deficiency Attenuates Pyroptosis Induced by Oxygen-glucose Deprivation/Reoxygenation in N2a Cells.

NOD like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis is strongly related to cerebral ischemia/reperfusion (I/R) injury. DDX3X, the DEAD-box family's ATPase/RNA helicase, promotes NLRP3 inflammasome activation. However, whether DDX3X deficiency attenuates NLRP3 inflammasome-mediated pyroptosis induced by cerebral I/R injury. This study investigated whether DDX3X deficiency attenuates NLRP3 inflammasomemediated pyroptosis in N2a cells after oxygen-glucose deprivation/ reoxygenation (OGD/R) treatment. In vitro model of cerebral I/R injury, mouse neuro2a (N2a) cells subjected to OGD/R were treated with the knockdown of DDX3X. Cell counting kit-8 (CCK-8) assay and Lactate dehydrogenase (LDH) cytotoxicity assay were conducted to measure cell viability and membrane permeability. Double immunofluorescence was performed to determine the pyroptotic cells. Transmission electron microscopy (TEM) was used to observe morphological changes of pyroptosis. Pyroptosis-associated proteins were analyzed by Western blotting. The OGD/R treatment reduced cell viability, increased pyroptotic cells and released LDH compared to the control group. TEM showed membrane pore formation of pyroptosis. Immunofluorescence showed that GSDMD was translocated from the cytoplasm to the membrane after OGD/R treatment. Western blotting showed that the expression of DDX3X, and pyroptosis-related proteins (NLRP3, cleaved-Caspase1, and GSDMD-N) were increased after OGD/R treatment. Nevertheless, DDX3X knockdown markedly improved cell viability and reduced LDH release, expression of pyroptosis-related proteins, and N2a cells pyroptosis. DDX3X knockdown significantly inhibited membrane pore formation and GSDMD translocation from cytoplasm to membrane. This research demonstrates for the first time that DDX3X knockdown attenuates OGD/R induced NLRP3 inflammasome activation and pyroptosis, which implies that DDX3X may become a potential therapeutic target for cerebral I/R injury.

Liu Y ，Gui Y ，Tang H ，Yu J ，Yuan Z ，Liu L ，Ma X ，Li C ... -  《-》

LDHA promotes osteoblast differentiation through histone lactylation.

Osteoblast cells tend to metabolize glucose to lactate via aerobic glycolysis during osteogenic differentiation. However, the function of lactate in this process is still elusive. As a newly discovered protein posttranslational modification, lactate-derived histone lactylation has been found to play important roles in gene regulation and have profound effects on diverse biological processes. Here, we found that the expression of lactate dehydrogenase A (LDHA), intracellular lactate, and histone lactylation levels were all gradually increased during osteogenic differentiation. Knockdown of LDHA impaired the formation of mineralized nodules and ALP activity. RNA-sequencing and subsequent validation experiments showed that JunB expression was decreased in LDHA knockdown cells. Mechanistically, knockdown of LDHA decreased histone lactylation mark enrichment on JunB promoter, and exogenous lactate treatment rescued this effect. Our study revealed a non-canonical function of lactate during osteogenic differentiation.

Nian F ，Qian Y ，Xu F ，Yang M ，Wang H ，Zhang Z ... -  《-》

Hepatocyte HSPA12A inhibits macrophage chemotaxis and activation to attenuate liver ischemia/reperfusion injury via suppressing glycolysis-mediated HMGB1 lactylation and secretion of hepatocytes.

Rationale: Liver ischemia-reperfusion (LI/R) injury is characterized by two interconnected phases: local ischemia that causes hepatic cell damage to release damage-associated molecular pattern (DAMPs), and DAMPs that recruit immune cells to elicit inflammatory cascade for further injury of hepatocytes. High-mobility group box 1 (HMGB1) is a representative DAMP. Studies in macrophages demonstrated that HMGB1 is secreted after lactylation during sepsis. However, whether lactylation mediates HMGB1 secretion from hepatocytes after LI/R is known. Heat shock protein A12A (HSPA12A) is an atypical member of HSP70 family. Methods: Gene expression was examined by microarray analysis and immunoblotting. The hepatic injury was analyzed using released ALT and AST activities assays. Hepatic macrophage chemotaxis was evaluated by Transwell chemotaxis assays. Inflammatory mediators were evaluated by immunoblotting. HMGB1 secretion was examined in exosomes or serum. HMGB1 lactylation was determined using immunoprecipitation and immunoblotting. Results: Here, we report that LI/R decreased HSPA12A expression in hepatocytes, while hepatocyte-specific HSPA12A overexpression attenuated LI/R-induced hepatic dysfunction and mortality of mice. We also noticed that hepatocyte HSPA12A overexpression suppressed macrophage chemotaxis to LI/R-exposed livers in vivo and to hypoxia/reoxygenation (H/R)-exposed hepatocytes in vitro. The LI/R-increased serum HMGB1 levels of mice and the H/R-increased HMGB1 lactylation and secretion levels of hepatocytes were also inhibited by hepatocyte HSPA12A overexpression. By contrast, HSPA12A knockout in hepatocytes promoted not only H/R-induced HMGB1 lactylation and secretion of hepatocytes but also the effects of H/R-hepatocytes on macrophage chemotaxis and inflammatory activation, while all these deleterious effects of HSPA12A knockout were reversed following hepatocyte HMGB1 knockdown. Further molecular analyses showed that HSPA12A overexpression reduced glycolysis-generated lactate, thus decreasing HMGB1 lactylation and secretion from hepatocytes, thereby inhibiting not only macrophage chemotaxis but also the subsequent inflammatory cascade, which ultimately protecting against LI/R injury. Conclusion: Taken together, these findings suggest that hepatocyte HSPA12A is a novel regulator that protects livers from LI/R injury by suppressing glycolysis-mediated HMGB1 lactylation and secretion from hepatocytes to inhibit macrophage chemotaxis and inflammatory activation. Therefore, targeting hepatocyte HSPA12A may have therapeutic potential in the management of LI/R injury in patients.

Du S ，Zhang X ，Jia Y ，Peng P ，Kong Q ，Jiang S ，Li Y ，Li C ，Ding Z ，Liu L ... -  《Theranostics》

Salvianolic Acid D Alleviates Cerebral Ischemia-Reperfusion Injury by Suppressing the Cytoplasmic Translocation and Release of HMGB1-Triggered NF-κB Activation to Inhibit Inflammatory Response.

Inflammatory response participates in the overall pathophysiological process of stroke. It is a promising strategy to develop antistroke drugs targeting inflammation. This study is aimed at investigating the therapeutic effect and anti-inflammatory mechanism of salvianolic acid D (SalD) against cerebral ischemia/reperfusion (I/R) injury. A rat middle cerebral artery occlusion/reperfusion (MCAO/R) injury model was established, and an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model was established in PC12 cells. Neurological deficit score, cerebral infarction, and edema were studied in vivo. Cell viability was achieved using the MTT method in vitro. The Bax, Bcl-2, cytochrome c, HMGB1, TLR4, TRAF6, NF-κB p65, p-NF-κB p65, and cleaved caspase-3 and -9 were tested via the Western blot method. Cytokines and cytokine mRNA, including TNF-α, IL-1β, and IL-6, were studied via ELISA and PCR methods. The translocation of HMGB1 and NF-κB were studied by immunofluorescence assay. The HMGB1/NeuN, HMGB1/GFAP, and HMGB1/Iba1 double staining was carried out to observe the localization of HMGB1 in different cells. Results showed that SalD alleviated neurological impairment, decreased cerebral infarction, and reduced edema in I/R rats. SalD improved OGD/R-downregulated PC12 cell viability. SalD also promoted Bcl-2 expression and suppressed Bax, cytochrome c, and cleaved caspase-3 and -9 expression. SalD decreased the intensity of TLR4, MyD88, and TRAF6 proteins both in vivo and in vitro, and significantly inhibited the NF-κB nuclear translocation induced by I/R and OGD/R. What's more, SalD inhibited HMGB1 cytoplasmic translocation in neurons, astrocytes, and microglia in both the cortex and hippocampus regions of I/R rats. In conclusion, SalD can alleviate I/R-induced cerebral injury in rats and increase the PC12 cell viability affected by OGD/R. The anti-inflammatory mechanism of SalD might result from the decreased nuclear-to-cytoplasmic translocation of HMGB1 and the inhibition on its downstream TLR4/MyD88/NF-κB signaling.

Zhang W ，Song J ，Li W ，Kong D ，Liang Y ，Zhao X ，Du G ... -  《-》