Puerarin induces Nrf2 as a cytoprotective mechanism to prevent cadmium-induced autophagy inhibition and NLRP3 inflammasome activation in AML12 hepatic cells.

Liver is the main target organ of cadmium (Cd) toxicity and puerarin (PU) has been shown to prevent Cd-induced hepatic cell damage via its antioxidant activity. Nrf2 acts as a critical regulator of cellular defense against various oxidative insults, but its role in the protection of PU against Cd-induced hepatic damage has not yet been clarified. Hereby, this study was designed to investigate the underlying mechanism using mouse hepatocyte line AML-12. Data firstly showed that Cd-inhibited Nrf2 pathway was markedly restored by PU treatment, assessed by Nrf2 nuclear translocation, protein levels of Keap1 and Nrf2 downstream target genes. Accordingly, Cd-reduced protein levels of antioxidant enzymes were significantly up-regulated by PU. Next, Nrf2 silencing cellular model was established to further elucidate the role of Nrf2 in the protection of PU against Cd-induced hepatic damage. Attenuation of Cd-induced autophagy inhibition and autophagosome accumulation by PU was remarkably countered by Nrf2 silencing. Moreover, alleviation of Cd-induced NLRP3 inflammasome activation by PU was distinctly prevented by Nrf2 knockdown, assessed by protein levels of NLRP3 inflammosome complex and downstream IL-18 and IL-1β production. Collectively, our data suggest that PU restores Cd-induced Nrf2 inhibition to prevent autophagy inhibition and NLRP3 inflammasome activation, providing novel insights into the protection of PU against Cd-induced hepatic cell damage.

Yu ZM ，Wan XM ，Xiao M ，Zheng C ，Zhou XL ... -  《-》

Puerarin reverses cadmium-induced lysosomal dysfunction in primary rat proximal tubular cells via inhibiting Nrf2 pathway.

Wang LY ，Fan RF ，Yang DB ，Zhang D ，Wang L ... -  《-》

Puerarin prevents cadmium-induced hepatic cell damage by suppressing apoptosis and restoring autophagic flux.

Cadmium (Cd) is a common heavy metal contamination that is highly toxic to liver. Puerarin (PU), a potent free radical scavenger, has been shown to exert cytoprotective effect in numerous pathological processes. However, whether PU affords protection against Cd-induced hepatotoxicity remains unclear to be known. Here, we aimed to investigate the protective effect of PU on Cd-induced hepatotoxicity in an immortalized mouse hepatocyte line, AML-12. First, Cd-induced cytotoxicity in AML-12 cells was obviously ameliorated by PU treatment. Also, Cd-induced apoptotic cell death was markedly alleviated by PU treatment, evidenced by two methods. Simultaneously, Cd-elevated malondialdehyde and reactive oxygen species levels were significantly reduced by PU administration, demonstrating the antioxidant effect of PU against Cd exposure. Moreover, Cd-induced blockage of autophagic flux in AML-12 cells was obviously restored by PU treatment, evidenced by immunoblot analysis of autophagy marker proteins and tandem fluorescent-tagged LC3 method. Resultantly, Cd-induced autophagosome accumulation was significantly alleviated by PU treatment. In conclusion, these observations demonstrate that PU treatment alleviates Cd-induced hepatic cell damage by inhibiting apoptosis and restoring autophagy activity, which is intimately related with its antioxidant activity.

Zhou XL ，Wan XM ，Fu XX ，Xie CG ... -  《-》

Puerarin inhibits hyperglycemia-induced inter-endothelial junction through suppressing endothelial Nlrp3 inflammasome activation via ROS-dependent oxidative pathway.

Recent studies indicate that vascular complications are closely related to diabetes mellitus; in particular, inflammatory-mediated endothelial dysfunction plays a crucial role in diabetes-induced cardiovascular diseases. Therefore, exploring effective methods to suppress endothelial dysfunction via inhibition of inflammatory responses is imperative. Puerarin (Pu), a flavonoid common in Pueraria, has been widely and successfully used to treat cardiovascular diseases in China for many years. However, information on its protective properties in hyperglycemia-induced vascular complications is insufficient. Hypothesis/Purpose: In this study, we investigate the protective effects of puerarin against high glucose-induced endothelial dysfunction and the underlying mechanism of the flavonoid. we investigated the protective effects of Pu against hyperglycemia-induced inter-endothelial junction by permeability and transendothelial electrical resistance (TEER) assay. In addition, changes in the Nlrp3 inflammasome activation via reactive oxygen species (ROS)-dependent oxidative pathway were investigated using western blot, immunofluorescence microscopy analyses and flow cytometry. ROS scavenger and Nlrp3 gene silencing were used to determine the roles of the ROS-Nlrp3 pathway involved in the molecular mechanism of Pu. Our findings demonstrate that puerarin inhibits high glucose-induced Nlrp3 inflammasome formation and activation, as shown by fluorescence confocal microscopy and Western blot. Puerarin decreases Nlrp3 protein, which is a critical factor necessary to form an inflammasome complex. We demonstrate that puerarin exerts anti-oxidation and ROS scavenged effects, similar to apocynin (APO). Interestingly, thioredoxin-interacting protein (TXNIP) protein and TXNIP binding to Nlrp3 markedly decreased with puerarin treatment. Together with these changes, puerarin could decrease high mobility group box 1 (HMGB1) release from mouse vascular endothelial cell (mMVECs). We also demonstrate the decreased expression of the tight junction proteins ZO-1/ZO-2, which are related to endothelial permeability after stimulation by high glucose in endothelial cells. Puerarin could recover the gap junction protein and decrease monolayer cell permeability in endothelial cells. In conclusion, we reveal a new protection mechanism of puerarin that inhibits Nlrp3 inflammasome activation and decreases subsequent caspase-1 activation, triggering the release of HMGB1 by reducing ROS generation. Our findings indicate that puerarin exhibits immense potential and specific therapeutic value in hyperglycemia-related cardiovascular disease and the development of innovative drugs.

Lian D ，Yuan H ，Yin X ，Wu Y ，He R ，Huang Y ，Chen Y ... -  《-》

Nrf2 inhibits NLRP3 inflammasome activation through regulating Trx1/TXNIP complex in cerebral ischemia reperfusion injury.

The nod-like receptor protein 3 (NLRP3) inflammasome has a critical role in inflammation damage in ischemic injury, and the activation of the inflammasome is closely related to the interaction with thioredoxin interacting protein (TXNIP), which dissociates from the thioredoxin1 (Trx1)/TXNIP complex under oxidative stress. However, the negative regulator of NLRP3 inflammasome activation has not been fully investigated. Nuclear factor erythroid 2-related factor 2 (Nrf2) takes on a critical part in the antioxidant stress system, that controls the driven genes of antioxidant response element (ARE). Activate Nrf2 could inhibit the activation of NLRP3 inflammasome in acute liver injury and severe lupus nephritis. We aimed to explore the protective effect of Nrf2 in inhibiting the NLPR3 inflammasome formulation through the Trx1/TXNIP complex in cerebral ischemia reperfusion (cerebral I/R) injury. Middle cerebral artery occlusion/reperfusion (MCAO/R) model was used to imitate ischemic insult. Nrf2 was activated by tert-butylhydroquinone (tBHQ) intraperitoneally (i.p.) injection (16.7mg/kg), Nrf2,Trx1 and NLRP3 siRNAs were infused into the left paracele (12μl per rat), protein and mRNA levels were assessed by Western blot, qRT-PCR. ELISA was used for IL-1β and IL-18 activity measurements. After upregulating Nrf2, the expression of TXNIP in cytoplasm, NLRP3 inflammasome, and downstream factors caspase-1, IL-18, and IL-1β were significantly reduced, and Nrf2 knockdown yielded the opposite results. Trx1 knockdown produced the same effect of Nrf2 inhibition and the protective effect of Nrf2 was mostly abolished. Our results suggested that Nrf2 acted as a protective regulator against NLRP3 inflammasome activation by regulating the Trx1/TXNIP complex, which could possibly represent an innovative insight into the treatment of ischemia and reperfusion injury.

Hou Y ，Wang Y ，He Q ，Li L ，Xie H ，Zhao Y ，Zhao J ... -  《-》