Pien-Tze-Huang, a Chinese patent formula, attenuates NLRP3 inflammasome-related neuroinflammation by enhancing autophagy via the AMPK/mTOR/ULK1 signaling pathway.

NLRP3 inflammasome is a key mediator in ischemic stroke-induced neuroinflammation and subsequent brain injury. Our previous study demonstrated the potent activity of Pien-Tze-Huang (PTH), a well-known Chinese patent formula, in reducing mitochondria-mediated neuronal apoptosis in cerebral ischemia/reperfusion impaired rats. This study aims to elucidate the mechanistic action of PTH related to neuroinflammation in LPS-induced BV2 microglial cells and cerebral ischemia/reperfusion impaired rats. BV2 cells were stimulated with LPS for 12 h and treated with PTH with various concentrations. Modulation by PTH of relevant genes (IL-6, IL-1β, IL-18, TNF-α, COX-2 and iNOS mRNA) and proteins (NLRP3 inflammasome, autophagy and AMPK/mTOR/ULK signaling) was analyzed by real-time PCR and western blot, respectively. Similar analyses were conducted in middle cerebral artery occlusion rat model including neurological deficit, infarct volume, microglial activation, and key genes and proteins in modulating autophagy and NLRP3. Our results showed that PTH significantly inhibited the production of key proinflammatory mediators and protein expressions of NLRP3 and caspase-1 p20 in LPS induced BV2 cells. It also enhanced the autophagy response by modulating the key autophagy proteins via AMPK/mTOR/ULK related pathway. The reduced inflammatory responses and NLRP3 expressions by PTH were partially blocked by the autophagy inhibitor (3-MA) and AMPK blocker (compound C). In rats, PTH significantly reduced infarct size, suppressed microglial activation, and improved neuron deficit. It also promoted autophagy and reduced NLRP3 activity. Our study demonstrated that PTH inhibited NLRP3 inflammasome-mediated neuroinflammation, which was associated with enhanced autophagy via AMPK/mTOR/ULK1 pathway in vitro and in vivo.

Huang Z ，Zhou X ，Zhang X ，Huang L ，Sun Y ，Cheng Z ，Xu W ，Li CG ，Zheng Y ，Huang M ... -  《-》

Qingkailing injection ameliorates cerebral ischemia-reperfusion injury and modulates the AMPK/NLRP3 Inflammasome Signalling pathway.

Ma C ，Wang X ，Xu T ，Yu X ，Zhang S ，Liu S ，Gao Y ，Fan S ，Li C ，Zhai C ，Cheng F ，Wang Q ... -  《BMC Complementary and Alternative Medicine》

Pien-Tze-Huang attenuates neuroinflammation in cerebral ischaemia-reperfusion injury in rats through the TLR4/NF-κB/MAPK pathway.

Zhang X ，Zhang Q ，Huang L ，Liu M ，Cheng Z ，Zheng Y ，Xu W ，Lu J ，Liu J ，Huang M ... -  《-》

Pien Tze Huang attenuated acetaminophen-induced liver injury by autophagy mediated-NLRP3 inflammasome inhibition.

Pien Tze Huang is a classic traditional Chinese medicinal product, used for inflammatory diseases as stated in Chinese Pharmacopoeia. In particular, it is effective in treating liver diseases and pro-inflammatory conditions. Acetaminophen (APAP) is a widely used analgesic drug, but its over-dose is associated with acute liver failure where the clinical approved antidote treatment is limited. Inflammation has been considered as one of the therapeutic targets against APAP-induced liver injury. We aimed to explore the therapeutic potential of Pien Tze Huang tablet (PTH) on protecting liver against APAP-induced liver injury through its strong anti-inflammatory pharmacological action. Wild-type C57BL/6 mice were given PTH (75, 150 and 300 mg/kg) by oral gavage 3 days before the APAP injection (400 mg/kg). The protective effect of PTH was assessed by aspartate aminotransferase (AST) and alanine transaminase (ALT) levels and pathological staining. The mechanisms underlying PTH's hepatoprotective effects were investigated in nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) knock-out (NLRP3-/-), over expression NLRP3 (oe-NLRP3) mice, and wild-type mice with the injection of autophagy inhibitor (3-methyladenine, 3-MA). APAP-exposed mice resulted in evident liver injury which was evidenced by hepatic necrosis and elevated levels of AST and ALT in the wild-type C57BL/6 mice. PTH dose-dependently reduced ALT, AST and upregulated autophagy activity. In addition, PTH significantly reduced elevated levels of proinflammatory cytokines and NLRP3 inflammasome. The liver protective effect of PTH (300 mg/kg) was still obvious in the oe-NLRP3 mice, however, it became insignificant in the NLRP3-/- mice. When PTH (300 mg/kg) was co-treated with 3-MA to the wild-type C57BL/6 mice, the NLRP3 inhibition were reversed when autophagy was blocked. PTH exerted a beneficial effect in protecting liver against APAP-induced liver injury. The underlying molecular mechanism was associated with the NLRP3 inflammasome inhibition which was likely driven by the upregulated autophagy activity. Our study underpins the traditional use of PTH in protecting liver through its anti-inflammatory action.

Zhao R ，Zhang Q ，Liu W ，Lin Y ，He Y ，Chang D ，Li S ，Xu W ，Lin Y ，Zheng Y ，Zhou X ，Huang M ... -  《-》

6-Gingerol protects against cerebral ischemia/reperfusion injury by inhibiting NLRP3 inflammasome and apoptosis via TRPV1 / FAF1 complex dissociation-mediated autophagy.

Our previous studies demonstrated that autophagy alleviates cerebral I/R injury by inhibiting NLRP3 inflammasome-mediated inflammation. 6-Gingerol, a phenolic compound extracted from ginger, was reported to possess potent antiapoptotic and anti-inflammatory activities and is associated with autophagy. However, the effects of 6-Gingerol in cerebral I/R injury have not been elucidated, and whether they involve autophagy-induced NLRP3 inflammasome inhibition remains unclear. Adult male Sprague-Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h, followed by reperfusion for 24 h. 6-Gingerol and 3-methyladenine (3-MA) were injected intraperitoneally, and si-TRPV1 was injected via the lateral ventricle. Cerebral infarct volume, brain edema, neurological deficits, HE and Nissl were used to evaluate the morphological and functional changes of brain tissue, respectively. TRPV1, FAF1, autophagy related (LC3II/I, P62, Beclin1), inflammation related (NLRP3, cleaved-caspase-1, caspase-1, cleaved-IL-1β, IL-1β, cleaved-IL-18, IL-18) and apoptosis related (Bcl-2, Bax, cleaved-caspase-3) proteins were assessed by Western blot, immunofluorescence staining and coimmunoprecipitation, respectively. Enzyme linked immunosorbent assay (ELISA) was used to evaluate the changes in the expression levels of interleukin-1 (IL-1β) and interleukin-18(IL-18), respectively. The degree of neuronal apoptosis was evaluated by TUNEL staining. Neuronal ultrastructure was examined by transmission electron microscopy. 6-Gingerol treatment significantly reduced cerebral infarct volume, improved brain edema and neurological scores, and reversed brain histomorphological damage after I/R injury. In addition, 6-Gingerol significantly reduced NLRP3 inflammasome-derived inflammation and neuronal apoptosis and upregulated autophagy. The autophagy inhibitor 3-MA rescued the effects of 6-Gingerol on the NLRP3 inflammasome and apoptosis. Moreover, the findings illustrated that 6-Gingerol inhibited autophagy-induced NLRP3 inflammasome activation and apoptosis through the dissociation of TRPV1 from FAF1. In brief, 6-Gingerol exerts antiapoptotic and anti-inflammatory effects via TRPV1/FAF1 complex dissociation-mediated autophagy during cerebral I/R injury. Therefore, 6-Gingerol may be an effective drug for the treatment of I/R injury.

Luo J ，Chen J ，Yang C ，Tan J ，Zhao J ，Jiang N ，Zhao Y ... -  《-》