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 ... -  《-》

Hepatoprotective effect of allicin against acetaminophen-induced liver injury: Role of inflammasome pathway, apoptosis, and liver regeneration.

Acetaminophen (APAP) overdose leads to liver injury. NLRP3 inflammasome is a key player in APAP-induced inflammation. Also, apoptosis and liver regeneration play an important role in liver injury. Therefore, we assessed allicin's protective effect on APAP-induced hepatotoxicity and studied its effect on NLRP3 inflammasome and apoptosis. Mice in the APAP group were injected by APAP (250 mg/kg, intraperitoneal). The allicin-treated group received allicin orally (10 mg/kg/d) during 7 days before APAP injection. Serum and hepatic tissues were separated 24 hours after APAP injection. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, alkaline phosphatase (ALP), and hepatic malondialdehyde (MDA) were assessed using the colorimetric method. Hepatic NLRP3 inflammasome, caspase-1, and interleukin-1β (IL-1β) were estimated using enzyme-linked immunosorbent assay. Hepatic Bcl-2 and Ki-67 were investigated by immunohistochemistry. APAP significantly increased AST, ALT, and ALP, whereas allicin significantly decreased their levels. Also, APAP significantly decreased albumin and allicin significantly improved it. APAP produced changes in liver morphology, including inflammation and massive coagulative necrosis. Allicin protected the liver from APAP-induced necrosis, apoptosis, and hepatocellular degeneration via increasing Bcl-2 and Ki-67 levels. APAP significantly increased the hepatic MDA, whereas allicin significantly prevented this increase. APAP markedly activated the NLRP3 inflammasome pathway and consequently increased the production of caspase-1 and IL-1β. Interestingly, we found that allicin significantly inhibited NLRP3 inflammasome activation, which resulted in decreased caspase-1 and IL-1β levels. Allicin has a hepatoprotective effect against APAP-induced liver injury via the decline of oxidative stress and inhibition of the inflammasome pathway and apoptosis. Therefore, allicin might be a novel tool to halt the progression of APAP-stimulated hepatotoxicity.

Samra YA ，Hamed MF ，El-Sheakh AR 《-》

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 ... -  《-》

Salidroside alleviates acetaminophen-induced hepatotoxicity via Sirt1-mediated activation of Akt/Nrf2 pathway and suppression of NF-κB/NLRP3 inflammasome axis.

Acetaminophen (APAP) overdose-induced hepatotoxicity is the most common cause of drug-induced liver injury worldwide, which is significantly linked to oxidative stress and sterile inflammation. Salidroside is the main active component extracted from Rhodiola rosea L., with anti-oxidative and anti-inflammatory activities. Herein, we investigated the protective effects of salidroside on APAP-induced liver injury and its underlying mechanisms. Pretreatment with salidroside reversed the impacts of APAP on cell viability, LDH release, and cell apoptosis in L02 cells. Moreover, the phenomena of ROS accumulation and MMP collapse caused by APAP were reverted by salidroside. Salidroside elevated the levels of nuclear Nrf2, HO-1, and NQO1. Using PI3k/Akt inhibitor LY294002 further confirmed that salidroside mediated the Nrf2 nuclear translocation through the Akt pathway. Pretreatment with Nrf2 siRNA or LY294002 markedly prevented the anti-apoptotic effect of salidroside. Additionally, salidroside reduced the levels of nuclear NF-κB, NLRP3, ASC, cleaved caspase-1, and mature IL-1β elevated by APAP. Moreover, salidroside pretreatment increased Sirt1 expression, whereas Sirt1 knock-down diminished the protective activities of salidroside, simultaneously reversing the up-regulation of the Akt/Nrf2 pathway and the down-regulation of NF-κB/NLRP3 inflammasome axis mediated by salidroside. We then used C57BL/6 mice to establish APAP-induced liver injury models and found that salidroside significantly alleviated liver injury. Furthermore, western blot analyses showed that salidroside promoted the Sirt1 expression, activated the Akt/Nrf2 pathway, and inhibited the NF-κB/NLRP3 inflammasome axis in APAP-treated mice. The findings of this study support a possible application of salidroside in the amelioration of APAP-induced hepatotoxicity.

Gao Z ，Zhan H ，Zong W ，Sun M ，Linghu L ，Wang G ，Meng F ，Chen M ... -  《-》

Baicalin promotes liver regeneration after acetaminophen-induced liver injury by inducing NLRP3 inflammasome activation.

Liver regeneration has become a new hotspot in the study of drug-induced liver injury (DILI). Baicalin has already been reported to alleviate acetaminophen (APAP)-induced acute liver injury in our previous study. This study aims to observe whether baicalin also promotes liver regeneration after APAP-induced liver injury and to elucidate its engaged mechanism. Baicalin alleviated APAP-induced hepatic parenchymal cells injury and enhanced the number of mitotic and proliferating cell nuclear antigen (PCNA)-positive hepatocytes in APAP-intoxicated mice. Baicalin increased hepatic PCNA and cyclinD1 expression in APAP-intoxicated mice. Baicalin induced the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, leading to the increased hepatic expression of interleukin-18 (IL-18) and IL-1β in APAP-intoxicated mice. The results in vitro demonstrated that IL-18 promoted the proliferation of human normal liver L-02 cells. Moreover, the baicalin-provided promotion on liver regeneration in APAP-intoxicated mice was diminished after the application of NLRP3 inhibitor MCC950 and the recombinant mouse IL-18 binding protein (rmIL-18BP). Baicalin induced the cytosolic accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), and increased the interaction between Nrf2 with Nlrp3, ASC and pro-caspase-1 in livers from APAP-intoxicated mice. Furthermore, the baicalin-provided NLRP3 inflammasome activation and promotion on liver regeneration after APAP-induced liver injury in wild-type mice were diminished in Nrf2 knockout mice. In conclusion, baicalin promoted liver regeneration after APAP-induced acute liver injury in mice via inducing Nrf2 accumulation in cytoplasm that led to NLRP3 inflammasome activation, and then caused the increased expression of IL-18, which induced hepatocytes proliferation.

Shi L ，Zhang S ，Huang Z ，Hu F ，Zhang T ，Wei M ，Bai Q ，Lu B ，Ji L ... -  《-》