Neutrophil extracellular traps promote acetaminophen-induced acute liver injury in mice via AIM2.

Excessive acetaminophen (APAP) can induce neutrophil activation and hepatocyte death. Along with hepatocyte dysfunction and death, NETosis (a form of neutrophil-associated inflammation) plays a vital role in the progression of acute liver injury (ALI) induced by APAP overdose. It has been shown that activated neutrophils tend to migrate towards the site of injury and participate in inflammatory processes via formation of neutrophil extracellular traps (NETs). In this study we investigated whether NETs were involved in hepatocyte injury and contributed to APAP-induced ALI progression. ALI mouse model was established by injecting overdose (350 mg/kg) of APAP. After 24 h, blood and livers were harvested for analyses. We showed that excessive APAP induced multiple programmed cell deaths of hepatocytes including pyroptosis, apoptosis and necroptosis, accompanied by significantly increased NETs markers (MPO, citH3) in the liver tissue and serum. Preinjection of DNase1 (10 U, i.p.) for two consecutive days significantly inhibited NETs formation, reduced PANoptosis and consequently alleviated excessive APAP-induced ALI. In order to clarify the communication between hepatocytes and neutrophils, we induced NETs formation in isolated neutrophils, and treated HepaRG cells with NETs. We found that NETs treatment markedly increased the activation of GSDMD, caspase-3 and MLKL, while pre-treatment with DNase1 down-regulated the expression of these proteins. Knockdown of AIM2 (a cytosolic innate immune receptor) abolished NETs-induced PANoptosis in HepaRG cells. Furthermore, excessive APAP-associated ALI was significantly attenuated in AIM2KO mice, and PANoptosis occurred less frequently. Upon restoring AIM2 expression in AIM2KO mice using AAV9 virus, both hepatic injury and PANoptosis was aggravated. In addition, we demonstrated that excessive APAP stimulated mtROS production and mitochondrial DNA (mtDNA) leakage, and mtDNA activated the TLR9 pathway to promote NETs formation. Our results uncover a novel mechanism of NETs and PANoptosis in APAP-associated ALI, which might serve as a therapeutic target.

PSTPIP2 ameliorates aristolochic acid nephropathy by suppressing interleukin-19-mediated neutrophil extracellular trap formation.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by herbal medicines. Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) play important roles in kidney injury and immune defense, respectively, but the mechanism underlying AAN regulation by PSTPIP2 and NETs remains unclear. We found that renal tubular epithelial cell (RTEC) apoptosis, neutrophil infiltration, inflammatory factor, and NET production were increased in a mouse model of AAN, while PSTPIP2 expression was low. Conditional knock-in of Pstpip2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. Conversely, downregulation of Pstpip2 expression promoted kidney injury. In vivo, the use of Ly6G-neutralizing antibody to remove neutrophils and peptidyl arginine deiminase 4 (PAD4) inhibitors to prevent NET formation reduced apoptosis, alleviating kidney injury. In vitro, damaged RTECs released interleukin-19 (IL-19) via the PSTPIP2/nuclear factor (NF)-κB pathway and induced NET formation via the IL-20Rß receptor. Concurrently, NETs promoted apoptosis of damaged RTECs. PSTPIP2 affected NET formation by regulating IL-19 expression via inhibition of NF-κB pathway activation in RTECs, inhibiting RTEC apoptosis, and reducing kidney damage. Our findings indicated that neutrophils and NETs play a key role in AAN and therapeutic targeting of PSTPIP2/NF-κB/IL-19/IL-20Rß might extend novel strategies to minimize Aristolochic acid I-mediated acute kidney injury and apoptosis.

Aristolochic acid nephropathy (or AAN for short) is a serious condition affecting the kidneys that is caused by certain traditional Chinese medicines containing a compound called aristolochic acid. This compound is known to have harmful effects on kidney tubular epithelial cells, causing increased inflammation and a form of controlled cell death called apoptosis, which can ultimately lead to organ failure. There is currently no effective treatment for AAN, highlighting the need for a deeper understanding of the mechanisms responsible. Previous studies have shown that immune cells called neutrophils infiltrate the kidneys and damage cells in the early stages of AAN. Neutrophils produce web-like structures called neutrophil extracellular traps, which have been identified as potentially contributing to the damage. A protein called PSTPIP2, which regulates inflammation, has also been shown to contribute to other types of kidney injury. To understand how these inflammatory factors might be involved in AAN, Du, Xu et al. genetically engineered mice to produce extra PSTPIP2 protein specifically in their kidneys. When given aristolochic acid, these mice displayed less kidney damage. Further studies of mouse kidney cells showed that PSTPIP2 protects the kidney by suppressing an inflammatory mechanism that leads to the production of neutrophil extracellular traps. By contrast, in models where PSTPIP2 levels were reduced, neutrophil extracellular traps were shown to cause both apoptosis and kidney injury. The findings of Du, Xu et al. show that neutrophil extracellular traps cause cell damage and apoptosis in a mouse model of AAN and that this action can be reduced by increasing the levels of the protein PSTPIP2. This sheds light on the inflammatory mechanisms behind the kidney damage caused by herbal medicines containing aristolochic acid. Additionally, it opens new avenues for studies aiming to find ways to treat AAN, suggesting that targeting PSTPIP2 could be a promising strategy.

Coiled-coil domain containing 25 (CCDC25) regulates cell proliferation, migration, and invasion in clear cell renal cell carcinoma by targeting the ILK-NF-κB signaling pathway.

Increasing evidence has demonstrated that the expression of coil domains containing 25 (CCDC25) in various malignancies is abnormally high. However, the potential regulatory role and mechanism of CCDC25 in the development of clear cell renal cell carcinoma (ccRCC) are still unclear. In this experiment, we combined in vitro experiments such as wound healing, CCK8, and transwell assay with in vivo experiments on tumor formation in nude mice to evaluate the effect of CCDC25 on the proliferation, migration, and invasion of renal cancer cells. In addition, we also used Western blotting and qPCR to evaluate the role of CCDC25 in activating the integrin-linked kinase (ILK)-NF-κB signaling pathway. Here, we demonstrate that compared to normal tissues and cell lines, CCDC25 is overexpressed in both human ccRCC tissues and cell lines. After CCDC25 knockdown, it has obvious inhibitory effect on the proliferation, migration, and invasion of cancer cells in vitro and in vivo. In contrast, CCDC25 overexpression promotes these effects. Additionally, we also discovered that CCDC25 interacts with ILK and coordinates the activation of the NF-κB signaling pathway downstream. Generally, our study suggests that CCDC25 plays a vital role in the development of ccRCC, which also means that it may be a potential therapeutic target for ccRCC.

Histone deacetylase-mediated silencing of PSTPIP2 expression contributes to aristolochic acid nephropathy-induced PANoptosis.

BACKGROUND AND PURPOSE: Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by using herbal medicines. Currently, no therapies are available to treat or prevent aristolochic acid nephropathy. Histone deacetylase (HDAC) plays a crucial role in the development and progression of renal disease. We tested whether HDAC inhibitors could prevent aristolochic acid nephropathy and determined the underlying mechanism. EXPERIMENTAL APPROACH: HDACs expression in the aristolochic acid nephropathy model was examined. The activation of PANoptosis of mouse kidney and renal tubular epithelial cell were assessed after exposure to HDAC1 and HDAC2 blockade. Kidney-specific knock-in of proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) mice were used to investigate whether PSTPIP2 affected the production of PANoptosome. KEY RESULTS: Aristolochic acid upregulated the expression of HDAC1 and HDAC2 in the kidneys. Notably, the HDAC1 and HDAC2 specific inhibitor, romidepsin (FK228, depsipeptide), suppressed aristolochic acid-induced kidney injury, epithelial cell pyroptosis, apoptosis and necroptosis (PANoptosis). Moreover, romidepsin upregulated PSTPIP2 in renal tubular epithelial cells, which was enhanced by aristolochic acid treatment. Conditional knock-in of PSTPIP2 in the kidney protected against aristolochic acid nephropathy. In contrast, the knockdown of PSTPIP2 expression in PSTPIP2-knock-in mice restored kidney damage and PANoptosis. PSTPIP2 function was determined in vitro using PSTPIP2 knockdown or overexpression in mouse renal tubular epithelial cells (mTECs). Additionally, PSTPIP2 was found to regulate caspase 8 in aristolochic acid nephropathy. CONCLUSION AND IMPLICATIONS: HDAC-mediated silencing of PSTPIP2 may contribute to aristolochic acid nephropathy. Hence, HDAC1 and HDAC2 specific inhibitors or PSTPIP2 could be valuable therapeutic agents for preventing aristolochic acid nephropathy.

Uncovering the role of cytoskeleton proteins in the formation of neutrophil extracellular traps.

Neutrophils are a type of lymphocyte involved in innate immune defense. In response to specific stimuli, these phagocytic cells undergo a unique form of cell death, NETosis, during which they release neutrophil extracellular traps (NETs) composed of modified chromatin structures decorated with cytoplasmic and granular proteins. Multiple proteins and pathways have been implicated in the formation of NETs. The cytoskeleton, an interconnected network of filamentous polymers and regulatory proteins, plays a crucial role in resisting deformation, transporting intracellular cargo, and changing shape during movement of eukaryotic cells. It may also have evolved to defend eukaryotic organisms against infection. Recent research focuses on understanding the mechanisms underlying NETs formation and how cytoskeletal networks contribute to this process, by identifying enzymes that trigger NETosis or interact with NETs and influence cellular behavior through cytoskeletal dynamics. An enhanced understanding of the complex relationship between the cytoskeleton and NET formation will provide a framework for future research and the development of targeted therapeutic strategies, and supports the notion that the long-lived cytoskeleton structures may have a lasting impact on this area of research.

Lymphocyte-Specific Protein Tyrosine Kinase Contributes to Spontaneous Regression of Liver Fibrosis may by Interacting with Suppressor of Cytokine Signaling 1.

Quiescent hepatic stellate cells (qHSCs), converted to myofibroblasts, produce fibrous scars, which is an essential event during liver fibrogenesis. Clinical and experimental fibrosis undergo remarkable regression when the underlying etiological agent is removed. Some myofibroblasts revert to an inactive phenotype (iHSCs) during the regression of fibrosis. However, the mechanisms underlying HSC activation and reversal remain unclear. The present study demonstrated that the expression of lymphocyte-specific protein tyrosine kinase (LCK) was increased in fibrotic livers but decreased after spontaneous recovery in vivo and in vitro, which was correlated with the expression of α-smooth muscle actin (α-SMA) and type I collagen (COL-1). Further investigation indicated that specific knockdown of LCK by a recombination adeno-associated virus 9 (rAAV9) in C57BL/6 mice ameliorated liver fibrosis. Co-incubation of TGF-ß1-induced HSC-T6 cells with LCK-siRNA inhibited cell proliferation and activation. Overexpression of LCK inhibited activated HSCs going to inactivated phenotype. Interestingly, we found that LCK may interact with suppressor of cytokine signaling 1 (SOCS1) and may influence the expression of p-JAK1 and p-STAT1/3. These data suggest that LCK may play a regulatory role in liver fibrosis by inhibiting SOCS1, indicating that LCK is a potential therapeutic target for liver fibrosis treatment.

Echinacoside from Cistanche tubulosa ameliorates alcohol-induced liver injury and oxidative stress by targeting Nrf2.

Cistanche tubulosa (Schrenk) Wight, named Guan hua Rou Cong-Rong in Chinese, is a traditional plant with liver, kidney, and intestine protective effects. Echinacoside (ECH) is its active constituent and has been found to have various biological effects, including antioxidative stress and anti-inflammatory effects. Liver injury caused by acetaminophen or CCL4 has been proven to benefit from ECH; however, the effects of ECH against alcoholic liver disease (ALD) remain unclear. This study was used to estimate the effect of echinacoside on nuclear factor erythroid 2-related factor 2 (Nrf2), which ameliorates ALD by inhibiting oxidative stress and cell apoptosis through affecting Nrf2.A mouse model of ALD was established with ethanol using hematoxylin and eosin (HE) staining, oiled staining, and biochemical indices. Alpha Mouse Liver 12 (AML-12) cells were induced with ethanol in vitro and analyzed using western blotting, flow cytometry, and biochemical assays. In the animal model of ALD, ECH dramatically reduced liver damage, as proven by the downregulation of aspartate aminotransferase (AST) and HE staining. In vitro, ECH distinctly reduced the damage caused by ethanol through the decreased expression of cleaved caspase-3 measured by western blotting. ECH significantly increased the activity of Nrf2 in vivo and in vitro. Nrf2 knockout may diminish the influence of ECH on ALD. Meanwhile, ECH also increased the expression of haem oxygenase-1 (HO-1) and glutamate-cysteine ligase catalytic subunit (GCLC), while it inhibited levels of oxidative stress and cell apoptosis. Our findings suggest that ECH protects against ethanol-induced liver injuries by alleviating oxidative stress and cell apoptosis by increasing the activity of Nrf2. Therefore, ECH is promising for the treatment of ALD.

Verbenalin attenuates hepatic damage and mitochondrial dysfunction in alcohol-associated steatohepatitis by regulating MDMX/PPARα-mediated ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Verbenalin is a major compound in Verbena officinalis L. Verbena officinalis L was first recorded in the 'Supplementary Records of Famous Physicians.' Verbenalin (VE) is its active constituent and has been found to have many biological effects, including anti-obesity, anti-inflammatory, and antioxidant activities, removing jaundice, and treating malaria. It could treat lump accumulation, dysmenorrhea, throat obstruction, edema, jaundice, and malaria. Palmitic acid (PA), oleic acid (OA), ethanol, and acetaminophen liver injuries have been proven to benefit from verbenalin. AIM OF THE STUDY: To study the effects of verbenalin on the prevention of alcoholic steatohepatitis (ASH) through the regulation of oxidative stress and mitochondrial dysfunction by regulating MDMX (Murine double minute X)/PPARα (Peroxisome proliferator-activated receptor alpha)-mediated ferroptosis. MATERIAL AND METHODS: C57BL/6 mice treated with alcohol followed by the Gao-Binge protocol were administered verbenalin by gavage simultaneously. The mitochondrial mass and morphology were visualized using TEM. AML-12 cells were stimulated with ethanol to mimic ASH in vitro. Western blotting, co-immunoprecipitation, and kit determination were simultaneously performed. The target protein of verbenalin was identified by molecular docking, and cellular thermal shift assay (CETSA) further confirmed its interactions. RESULTS: Verbenalin alleviates oxidative stress and ferroptosis in alcohol-associated steatohepatitis. To elucidate the molecular mechanism by which verbenalin inhibits abnormal mitochondrial dysfunction, molecular docking was performed, and MDMX was identified as the target protein of verbenalin. CETSA assays revealed a specific interaction between MDMX and verbenalin. Co-immunoprecipitation demonstrated that PPARα played a critical role in promoting the ability of MDMX to affect ferroptosis. Verbenalin regulates MDMX/PPARα-mediated ferroptosis in AML-12 cells. CONCLUSION: Verbenalin regulates ferroptosis and highlights the therapeutic potential of verbenalin and ferroptosis inhibition in reducing alcoholic steatohepatitis.

Hastatoside attenuatescarbon tetrachloride-induced liver fibrosis by targeting glycogen synthase kinase-3ß.

BACKGROUND: Hastatoside is an iridoid glycoside extracted from the herb, Verbena officinalis, that exerts various pharmacological effects, including anti-inflammatory, sleep-promoting, and analgesic effects. However, only a few studies have reported the efficacy of hastatoside in liver fibrosis. Liver fibrosis is a pathophysiological process, and its persistence can seriously affect the quality of life and well-being of the patients. HYPOTHESIS/PURPOSE: This study aimed to investigate the role of hastatoside on liver fibrosis and its possible underlying mechanisms. METHODS: C57BL/6 J mice with carbon tetrachloride (CCl4)-induced hepatic fibrosis were used as the in vivo models. Histological features of the liver were observed using Masson's trichrome and hematoxylin-eosin staining. Alanine aminotransferase and aspartate aminotransferase levels and the hepatic fibrosis indices (type 3 procollagen, laminin, and hyaluronic acid) were measured using corresponding assay kits. LX-2 human hepatic stellate cells (HSCs) stimulated with the transforming growth factor ß1 were used as the vitro models. Transfection of the glycogen synthase kinase (GSK)-3ß small interfering RNA (siRNA) and ß-catenin plasmids was also performed in vitro. Protein levels of GSK-3ß, phospho-GSK-3ß (Ser 9), α-smooth muscle actin, collagen type I alpha 1, c-Myc, cyclin D1, and ß-catenin were determined via western blotting. Moreover, the p-GSK-3ß:GSK-3ß ratio was calculated to determine the GSK-3ß activity. RESULTS: Hastatoside prevented CCl4-induced liver injury and histological damage. It inhibited the upregulation of α-SMA and Col1α1 levels in a CCl4-induced mouse hepatic fibrosis model. In vitro, hastatoside inhibited the proliferation and activation of HSCs by decreasing the expression levels of cyclin D1 and c-Myc and the proportion of LX-2 cells activated in the G0/G1 phase. Molecular docking results showed that hastatoside bound to GSK-3ß. Hastatoside significantly increased the GSK-3ß activity and inhibited the downstream effector expression of ß-catenin. CONCLUSION: These findings suggest that hastatoside can bind to GSK-3ß and promote its activity, while inhibiting the GSK-3ß downstream effector expression of ß-catenin, thereby inhibiting the activation and proliferation of HSCs, which further prevents the development of liver fibrosis. These results provide innovative insights into the underlying liver fibrosis. Moreover, hastatoside is a potential anti-fibrosis monomer that can potentially be used for the treatment of liver fibrosis.

Downregulating PDPK1 and taking phillyrin as PDPK1-targeting drug protect hepatocytes from alcoholic steatohepatitis by promoting autophagy.

The health risk stemming from drinking alcohol is serious, sometimes even life-threatening. Alcoholic steatohepatitis (ASH) is a critical stage leading to cirrhosis and end-stage liver disease. However, its pathogenesis is still far from clearly understood, and a treatment that is widely recognised as effective has not been discovered. Interestingly, PDPK1,3-phosphoinositide-dependent protein kinase 1, also known as PDK1, was observed to be obviously increased in the ASH model by our researchers. We also investigated the protective role of autophagy in ASH. Here, we studied the function of PDPK1 and found an efficient treatment to alleviate symptoms by targeting PDPK1 in ASH. In our study, PDPK1 affected hepatocyte self-healing by inhibiting autophagy. Both inhibiting PDPK1 and the phosphorylation of PDPK1 (ser241) could protect hepatocytes from suffering heavy alcoholic hepatitis.

Betulinic acid prevents liver fibrosis by binding Lck and suppressing Lck in HSC activation and proliferation.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum japonicum Thunb. ex Murray (Hypericaceae), named 'Tianjihuang' is a traditional Chinese medicine with hepatoprotective, antibacterial, and antitumour effects. Betulinic acid (BA) is its active constituent and has been found to have a number of biological effects, including antiviral, anti-inflammatory, and anti-malarial therapeutic properties. Non-alcoholic fatty liver disease and acute alcoholic liver injury have both been proven to benefit from BA. BA's effects and mechanism on liver fibrosis are still unknown. AIM OF THE STUDY: The purpose of this study was to explore the influence of BA on lymphocyte-specific protein tyrosine kinase (Lck), a non-receptor Src family kinase, that reduces liver fibrosis by inhibiting the phosphorylation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways through the interaction of Lck and SOCS1. MATERIALS AND METHODS: A liver fibrosis model was established in vivo with CCl4 using haematoxylin and eosin (HE) staining, Masson staining, immunohistochemical staining, and immunofluorescence staining. Hepatic stellate cells were induced with transforming growth factor (TGF)-ß1 in vitro, using Western blotting, immunofluorescence staining, and a cell scratch assay. RESULTS: In a CCl4-induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, BA markedly reduced fibrosis, as demonstrated by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in vivo and in vitro. BA significantly suppressed the activity and expression of Lck in vitro. Overexpression of Lck may diminish the effect of BA on liver fibrosis. In vitro, BA also greatly increased the expression of suppressor of cytokine signalling 1 (SOCS1) while it considerably inhibited the expression of p-JAK and p-STAT1. CONCLUSIONS: These findings suggest that BA promotes the expression of SOCS1 by the inhibiting the interaction between Lck and SOCS1, followed by the inhibition of JAK/STAT phosphorylation to prevent the progression of liver fibrosis. Therefore, BA could be used as a promising natural supplement for the treatment of liver fibrosis.

Tubular epithelial cell-to-macrophage communication forms a negative feedback loop via extracellular vesicle transfer to promote renal inflammation and apoptosis in diabetic nephropathy.

Background: Macrophage infiltration around lipotoxic tubular epithelial cells (TECs) is a hallmark of diabetic nephropathy (DN). However, how these two types of cells communicate remains obscure. We previously demonstrated that LRG1 was elevated in the process of kidney injury. Here, we demonstrated that macrophage-derived, LRG1-enriched extracellular vesicles (EVs) exacerbated DN. Methods: We induced an experimental T2DM mouse model with a HFD diet for four months. Renal primary epithelial cells and macrophage-derived EVs were isolated from T2D mice by differential ultracentrifugation. To investigate whether lipotoxic TEC-derived EV (EVe) activate macrophages, mouse bone marrow-derived macrophages (BMDMs) were incubated with EVe. To investigate whether activated macrophage-derived EVs (EVm) induce lipotoxic TEC apoptosis, EVm were cocultured with primary renal tubular epithelial cells. Subsequently, we evaluated the effect of LRG1 in EVe by investigating the apoptosis mechanism. Results: We demonstrated that incubation of primary TECs of DN or HK-2 mTECs with lysophosphatidyl choline (LPC) increased the release of EVe. Interestingly, TEC-derived EVe activated an inflammatory phenotype in macrophages and induced the release of macrophage-derived EVm. Furthermore, EVm could induce apoptosis in TECs injured by LPC. Importantly, we found that leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EVe activated macrophages via a TGFßR1-dependent process and that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-enriched EVm induced apoptosis in injured TECs via a death receptor 5 (DR5)-dependent process. Conclusion: Our findings indicated a novel cell communication mechanism between tubular epithelial cells and macrophages in DN, which could be a potential therapeutic target.

Macrophage-derived, LRG1-enriched extracellular vesicles exacerbate aristolochic acid nephropathy in a TGFßR1-dependent manner.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by some herbal medicines, but treatment remains ineffective. We previously found that leucine-rich α-2-glycoprotein 1 (LRG1), which regulates cellular processes, plays an important role in a kidney injury model. However, the underlying mechanism by which LRG1 regulates AAN is still unknown. In this study, we established an AAN model in vivo, a coculture system of macrophages and TECs, and a macrophage/TEC conditioned media culture model in vitro. We found that macrophage infiltration promoted injury, oxidative stress, and apoptosis in TECs. Furthermore, the role of macrophages in AAN was dependent on macrophage-derived extracellular vesicles (EVs). Importantly, we found that macrophage-derived, LRG1-enriched EVs induced TEC injury and apoptosis via a TGFßR1-dependent process. This study may help design a better therapeutic strategy to treat AAN patients.

Sennoside A alleviates inflammatory responses by inhibiting the hypermethylation of SOCS1 in CCl4-induced liver fibrosis.

Liver fibrosis is the consequence of chronic liver injury and is a major challenge to global health. However, successful therapy for liver fibrosis is still lacking. Sennoside A (SA), a commonly used clinical stimulant laxative, is reported to improve hepatic disease, but the underlying mechanisms remain largely elusive. Here, we show for the first time that SA enhanced suppressor of cytokine signaling 1 (SOCS1) expression in a DNA methyltransferase 1 (DNMT1)-dependent manner and thereby attenuated liver fibrosis. Consistently, SA inhibited the expression of the liver fibrogenesis markers α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) and suppressed inflammatory responses in vivo and in vitro. Coculture experiments with macrophages/hepatic stellate cells (HSCs) revealed that SA suppressed HSC proliferation by downregulating proinflammatory cytokines in macrophages. Mechanically, SA promoted the aberrant expression of SOCS1 in liver fibrosis. However, blocking SOCS1 expression weakened the inhibitory effect of SA on HSC proliferation, indicating that SOCS1 may play an important role in mediating the antifibrotic effect of SA. Furthermore, SA inhibited DNMT1-mediated SOCS1 and reduced HSC proliferation by inhibiting inflammatory responses in carbon tetrachloride (CCl4) -induced liver fibrosis.

Gypenoside XLIX protects against acute kidney injury by suppressing IGFBP7/IGF1R-mediated programmed cell death and inflammation.

BACKGROUND: Acute kidney injury (AKI), characterised by excessive inflammatory cell recruitment and programmed cell death, has a high morbidity and mortality; however, effective and specific therapies for AKI are still lacking. OBJECTIVE: This study aimed to evaluate the renoprotective effects of gypenoside XLIX (Gyp XLIX) in AKI. METHODS: The protective effects of Gyp XLIX were tested in two AKI mouse models established using male C57BL/6 mice (aged 6-8 weeks) by a single intraperitoneal injection of cisplatin (20 mg/kg) or renal ischemia-reperfusion for 40 min. Gyp XLIX was administered intraperitoneally before cisplatin administration or renal ischemia-reperfusion. Renal function, tubular injury, renal inflammation and programmed cell death were evaluated. In addition, the renoprotective effects of Gyp XLIX were also evaluated in cisplatin- or hypoxia-treated tubular epithelial cells. The mechanisms underlying these effects were then explored using RNA sequencing. RESULTS: In vivo, Gyp XLIX substantially suppressed the increase in serum creatinine and blood urea nitrogen levels. Moreover, tubular damage was alleviated by Gyp XLIX as shown by periodic acid-Schiff staining, electron microscopy and molecular analysis of KIM-1. Consistently, we found that Gyp XLIX suppressed renal necroptosis though the RIPK1/RIPK3/MLKL pathway. The anti-inflammatory and antinecroptotic effects were further confirmed in vitro. Mechanistically, RNA sequencing showed that Gyp XLIX markedly suppressed the levels of IGF binding protein 7 (IGFBP7). Co-immunoprecipitation and western blot analysis further showed that Gyp XLIX reduced the binding of IGFBP7 to IGF1 receptor (IGF1R). Additionally, picropodophyllin, an inhibitor of IGF1R, abrogated the therapeutic effects of Gyp XLIX on cisplatin-induced renal cell injury; this finding indicated that Gyp XLIX may function by activating IGF1R-mediated downstream signalling Additionally, we also detected the metabolic distribution of Gyp XLIX after injection; Gyp XLIX had a high concentration in the kidney and exhibited a long retention time. These findings may shed light on the application of Gyp XLIX for AKI treatment clinically. CONCLUSION: Gyp XLIX may serve as a potential therapeutic agent for AKI treatment via IGFBP7/ IGF1R-dependent mechanisms.

The permeability characteristics and interaction of main components from Si-Ni-San in a MDCK epithelial cell monolayer model.

1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism.

PSTPIP2 inhibits cisplatin-induced acute kidney injury by suppressing apoptosis of renal tubular epithelial cells.

Cisplatin (CP) is an effective chemotherapeutic agent widely used in the treatment of various solid tumours. However, CP nephrotoxicity is an important limitation for CP use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Recently, we identified a specific role of proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) in cisplatin-induced AKI. PSTPIP2 was reported to play an important role in a variety of diseases. However, the functions of PSTPIP2 in experimental models of cisplatin-induced AKI have not been extensively studied. The present study demonstrated that cisplatin downregulated the expression of PSTPIP2 in the kidney tissue. Administration of AAV-PSTPIP2 or epithelial cell-specific overexpression of PSTPIP2 reduced cisplatin-induced kidney dysfunction and inhibited apoptosis of renal tubular epithelial cells. Small interfering RNA-based knockdown of PSTPIP2 expression abolished PSTPIP2 regulation of epithelial cell apoptosis in vitro. Histone acetylation may impact gene expression at the epigenetic level, and histone deacetylase (HDAC) inhibitors were reported to prevent cisplatin-induced nephrotoxicity. The UCSC database was used to predict that acetylation of histone H3 at lysine 27 (H3K27ac) induces binding to the PSTPIP2 promoter, and this prediction was validated by a ChIP assay. Interestingly, an HDAC-specific inhibitor (TSA) was sufficient to potently upregulate PSTPIP2 in epithelial cells. Histone acetylation-mediated silencing of PSTPIP2 may contribute to cisplatin nephrotoxicity. PSTPIP2 may serve as a potential therapeutic target in the prevention of cisplatin nephrotoxicity.

Sennoside A prevents liver fibrosis by binding DNMT1 and suppressing DNMT1-mediated PTEN hypermethylation in HSC activation and proliferation.

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.

Rutaecarpine derivative Cpd-6c alleviates acute kidney injury by targeting PDE4B, a key enzyme mediating inflammation in cisplatin nephropathy.

Acute kidney injury (AKI), characterized by a rapid decline in renal function, is triggered by an acute inflammatory response that leads to kidney damage. An effective treatment for AKI is lacking. Using in vitro and in vivo AKI models, our laboratory has identified a series of anti-inflammatory molecules and their derivatives. In the current study, we identified the protective role of rutaecarpine (Ru) on renal tubules. We obtained a series of 3-aromatic sulphonamide-substituted Ru derivatives exhibiting enhanced renoprotective and anti-inflammatory function. We identified Compound-6c(Cpd-6c) as having the best activity and examined its protective effect against cisplatin nephropathy both in vivo and in vitro in cisplatin-stimulated tubular epithelial cells (TECs). Our results showed that Cpd-6c restored renal function more effectively than Ru, as evidenced by reduced blood urea nitrogen and serum creatinine levels in mice. Cpd-6c alleviated tubular injury, as shown by PAS staining and molecular analysis of kidney injury molecule-1 (KIM-1), with both prevention and treatment protocols in cisplatin-treated mice. Moreover, Cpd-6c decreased kidney inflammation, oxidative stress and programmed cell death. These results have also been confirmed in cisplatin-treated TECs. Using web-prediction algorithms, molecular docking, and cellular thermal shift assay (CETSA), we identified phosphodiesterase 4B (PDE4B) as a Cpd-6c target. In addition, we firstly found that PDE4B was up-regulated significantly in the serum of AKI patients. After identifying the function of PDE4B in cisplatin-treated tubular epithelial cells by siRNA transfection or PDE4 inhibitor rolipram, we showed that Cpd-6c treatment did not protect against cisplatin-induced injury in PDE4B knockdown TECs, thus indicating that Cpd-6c exerts its renoprotective and anti-oxidative effects via the PDE4B-dependent pathway. Collectively, Cpd-6c might serve as a potential therapeutic agent for AKI and PDE4B may be highly involved in the initiation and progression of AKI.