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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 TGFβR1-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.

Alcoholic steatohepatitis (ASH) is one of the predominant causes of liver-related morbidity and mortality worldwide. However, effective therapy for ASH is still lacking. Notably, increasing evidence indicates ferroptosis may counteract the injury of the ASH. We recently identified a novel strategy for attenuating ASH by an effective adjuvant via ferroptosis through targeting MDMX. Verbenalin, which is a major compound in Verbena officials L (Verbenaceae), is generally recognized as a kind of safe food by the U.S. Food and Drug Administration. In this study, we found for the first time that Verbenalin is protective against alcoholic liver injury through transmission electron microscopy, Fe2+ content detection and other research methods. Its effect can not only inhibit oxidative stress such as MDA, SOD, GSH, ROS, 4-HNE and other related indicators, but also inhibit the ferroptosis of hepatocytes caused by alcohol. It was further confirmed by computer docking that verbenalin was targeted to inhibit MDMX activity, promote PPAR-alpha activation, and inhibit alcohol-induced ferroptosis in hepatocytes. Hence, Verbenalin might be employed as a promising natural supplement for alcoholic liver injury drug therapy.

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.

Quiescent hepatic stellate cells (qHSCs), activated to myofibroblasts, produce the fibrous scar is an essential event during liver fibrogenesis. Clinical and experimental fibrosis undergoes a remarkable regression when the underlying etiological agent is removed. Some myofibroblasts revert to an inactive phenotype (iHSCs) during regression of fibrosis. However, the mechanism of HSCs activation and reversal is still obscure. The present study demonstrated the expression of Lymphocyte-specific protein tyrosine kinase (LCK) was increased in fibrotic liver but decreased after spontaneous recovery in vivo and in vitro, and this 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 hepatic-adeno-associated virus (AAV9) in C57BL/6 mice ameliorated liver fibrosis. Co-incubation of TGF-β1-induced HSC-T6 with LCK-siRNA inhibited the cell proliferation and activation. Over-expression of LCK hindered MDI-induced HSC-T6 inactivation. Interesting we found LCK interact with suppressor of cytokine signaling 1 (SOCS1) and influenced the expression of p-JAK1 and p-STAT1/3. And LCK has a nagative effect of SOCS1 in liver fibrosis. These data suggest that LCK may play a regulatory role in liver fibrosis by inhibiting SOCS1 and implied LCK as a potential therapeutic target for liver fibrosis treatment.

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.This study aimed to investigate the role of hastatoside on liver fibrosis and its possible underlying mechanisms.C57BL/6 J mice with carbon tetrachloride (CClHastatoside prevented CClThese findings suggest that hastatoside can bind to GSK-3β and promote its activity, while inhibiting the GSK-3β downstream effector expression of β-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.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by some herbal medicines, but treatment remains ineffective. We previously found NADPH oxidases 4 (NOX4), which regulates oxidative stress, play an important role in kidney injury model. However, its regulatory mechanism of action in AAN is still obscure. In this study, we established AAN model in vivo, a co-culture system of macrophage and TEC, and macrophage/TEC conditioned media culture model in vitro respectively. We found macrophages infiltration promoted injury，oxidative stress and apoptosis of TEC. Furthermore, the role of macrophage in AAN was dependent on macrophages-derived EV. Importantly, we found that macrophages-derived, Leucine-rich α-2-glycoprotein 1(LRG1)-enriched EV induced TEC injury and apoptosis of via a TGFβR1-dependent process. Mechanistically, macrophages-derived, LRG1-enriched EV mediating TECs injury by upregulating NOX4 in AAN model. This study may help design a better therapeutic strategy to treat AAN patients.

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 (CCl

4-Methylcoumarin-[5,6–g]-hesperetin (4-MCH) is a hesperidin derivative produced by the structural modification of hesperetin. Alcoholic hepatitis (AH) is the origin of many serious liver diseases that are accompanied by hepatic inflammation. In this study, we detected the anti-inflammatory activity of 4-MCH in EtOH fed mice and examined the potential molecular mechanism of this activity. We found that 4-MCH suppressed the release of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in primary liver macrophages isolated from mice and in EtOH-treated RAW264.7 cells. In addition, we showed that the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) was down-regulated in vivo and in vitro in AH. Furthermore, 4-MCH acted as an activator of PPAR-γ, which could therefore ameliorate the inhibitory effects of EtOH on the expression of PPAR-γ. The impairment of PPAR-γ function (T0070907 or PPAR-γ siRNA treatment) resulted in greater inflammation than that in the control group. Conversely, over-expression of PPAR-γ further reduced the release of inflammatory cytokines from EtOH-stimulated RAW264.7 cells. Additional investigations showed that 4-MCH significantly inhibited the phosphorylation of p65. Collectively, these results indicate that 4-MCH alleviated the inflammatory reaction through PPAR-γ activation via the NF-κB-p65 signaling pathway, which regulates the expression of IL-6 and TNF-α in AH.

Rheumatoid arthritis (RA) is one of the chronic systemic autoimmune diseases that cardinally affect the joints. Many people all over the world suffer from the disease. Fibroblast-like synoviocytes (FLSs) play a significant role in the occurrence and development of RA. The long noncoding RNA maternally expressed gene 3 (MEG3) is an imprinted gene, which participates in various cancers as a tumor suppressor. Previous studies have shown that nucleotide oligomerization domain (NOD)-like receptors 5 (NLRC5) plays a key role in inflammatory and autoimmune diseases. Nonetheless, we know very little about the biofunctionality of MEG3 during the development of RA. In this paper, we used complete Freund's adjuvant (CFA)-induced rats as RA animal models. The level of MEG3 significantly reduced in CFA-induced synovial tissues and FLSs, whereas the NLRC5 levels were increased. Enforced expression of MEG3 may be responsible for the decreased level of NLRC5 and inflammatory cytokine level. The results of methylation-specific PCR suggested that the MEG3 gene promoter was significantly methylated in CFA-induced synovial tissues and FLSs. More important, hypermethylation of MEG3 promoter could be inhibited by 5-aza-2-deoxycytidine (5-azadC; methylation inhibitor). Besides, the expression of NLRC5 significantly decreased followed by 5-azadc. Furthermore, DNA methyltransferases 1 (DNMT1) increased in CFA-induced synovial tissues and cells. These results indicated that MEG3 regulates RA by targeting NLRC5 potentially.

Background: Liver fibrosis is characterized by extensive deposition of extracellular matrix (ECM) components in the liver. RCAN1 (regulator of calcineurin 1), an endogenous inhibitor of calcineurin (CaN), is required for ECM synthesis during hypertrophy of various organs. However, the functional role of RCAN1 in liver fibrogenesis has not yet been addressed. Methods: We induced experimental liver fibrosis in mice by intraperitoneal injection of 10 % CCl4 twice a week. To investigate the functional role of RCAN1.4 in the progression of liver fibrosis, we specifically over-expressed RCAN1.4 in mice liver using rAAV8-packaged RCAN1.4 over-expression plasmid. Following the establishment of the fibrotic mouse model, primary hepatic stellate cells were isolated. Subsequently, we evaluated the effect of RCAN1.4 on hepatic fibrogenesis, hepatic stellate cell activation, and cell survival. The biological role and signaling events for RCAN1 were analyzed by protein-protein interaction (PPI) network. Bisulfite sequencing PCR (BSP) was used to predict the methylated CpG islands in the RCAN1.4 gene promoter. We used the chromatin immunoprecipitation (ChIP assay) to investigate DNA methyltransferases which induced decreased expression of RCAN1.4 in liver fibrosis. Results: Two isoforms of RCAN1 protein were expressed in CCl4-induced liver fibrosis mouse model and HSC-T6 cells cultured with transforming growth factor-beta 1 (TGF-β1). RCAN1 isoform 4 (RCAN1.4) was selectively down-regulated in vivo and in vitro. The BSP analysis indicated the presence of two methylated sites in RCAN1.4 promoter and the downregulated RCAN1.4 expression levels could be restored by 5-aza-2'-deoxycytidine (5-azadC) and DNMTs-RNAi transfection in vitro. ChIP assay was used to demonstrate that the decreased RCAN1.4 expression was associated with DNMT1 and DNMT3b. Furthermore, we established a CCl4-induced liver fibrosis mouse model by injecting the recombinant adeno-associated virus-packaged RCAN1.4 (rAAV8-RCAN1.4) over-expression plasmid through the tail vein. Liver- specific-over-expression of RAN1.4 led to liver function recovery and alleviated ECM deposition. The key protein (a member of the NFAT family of proteins) identified on PPI network data was analyzed in vivo and in vitro. Our results demonstrated that RCAN1.4 over-expression alleviates, whereas its knockdown exacerbates, TGF-β1-induced liver fibrosis in vitro in a CaN/NFAT3 signaling-dependent manner. Conclusions: RCAN1.4 could alleviate liver fibrosis through inhibition of CaN/NFAT3 signaling, and the anti-fibrosis function of RCAN1.4 could be blocked by DNA methylation mediated by DNMT1 and DNMT3b. Thus, RCAN1.4 may serve as a potential therapeutic target in the treatment of liver fibrosis.

Background/Aims: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease with unclear molecular mechanisms. Our study intended to identify potential long non-coding RNAs (lncRNAs) and genes, and to determine the potential molecular mechanisms of NAFLD pathogenesis. Methods: The microarrays of GSE24031 and GSE57425 were downloaded from the Gene Expression Omnibus database. GSE24031 included 4 control and 4 model mice and GSE57425 included 3 control and 3 model mice on the basis of GPL1261 platform. Differentially expressed lncRNAs and mRNAs between control and NAFLD liver tissue were calculated. Gene ontology (GO), pathway enrichment analyses, co-expression network and PPI were performed to analyze the biological roles and pathways for the differentially expressed lncRNAs and mRNAs. Non-alcoholic steatohepatitis (NASH) rats were further chosen to investigate the key protein identified based on co-expression network and protein-protein interaction (PPI) network data. Results: A total of 6 significantly up-regulated and 39 down-regulated lncRNAs, 340 up-regulated and 281 down-regulated mRNAs were identified. LncRNA-mRNA co-expression network were analyzed to show a total of 16 key lncRNAs (node degree > 10) in NAFLD samples compared to control tissues. Three key protein identified on co-expression network and protein-protein interaction (PPI) network data were verified in NASH in vivo. The protein level of ATP-citrate lyase (Acly) was significantly increased while lncNONMMUT010685 and NONMMUT050689 in NAFLD samples, whose regulator gene was x-box binding protein 1 (XBP1) and receptor-interacting protein 1 kinase (RIPK1) respectively, were gradually reduced in NASH. Conclusion: In summary, we found a set of lncRNAs and mRNAs differentially expressed in the development of NAFLD. LncRNA Ttc39aos1 and Acly, may be crucial biomarkers for NAFLD. LncRNA NONMMUT010685 and NONMMUT050689, the regulator of XBP1 gene and RIPK1 gene respectively, played important roles in the development of NAFLD.

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.

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.

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-β1-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-β1-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.

Nephrotoxicity is a major side effect of cisplatin (CP)- and platinum-related chemotherapy, and inflammation contributes to disease pathogenesis. Interleukin-9 (IL-9) is a pleiotropic cytokine associated with inflammation. Here, we investigated the key role of IL-9 as a regulator of protective mechanisms in CP-induced acute kidney injury (AKI). We observed that IL-9 was decreased not only in a CP-induced AKI mouse model but also in THP-1 and RAW264.7 cell lines. Seventy-two hours post-CP injection, renal dysfunction and tubule injury were significantly attenuated in IL-9 overexpression adeno-associated virus 9 (AAV9)-treated mice. The levels of serum urea, serum creatinine, kidney injury molecule-1 (KIM-1), and histological damage were partially diminished following treatment with IL-9. The renoprotective effects of IL-9 may be attributed to the regulation of cytokines, and we found that IL-9 acted on macrophages in a regulatory manner, promoting an anti-inflammatory phenotype. Furthermore, IL-9 enhanced the suppression of macrophage-driven renal inflammation. Inhibition of H3K27 acetylation orchestrated IL-9-mediated renoprotection in CP-induced AKI. Thus, our findings indicate novel and potent anti-inflammatory properties of IL-9 that confer preservation of kidney function and structure in CP-induced AKI, which may counteract kidney disease procession.

The incidence and severity of acute kidney injury (AKI) is increased yearly in diabetic patients. Although the mechanisms for this remain unclear, the prevention of AKI in diabetic nephropathy is feasible and of value. As we detected highly activation of TGF-β/Smad3 signaling in both human biopsy and mouse model of diabetic nephropathy, we hypothesized that Smad3 activation in diabetic kidneys may increase AKI sensitivity. We tested our hypothesis in vitro using TGF-β type II receptor (TGF-βRII) disrupted tubular epithelial cells (TECs) and in vivo in mice with streptozotocin (STZ)-induced diabetic nephropathy before the induction of ischemia/reperfusion (I/R) injury. We found that high glucose (HG)-cultured TECs showed increased inflammation, apoptosis and oxidative stress following hypoxia/reoxygenation (H/R) injury. Disruption of TGF-βRII attenuated cell injury induced by H/R in HG-treated TECs. Consistently, Smad3 knockdown in diabetic kidney attenuated I/R-induced AKI. Mechanistically, Smad3 binds to p53 and enhances p53 activity in cells treated with HG and H/R, which may lead to TECs apoptosis. Additionally, ChIP assay showed that Smad3 bound with the promoter region of NOX4 and induced ROS production and inflammation. In conclusion, our results demonstrate that Smad3 promotes AKI susceptibility in diabetic mice by interacting with p53 and NOX4., Graphical abstract Image 1, Highlights • Smad3 activation in diabetic kidneys may increase AKI sensitivity. • Blockade of Smad3 in diabetic kidney may both prevent AKI and CKD progression. • Smad3 interacts with p53 to enhance TECs apoptosis. • Smad3 binds with promoter region of NOX4 to induce ROS production and inflammation.

Here, we report a novel hole-transporting organic salt (HTOS) based on (4'-(bis(4-methoxyphenyl)amino)-[1,1'-biphenyl]-4-yl)methanaminium bromide, which facilely forms a hole-transporting 2D perovskite on the top surface of 3D perovskite via a spin-coating procedure. This capping 2D perovskite can significantly reduce interface trap densities and enhance hole-extracting abilities of heterojunction region between 3D perovskite and hole-transporting layer (HTL), owing to its spatially and potentially confined hole-charge carriers, simultaneously. This interfacial HTOS induced 2D perovskite can effectively suppress nonradiative recombination, which leads to an increase in the photovoltage of perovskite solar cells (PSC) to more than 1.20 V. As a result, the n-i-p planar PSC can achieve a champion power conversion efficiency of 20.71% as well as excellent operational stability exhibiting 92% of initial efficiency after 500 h continuous illumination without encapsulation. This work provides a facile approach to fabricate hole-transporting surface-2D and light-harvesting bulk-3D hierarchy perovskite structure, which delivers significantly enhanced efficiency and stability of PSCs.

Acute kidney injury (AKI), characterized by aggressive inflammatory responses and destruction of renal resident cells, can cause abrupt kidney dysfunction. To date, effective therapy for AKI is lacking. In this study, we evaluated the renoprotective effect of wogonin, an herbal active compound, using a cisplatin-induced AKI mouse model. In vivo results show that wogonin substantially suppressed the increased levels of serum creatinine and blood urea nitrogen (BUN) almost to the normal level. Wogonin also attenuated tubular damage, shown by PAS staining, electron microscopy and molecular analysis of KIM-1. In addition, wogonin suppressed kidney inflammation as indicated by a >60% decrease in macrophage infiltration, a >50% reduction in inflammatory cytokine production and inhibited NF-κB activation in the injured kidney. Mechanistically, molecular docking results show that wogonin effectively inhibited RIPK1 by occupying the ATP-binding pocket of the enzyme, which is a key regulator of necroptosis. Moreover, inhibition of RIPK1, or RIPK3, reversed the protective effects of wogonin in cisplatin-treated HK2 cells, indicating wogonin works in a RIPK1/RIPK3-dependent manner. Surprisingly, wogonin enhanced the anti-proliferative effect of cisplatin on human hepatoma HepG2 cells. Thus, our findings suggest wogonin may be a renoprotective adjuvant for cisplatin-based anticancer therapy.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease, in which pathogenesis is not clear. Many research demonstrated that fibroblast-like synoviocytes (FLSs) play a key role in RA pathogenesis, join in the cartilage injury and hyperplasia of the synovium, and contribute to the release of inflammatory cytokines. We used adjuvant arthritis (AA) rats as RA animal models. The methyl-CpG-binding protein 2 (MeCP2) enables the suppressed chromatin structure to be selectively detected in AA FLSs. Overexpression of this protein leads to an increase of integral methylation levels. Some research has confirmed the hedgehog (Hh) signaling pathway plays an important role in RA pathogenesis; furthermore, patched 1 (PTCH1) is a negative fraction of Hh signaling pathway. We used 5-aza-2'-deoxycytidine (5-azadc) as DNA methylation inhibitor. In our research, we found MeCP2 reduced PTCH1 expression in AA FLSs; 5-azadc obstructed the loss of PTCH1 expression. 5-Azadc, treatment of AA FLSs, also blocks the release of inflammatory cytokines. In order to probe the potential molecular mechanism, we assumed the epigenetic participation in the regulation of PTCH1. Results demonstrated that PTCH1 hypermethylation is related to the persistent FLS activation and inflammation in AA rats. Knockdown of MeCP2 using small-interfering RNA technique added PTCH1 expression in AA FLSs. Our results indicate that DNA methylation may offer molecule mechanisms, and the reduced PTCH1 methylation level could regulate inflammation through knockdown of MeCP2. Graphical Abstract PTCH1 is an inhibitory protein of the Hedgehog signaling pathway. Increased expression of PTCH1 can inhibit the expression of Gli1 and Shh, thereby inhibiting the activation of Hedgehog signaling pathway. Inactivated Hedgehog signaling pathway inhibits the secretion of IL-6 and TNF-α. MeCP2 mediates hypermethylation of PTCH1 gene and decreases the expression of PTCH1 protein, thus activating Hedgehog signaling pathway and increasing secretion of IL-6 and TNF-α.

Liver fibrosis, resulting from chronic and persistent injury to the liver, is a worldwide health problem. Advanced liver fibrosis results in cirrhosis, liver failure and even hepatocellular cancer (HCC), often eventually requiring liver transplantation, poses a huge health burden on the global community. However, the specific pathogenesis of liver fibrosis remains not fully understood. Numerous basic and clinical studies have provided evidence that epigenetic modifications, especially DNA methylation, might contribute to the activation of hepatic stellate cells (HSCs), the pivotal cell type responsible for the fibrous scar in liver. Here, reduced representation bisulfite sequencing (RRBS) and bisulfite pyrosequencing PCR (BSP) analysis identified hypermethylation status of Septin9 (Sept9) gene in liver fibrogenesis. Sept9 protein was dramatically decreased in livers of CCl4-treated mice and immortalized HSC-T6 cells exposed to TGF-β1. Nevertheless, the suppression of Sept9 could be blocked by DNMT3a-siRNA and DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-azadC). Overexpressed Sept9 attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and Col1a1, accompanied by up-regulation of cell apoptosis-related proteins. Conversely, RNAi-mediated silencing of Sept9 enhanced accumulation of extracellular matrix. These observations suggested that Sept9 contributed to alleviate liver fibrosis might partially through promoting activated HSCs apoptosis and this anti-fibrogenesis effect might be blocked by DNMT-3a mediated methylation of Sept9. Therefore, pharmacological agents that inhibit Sept9 methylation and increase its expression could be considered as valuable treatments for liver fibrosis.

Alcohol consumption causes renal injury and compromises kidney function. The underlying mechanism of the alcoholic kidney disease remains largely unknown. In the present study, an alcoholic renal fibrosis animal model was first employed which mice received liquid diet containing alcohol for 4 to 12 weeks. The Masson’s Trichrome staining analysis showed that kidney fibrosis increased at week 8 and 12 in the animal model that was further confirmed by albumin assay, Western blot, immunostaining and real-time PCR of fibrotic indexes (collagen I and α-SMA). In vitro analysis also confirmed that alcohol significantly induced fibrotic response (collagen I and α-SMA) in HK2 tubular epithelial cells. Importantly, both in vivo and in vitro studies showed alcohol treatments decreased Smad7 and activated Smad3. We further determined how the alcohol affected the balance of Smad7 (inhibitory Smad) and Smad3 (regulatory Smad). Genome-wide methylation sequencing showed an increased DNA methylation of many genes and bisulfite sequencing analysis showed an increased DNA methylation of Smad7 after alcohol ingestion. We also found DNA methylation of Smad7 was mediated by DNMT1 in ethyl alcohol (EtOH)-treated HK2 cells. Knockdown of Nox2 or Nox4 decreased DNMT1 and rebalanced Smad7/Smad3 axis, and thereby relieved EtOH-induced fibrotic response. The inhibition of reactive oxygen species by the intraperitoneal injection of apocynin attenuated renal fibrosis and restored renal function in the alcoholic mice. Collectively, we established novel in vivo and in vitro alcoholic kidney fibrosis models and found that alcohol induces renal fibrosis by activating oxidative stress-induced DNA methylation of Smad7. Suppression of Nox-mediated oxidative stress may be a potential therapy for long-term alcohol abuse-induced kidney fibrosis.

Via promoting synovitis, pannus growth and cartilage/bone destruction, fibroblast-like synovial cells (FLSs) play a significant role in the pathogenesis of rheumatoid arthritis (RA). In our study, rats were induced with complete freund's adjuvant (CFA) to be animal models for studying the RA pathogenesis. Microtubule-associated Serine/Threonine-protein kinase 3 (MAST3) has been documented to play a critical role in regulating the immune response of IBD (Inflammatory bowel disease) and involved in the process of cytoskeleton organization, intracellular signal transduction and peptidyl-serine phosphorylation, but its role in the progression of RA remains unknown and is warranted for investigation. So, we tried our best to investigate the mechanism and signaling pathway of MAST3 in RA progression. In the synovial tissue and FLSs of AA rats, we have found that MAST3 was significantly up-regulated than normal. Furthermore, MAST3 overexpression could promote proliferation and inflammatory response of FLSs. In the aspect of mechanism, we discovered that the expression of MAST3 might involve in NF-κB signaling pathway in RA. On the whole, our results suggested that MAST3 might promote the proliferation and inflammation of FLSs by regulating NF-κB signaling pathway.

Acute kidney injury (AKI) is a destructive clinical condition induced by multiple insults including ischemic reperfusion, nephrotoxic drugs and sepsis. It is characterized by a sudden decline in renal function, in addition to excessive inflammation, oxidative stress and programmed cell death of renal tubular epithelial cells. RIPK1-mediated necroptosis plays an important role in AKI. In the present study, we evaluated the treatment effects of Compound-71 (Cpd-71), a novel RIPK1 inhibitor, by comparing with Necrostatin-1 (Nec-1), a classic RIPK1 inhibitor, which has several drawbacks like the narrow structure–activity relationship (SAR) profile, moderate potency and non-ideal pharmacokinetic properties, in vivo and in vitro. Our results showed that pretreatment of Cpd-71 attenuated cisplatin-induced renal injury, restored renal function and suppressed renal inflammation, oxidative stress and cell necroptosis. In addition, Cpd-71 inhibited renal damage while reducing the up-regulated serum creatinine (Cr) and blood urea nitrogen (BUN) levels in established AKI mice model. Consistently, we confirmed that Cpd-71 exhibited more effectively suppressive effect on cisplatin-induced renal tubular cell necroptosis than Nec-1, by physically binding to the allosteric type III ligand binding site of RIPK1, thereby reduced RIPK1 kinase activity, RIPK1/RIPK3 complex formation and phosphor-MLKL membrane translocation by molecular docking, Western blot, co-immunoprecipitation and cellular thermal shift assay (CETSA). Taken together, we currently showed that targeting RIPK1 with Cpd-71 may serve as a promising clinical candidate for AKI treatment.

Nephrotoxicity limits the use of cisplatin (CP) in cancer therapy; however, current clinical measures of renal health do not reflect low degrees of kidney injury. Therefore, discovering new biomarkers for CP-induced acute kidney injury (AKI) is essential for monitoring kidney health during therapy. To identify novel candidate biomarkers in urine for reduced renal function due to CP therapy, we conducted a pilot study on cancer patients eligible for CP treatment. Urine from 30 patients was collected before (baseline) and after 3d of intravenous CP infusion. Urine samples were subjected to Isobaric Tag for Relative Absolute Quantitation (iTRAQ) analysis. Biological roles and pathways for the proteins with altered urine concentrations were identified using bioinformatic tools ITRAQ analysis detected 1411 proteins, 12 of which showed significantly altered levels. Growth differentiation factor-15 (GDF15), leucine-rich alpha-2-glycoprotein 1 (LRG1), and secreted phosphoprotein 1 (SPP1/ osteopontin, OPN) were identified as potential candidate markers by proteomic analysis and were validated by ELISA in another 30 patients and in a CP-induced AKI mouse model. Therefore, GDF15, LRG1, and SPP1 may be applied as novel candidate urinary markers of kidney injury after cisplatin treatment. These findings may facilitate the development of new methods to monitor kidney function, particularly in CP-based chemotherapy. SIGNIFICANCE: Cisplatin is pivotal for cancer treatment; however, nephrotoxicity limits its use. Clinical measures for renal health are not reflective of early signs of acute kidney injury. Thus, new indicators of the state of renal health following cisplatin treatment will have to be discovered. This study reports the use of proteomics to mine for candidate markers of kidney injury in the urine of patients undergoing treatment with cisplatin. Results were experimentally validated using patient urine and a mouse model of cisplatin-induced acute kidney injury. The novel candidate biomarkers reported in this study may be used for the non-invasive monitoring of renal health and in mitigating the side-effects of cisplatin during the course of cancer treatment.

Macrophages are heterogeneous and plastic cells which are able to undergo dynamic transition between M1 and M2 polarized phenotypes in response to the microenvironment signals. However, the underlying molecular mechanisms of macrophage polarization are still obscure. In the current study, it was revealed that miR-146a might play a pivotal role in macrophage polarization. As our results indicated, miR-146a was highly expressed in M2 macrophages rather than M1 macrophages. Over-expression of miR-146a resulted in significantly decreased production of pro-inflammatory cytokines including iNOS and TNF-α in M1 macrophages, while increased production of M2 marker genes such as Arg1 and CD206 in M2 macrophages. In contrast, knockdown of miR-146a promoted M1 macrophage polarization but diminished M2 macrophage polarization. Mechanistically, it was revealed that miR-146a modulated macrophage polarization by targeting Notch1. Of note, PPARγ was responsible as another target for miR-146a-mediated macrophage polarization. Taken together, it was suggested that miR-146a might serve as a molecular regulator in macrophage polarization and is a potential therapeutic target for inflammatory diseases.

A sensitive and reliable HPLC-MS/MS method was developed and validated for the determination of free (unconjugated), glucuronidated, sulfated, and total (free and conjugated) HDND-7 in rat plasma and tissues. Plasma and tissues samples were treated prior to and after the enzyme hydrolysis. Chromatographic separation was achieved on a Phenomenex Luna C18 column (150 × 4.6mm, 3 μm), using isocratic mobile phase consisting of 0.1% formic acid-acetonitrile (50:50, v/v) at a flow rate of 300 μl/min. The detection was performed on a triple quadruple tandem mass spectrometer using positive electrospray ionization (ESI) source with a chromatographic run time of 5.0 min. The detection was operated by multiple reaction monitoring (MRM) of the transitions of m/z 429.3 → 223.9 for HDND-7 and 272.9 → 152.9 for naringenin (IS), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The calibration curves for plasma and tissues were linear over a wide concentration range of 0.02-40 μg/ml with a lower limit of quantification (LLOQ) of 0.02 μg/ml. Mean extraction recoveries in plasma and tissues ranged from 87.4 to 97.1% and from 54.2 to 70.5%, respectively. The intra- and inter-day precision values were below 15% and the accuracy was within ± 15%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study following oral doses of 25, 50 and 100mg/kg and intravenous dose of 25mg/kg, and tissue distribution study following oral dose of 50mg/kg.

Rapid processing technologies of perovskite solar cells (PSCs) offer an exciting approach to raise the rate of production. Herein, a rapid microwave‐annealing process (MAP) is reported to replace the traditional hotplate annealing process (HAP) and the processing period of perovskite is reduced to less than 1 min. Benefiting from the penetrability and simultaneity of microwave irradiation, the MAP method can effectively eliminate miscellaneous phases and thus achieve >1 µm large‐size crystal grains in perovskite films. These MAP treated perovskite films exhibit pure crystalline phase, long charge‐carrier lifetime, and low defect density, which can substantially improve the PSC efficiency without requiring an additional enhancer/passivation layer. The inverted planar PSCs present enhanced power conversion efficiency from 18.33% (HAP) to 21.59% (MAP) and good stability of >1000 h lifetime without encapsulation under ambient conditions. In addition, MAP can be applied to a large‐size (10 cm × 10 cm) perovskite film fabrication as well as a broader tolerance in environmental temperature and precursor concentration, making it a reliable method for repeatably practical fabrication of perovskite photovoltaics., Microwave irradiation can accelerate the annealing period of perovskite films from the minute to second scale. The microwave annealed perovskite possesses >1 µm crystal grain size, eliminates miscellaneous phases, and facilitates >21% power‐conversion efficiency.

ABSTRAT Alcoholic liver disease (ALD) and its complication continued to be a major health problem throughout the world. Increasing evidence suggests that microRNA (miRNA) that regulate apoptosis, inflammation and lipid metabolism are affected by alcohol in ALD. MiR-200a has emerged as a major regulator in several liver diseases, but its role in ALD has not been elucidated. The aim of this study is to figure out the biological function of miR-200a in ALD and to explore its underlying mechanism. The expression pattern of miR-200a were analyzed in vitro and in vivo, we showed that miR-200a was up-regulated in ALD in AML-12 and primary hepatocyte. We then examined it's effect on cell apoptosis and identified zinc finger E-box binding homeobox 2 (ZEB2; also known as SIP1) as a direct target gene of miR-200a. Furthermore, reintroduction of ZEB2 could reverse the pro-apoptosis of miR-200a on AML-12. Taken together, our study demonstrated that miR-200a regulates the apoptosis of hepatocyte in ALD by directly target ZEB2, both of which could serve as new therapeutic targets for ALD.

Hepatic stellate cell (HSC) activation is a significant event in the development of liver fibrosis. Promoting the activated HSCs apoptosis contributes to the reversal of liver fibrosis. Autophagy is considered to be critical for many cellular and pathological processes including liver fibrosis. Transient receptor potential vanilloid 4 (TRPV4), another member of the transient receptor potential (TRP) channel, is proved to be a vital modulator in regulating HSC proliferation during liver fibrosis. However, the precise mechanism of TRPV4 on HSC apoptosis is still unclear. Here, we explored the role of TRPV4 in regulating HSC-T6 cell apoptosis. Our study detected that the expressions of TRPV4 mRNA and protein were dramatically increased in HSC-T6 in response to TGF-β1 stimulation by qRT-PCR and Western blot. Moreover, the HSC-T6 transfected with si-TRPV4 increased apoptosis and inhibited autophagy. In addition, the HSC-T6 treated with 4α-phorbol 12,13-didecanoate results in suppression of apoptosis and increase of autophagy. Furthermore, we indicated that TRPV4 induces autophagy by regulating AKT signaling pathway. In addition, we found that blockade of autophagy by chemical antagonists chloroquine (CQ) leads to increased apoptosis. Furthermore, blocking autophagy by CQ did not lead to a distinct change with or without TRPV4 over-expression. These results indicated that TRPV4 could inhibit HSCs apoptosis partially by regulating autophagy-dependent AKT signaling pathway activation.

Long noncoding RNAs (lncRNAs) are being increasingly recognized as major players in governing fundamental biological processes through diverse mechanisms. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a lncRNA correlated with several human cancers. Recently, the methylation-dependent downregulation of MEG3 has been described in liver cancers. However, its biological functional role in liver fibrosis remains unknown. In our study, MEG3 levels were remarkably decreased in CCl4-induced mouse liver fibrosis models and human fibrotic livers as demonstrated by real-time quantitative PCR. Moreover, the expression of MEG3 was downregulated in human hepatic stellate cell lines LX-2 cells in response to transforming growth factor-β1 (TGF-β1) stimulation in dose and time-dependent manner. Enforced expression of MEG3 in LX-2 cells inhibited TGF-β1-induced cell proliferation, while promoting cell apoptosis. In addition, hypermethylation of MEG3 promoter was identified by methylation-specific PCR and MEG3 expression was robustly increased by the inhibition of methylation with either 5-aza-2-deoxycytidine (5-azadC), or siRNA to DNA methyltransferase 1 (DNMT1) in TGF-β1-induced LX-2 cells. More importantly, overexpression of MEG3 could activate p53 and mediate cytochrome c release, subsequently leading to caspase-3-dependent apoptosis in TGF-β1-treated LX-2 cells. These findings suggested that MEG3 may play an important role in stellate cell activation and liver fibrosis progression and act as a novel potential therapeutic target for liver fibrosis.

Background Chemotherapy has been widely used to treat cancer, but the appearance of multidrug resistance (MDR) is the biggest obstacle to successful chemotherapy. One of the conventional mechanisms of MDR is overexpression of ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp/ABCB1) and multidrug resistance-associated proteins (MRPs/ABCCs) that limits the prolonged and efficient use of chemotherapeutic drugs. To enhance the chemosensitivity of tumor cells, attentions have been focused on effective MDR modulators. Purpose This study aimed to investigate the reversal effect of quercetin on MDR, and explored its mechanism of action in vitro. Study design/methods The effect and mechanism of quercetin on MDR was examined by using MTT assay, flow cytometry, real-time PCR and western blot analysis in human hepatocellular carcinoma cells. Results Our data found that the intracellular accumulation of rhodamine-123 (Rh123) and doxorubicin (ADR) were increased, the sensitivity of BEL/5-FU cells to chemotherapeutic drugs were increased, and the expressions of ABCB1, ABCC1 and ABCC2 were all down-regulated, which indicated that the functions and expressions of ABCB1, ABCC1 and ABCC2 efflux pump were inhibited by quercetin treatment. Moreover, the suppression of ABCB1, ABCC1 and ABCC2 by quercetin was dependent on the FZD7 through the Wnt/β-catenin pathway. Further research revealed that reduction of FZD7 by RNA interference (siFZD7) enhanced the sensitivity to chemotherapeutic drugs, increased the cellular accumulation of Rh123 and ADR, and induced inhibitory effects on the expression of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin, similar to quercetin. In the meanwhile, overexpression of FZD7 showed the inversely effect on the expressions. Interesting, it was confirmed that quercetin could inhibit the expression levels of FZD7, ABCB1, ABCC1, ABCC2 and β-catenin in BEL-7402 cells; furthermore, treatment by quercetin combined with siFZD7 in BEL/5-FU cells, the expressions of these genes were effectively decreased in comparison to quercetin combined with siRNA negative control (sncRNA). Conclusion Overall, these data suggested the effectiveness of using quercetin, at least in part, via inhibiting FZD7 to combat chemoresistance and showed that quercetin could be developed into an efficient natural sensitizer for resistant human hepatocellular carcinoma.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease and the pathogenesis remains unclear. Previous studies suggested that fibroblast-like synoviocytes (FLSs) play an important role in RA pathogenesis, including the injury of cartilage, the hyperplasia of the synovium and the release of inflammatory cytokines. We used complete Freund’s adjuvant (CFA) induced rats as animal models for studying the RA pathogenesis. NLRC5 as the largest member of the NLR family has been reported to play a critical role in regulating immune responses. Increasing evidence suggests that NLRC5 is an pivotal negative modulator of inflammatory pathways. We investigated the mechanisms and signaling pathways of NLRC5 in RA progression. Significantly increased expression of NLRC5 was found in AA rats synovial tissues and cells. And high expression of inflammatory cytokine and cell proliferation of FLSs accompanied with NLRC5 overexpression, but inhibited in cells with NLRC5 silencing treatment. Interestingly, we found that overexpression of NLRC5 also coordinated the activation of NF-κB signaling pathway. These results suggested that NLRC5 promotes RA progression via the NF-κB signaling pathway potentially.

A flavonoid hesperetin is reported to have a variety of biological activities, including anticancer, antiviral, antioxidant, neuroprotective and anti-inflammatory properties. Thirty-one novel hesperetin derivatives were designed, synthesized and evaluated for anti-inflammatory activity using RAW264.7 cells and CCl4-induced acute liver injury model. Among these compounds, 5b displayed the excellent anti-inflammatory activity on decreasing NO, IL-6 and TNF-α both in vitro and vivo. In addition, 5b could also reduce the release of NO, IL-6 and TNF-α production by LPS stimulated RAW 264.7 cell through MAPK and NF-κB signaling pathway in a concentration dependent manner. From in vivo study, it was also observed that 5b attenuated liver histopathologic changes in mouse models.