Identification of stable housekeeping genes in mouse liver for studying carbon tetrachloride-induced injury and cellular senescence.

Acute liver injury (ALI) presents a challenging problem worldwide, prompting extensive research efforts. Cellular senescence has been found to be induced following ALI, and targeting cellular senescence has shown therapeutic potential. Therefore, understanding the expression of senescence-related genes in ALI can help to explore pathogenesis and treatment of this common disease. Carbon tetrachloride (CCl4) is commonly used to study ALI. Although polymerase chain reaction (PCR) is a convenient and economical molecular biology technique widely used in basic medicine, research on selecting suitable reference genes to obtain objective and reproducible PCR data is scarce. Moreover, evidence supporting the choice of reference genes for experimental studies of CCl4-induced ALI and hepatic senescence in mice is limited. In this study, we obtained murine livers at four time points (0, 12, 24, and 48 h) following CCl4 treatment. We used five algorithms (geNorm, BestKeeper, NormFinder, delta Ct, and RefFinder) to rank 12 candidate genes in real-time reverse-transcription quantitative PCR (RT-qPCR) experiments. Focusing on cellular senescence in this model, we adopted four senescence-associated secretory phenotype (SASP) genes (Il6, Il1b, Ccl2, and Ccl5) as target genes. Our results confirmed Gapdh and Tbp as suitable reference genes in murine CCl4-induced ALI models. Furthermore, we provide a table of published studies recommending reference genes for various liver disease models. This study provides a valuable reference for enhancing the reliability and reproducibility of ALI molecular findings.

LC-QTOF-MS/MS metabolic profiling and hepatoprotective effects of Litsea monopetala bark methanol extract against liver injury in rats and HepG2 cells.

Fresh stem bark decoction of Litsea monopetala has been practiced for the treatment of jaundice and other liver disorders by the tribal communities of Thakht-e-Sulaiman hills from West Pakistan. As per the folkloric claim, this study aims to identify the phytoconstituents and evaluate the hepatoprotective action of stem bark methanol extract of L. monopetala (LMME). The in-vitro hepatoprotective effect of L. monopetala was performed by H2O2-induced toxicity in the HepG2 cell line and in-vivo by cclt;sub>4-induced hepatotoxicity in Wistar albino rats taking Silymarin as standard drug. Phytoconstituents were identified using LC-QTOF-MS analysis followed by in-silico docking and validation. Molecular docking interactions between identified compounds of L. monopetala and two target proteins, namely 1VJY and 5HYK were presented. In this study, treatment with LMME at 100 µg/mL showed 67.73 % cell viability as compared to H2O2 (100 µM) treated alone i.e., 18.55 % in the HepG2 cell line. In-vivo treatment of LMME reversed the altered serum biochemical parameters and reduced the inflammatory response similar to that of the Silymarin-treated group supported by histopathological investigation. This research reveals that L. monopetala is a rich source of flavonoids and phenols which supports its hepatoprotective effects and is proposed for its usage as a promising hepatoprotective agent after controlled trials.

Decompensated MASH-Cirrhosis Model by Acute and Toxic Effects of Phenobarbital.

Metabolic dysfunction-associated Steatohepatitis (MASH), is a prominent cause for liver cirrhosis. MASH-cirrhosis is responsible for liver complications and there is no specific treatment. To develop new therapeutic approaches, animal models are needed. The aim of this study was to develop a fast animal model of MASH-cirrhosis in rats reflecting the human disease. Carbon tetrachloride (CCl4) injections in combination with a high-fat Western diet (WD) were used to induce MASH-cirrhosis. To accelerate liver injury, animals received phenobarbital (PB) in their drinking water using two different regimens. Rats developed advanced MASH-cirrhosis characterized by portal hypertension, blood biochemistry, hepatic ballooning, steatosis, inflammation and fibrosis. Importantly, rats receiving low-dose PB for the long term (LT) showed ascites after 6 weeks, whereas rats with high-dose short-term (ST) PB developed ascites after 8 weeks. ST- and LT-treated rats showed increased portal pressure (PP) and decreased mean arterial pressure (MAP). Of note, hepatocyte ballooning was only observed in the LT group. The LT administration of low-dose PB with CCl4 intoxication and WD represents a fast and reproducible rat model mimicking decompensated MASH-cirrhosis in humans. Thus, CCl4 + WD with LT low-dose phenobarbital treatment might be the preferred rat animal model for drug development in MASH-cirrhosis.

Arylacetamide deacetylase regulates hepatic iron homeostasis to protect against carbon tetrachloride-induced ferroptosis.

Arylacetamide deacetylase (AADAC) catalyzes the hydrolysis of small molecules containing ester and amide bonds. Recently, it has been reported that AADAC can suppress reactive oxygen species production in cancer cells. This study aimed to elucidate the possibility that AADAC protects against drug-induced liver injury accompanied by oxidative stress and to explore its molecular mechanisms. Intraperitoneal administration of carbon tetrachloride induced significantly more severe liver injury in Aadac knockout (KO) mice (plasma alanine aminotransferase level: 19,381 ± 10,578 U/L) than in wild-type (WT) mice (7219 ± 4729 U/L). More severe liver injury in Aadac KO mice was accompanied by higher hepatic malondialdehyde and antioxidant gene mRNA levels than those in WT mice. The increase in plasma alanine aminotransferase levels in Aadac KO mice was substantially suppressed by pretreatment with the ferroptosis inhibitors deferoxamine or ferrostatin-1, suggesting that Aadac deficiency increases susceptibility to ferroptosis. Immunoprecipitation followed by proteomic analysis revealed that AADAC interacts with ceruloplasmin (CP), which oxidizes ferrous iron to ferric iron. Hepatic CP activity was significantly lower in Aadac KO mice than that in WT mice, resulting in elevated hepatic ferrous iron levels in Aadac KO mice. Overexpression of human AADAC in Huh-7 cells significantly attenuated carbon tetrachloride-induced cytotoxicity by suppressing ferrous iron accumulation, suggesting that AADAC interacts with CP to suppress hepatic ferrous iron accumulation. The hepatoprotective role of Aadac in ferroptosis was also observed in mice with acetaminophen-induced liver injury. This study demonstrates a novel function of AADAC in protecting against ferroptosis induced by hepatotoxicants, carbon tetrachloride and acetaminophen.

In-Depth Proteomic Analysis Reveals Phenotypic Diversity of Macrophages in Liver Fibrosis.

Macrophages make up a heterogeneous population of immune cells that exhibit diverse phenotypes and functions in health and disease. Although macrophage epigenomic and transcriptomic profiles have been reported, the proteomes of distinct macrophage populations under various pathological conditions remain largely elusive. Here, we employed a label-free proteomic approach to characterize the diversity of the hepatic macrophage pool in an experimental model of CCl4-induced liver fibrosis. We found a decrease in the proportion of liver resident embryo-derived KCs (EmKCs), and a drastic increase in the proportion of monocyte-derived KCs (MoKCs) and CLEC2-Macs. Proteomic profiling revealed that MoKCs largely resembled EmKCs, whereas CLEC2-Macs exhibited greater proteomic alternations compared with EmKCs, suggesting two distinct destinations for monocyte differentiation during liver fibrosis. Furthermore, CLEC2-Macs were characterized by increased expression of proteins associated with inflammatory response, antigen processing and presentation processes, which may be involved in the pathogenesis of liver fibrosis. Collectively, our study provides insights into the considerable heterogeneity within the hepatic macrophage pool during liver fibrosis.

LHPP deficiency aggravates liver fibrosis through TGF-ß/Smad3 signaling.

Liver fibrosis is characterized by a wound-healing response and may progress to liver cirrhosis and even hepatocellular carcinoma. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a tumor suppressor that participates in malignant diseases. However, the role of LHPP in liver fibrosis has not been determined. Herein, the function and regulatory network of LHPP were explored in liver fibrosis. The expression of LHPP in human and murine fibrotic liver tissues was assessed via immunohistochemistry and Western blot analysis. In addition, liver fibrosis was induced in wild-type (WT) and LHPP-/- (KO) mice after carbon tetrachloride (CCl4) or thioacetamide (TAA) treatment. The effect of LHPP was systematically assessed by using specimens acquired from the above murine models. The functional role of LHPP was further explored by detecting the pathway activity of TGF-ß/Smad3 and apoptosis after interfering with LHPP in vitro. To explore whether the function of LHPP depended on the TGF-ß/Smad3 pathway in vivo, an inhibitor of the TGF-ß/Smad3 pathway was used in CCl4-induced WT and KO mice. LHPP expression was downregulated in liver tissue samples from fibrosis patients and fibrotic mice. LHPP deficiency aggravated CCl4- and TAA-induced liver fibrosis. Moreover, through immunoblot analysis, we identified the TGF-ß/Smad3 pathway as a key downstream pathway of LHPP in vivo and in vitro. The effect of LHPP deficiency was reversed by inhibiting the TGF-ß/Smad3 pathway in liver fibrosis. These results revealed that LHPP deficiency exacerbates liver fibrosis through the TGF-ß/Smad3 pathway. LHPP may be a potential therapeutic target in hepatic fibrosis.

Beta-caryophyllene attenuates experimental hepatocellular carcinoma through downregulation of oxidative stress and inflammation.

Hepatocellular carcinoma (HCC) is caused by various factors including toxic substances and xenobiotics. Numerous treatment strategies are used to address toxicity to the liver and HCC, yet their adverse effects are drawbacks. This study aimed to assess the effect of DEN/CCl4 on morphological changes in the liver, body weight, tumor incidence, and hematological tumor incidence, hematological parameters, hepatic markers, and histopathological analysis in mice following a preventive measure by using ß-caryophyllene (BCP). Adult Balb/c mice were administered a single dose of DEN 1-mg/kg body weight and 0.2-mL CCl4/kg body weight intraperitoneal twice a week (i.p.) for 22 weeks. BCP was treated in one group of mice at 30-mg/kg body weight, intraperitoneal, for 7 weeks. BCP alone was treated in one group of mice at 300-mg/kg body weight intraperitoneal for 22 weeks. DEN/CCl4 caused a reduction in mice's body weight, which was significantly attenuated by BCP administration. BCP supplementation attenuated the tumor incidence DEN/CCl4 (100%) to about 25%. DEN/CCl4 caused alterations in the hematological parameters, serum total protein albumin globulin, A/G ratio, liver function markers (AST, ALT, ALP, GGT, ACP, and bilirubin), and lipid profile markers that were significantly reinstated by BCP administration. Oxidative stress markers (MDA, SOD, CAT, NO, LDH, and GST) were reduced by DEN/CCl4, which were significantly increased in BCP-treated groups. The liver histopathology alterations caused by DEN/CCl4 were amended considerably by BCP treatment. Immunohistochemical studies suggest that AFP, caspase-3, and COX-2 were chronically overexpressed in DEN/CCl4-exposed mice, notably attenuated by BCP administration. BCP suppressed tumor incidence by downregulating inflammation and inducing caspase-3-mediated apoptosis. Conclusively, BCP appears to be a potent natural supplement capable of repressing liver inflammation and carcinoma through the mitigation of oxidative stress and inflammation pathways.

The role of forkhead box M1-methionine adenosyltransferase 2 A/2B axis in liver inflammation and fibrosis.

Methionine adenosyltransferase 2 A (MAT2A) and MAT2B are essential for hepatic stellate cells (HSCs) activation. Forkhead box M1 (FOXM1) transgenic mice develop liver inflammation and fibrosis. Here we examine if they crosstalk in male mice. We found FOXM1/MAT2A/2B are upregulated after bile duct ligation (BDL) and carbon tetrachloride (CCl4) treatment in hepatocytes, HSCs and Kupffer cells (KCs). FDI-6, a FOXM1 inhibitor, attenuates the development and reverses the progression of CCl4-induced fibrosis while lowering the expression of FOXM1/MAT2A/2B, which exert reciprocal positive regulation on each other transcriptionally. Knocking down any of them lowers HSCs and KCs activation. Deletion of FOXM1 in hepatocytes, HSCs, and KCs protects from BDL-mediated inflammation and fibrosis comparably. Interestingly, HSCs from Foxm1Hep-/-, hepatocytes from Foxm1HSC-/-, and HSCs and hepatocytes from Foxm1KC-/- have lower FOXM1/MAT2A/2B after BDL. This may be partly due to transfer of extracellular vesicles between different cell types. Altogether, FOXM1/MAT2A/MAT2B axis drives liver inflammation and fibrosis.

FoxD1 expression identifies a distinct subset of hepatic stellate cells involved in liver fibrosis.

Hepatic stellate cells (HSCs) are pericytes of the liver responsible for liver fibrosis and cirrhosis, which are the end stages of chronic liver diseases. TGF-ß activates HSCs, leading to the differentiation of myofibroblasts in the process of liver fibrosis. While the heterogeneity of HSCs is appreciated in the fibrotic liver, it remains elusive which HSC subsets mainly contribute to fibrosis. Here, we show that the expression of the pericyte marker FoxD1 specifically marks a subset of HSCs in FoxD1-fate tracer mice. HSCs fate-mapped by FoxD1 were preferentially localized in the portal and peripheral areas of both the homeostatic and fibrotic liver induced by carbon tetrachloride. Furthermore, the deletion of Cbfß, which is necessary for TGF-ß signaling, in FoxD1-expressing cells ameliorated liver fibrosis. Thus, we identified an HSC subset that preferentially responds to liver injuries.

A peptide alleviated oxidative damages in the L02 cells and mice liver.

The liver is vitally metabolic for a multitude of biochemical reactions. Consequently, it generates many free radicals and reactive oxygen species, rendering it more susceptible to oxidative stress-induced damage. Oxidative stress represents a pivotal factor in the pathogenesis of liver diseases. We screened some antioxidant peptides previously. Here we investigated whether the peptides could attenuate oxidative damage with APPH in L02 cells. The results showed that one of the peptides, sequence FETLMPLWGNK, could decrease the excessive reactive oxygen species, increase antioxidant enzyme activity and protect mitochondrial function, reduce the ratio of apoptosis and S phase cycle arrest, and improve the survival rate of L02 cells damaged by APPH compared to cells of the control group. Then the peptide was evaluated in mice that CCl4 injured. We found that CCl4-injured mice had significantly increased serum inflammatory factors and liver injury markers, a large number of inflammatory cell infiltration, and local necrosis in the liver. The peptide could reduce inflammation, and improve liver pathological changes. This phenomenon may be associated with the activation of the Nrf2 signaling pathway. Concurrently, the peptide protects the liver by regulating the expression of proteins related to the mitochondrial apoptosis pathway (p53, Bax, Bcl-2, and Caspase3) and mitophagy-related proteins (PINK1, Parkin, and AMPKα). Therefore, the results indicated that the peptide is an active substance with antioxidant activity and anti-inflammatory effects.

Alterations in liver triglyceride profiles in CCl4-induced liver regeneration.

Lipid metabolism, particularly triglyceride (TG) metabolism, is crucial for liver regeneration. During the early phase of liver regeneration, the liver temporarily accumulates a substantial amount of TG-dominated lipids. However, the specific composition of the TG profile during this phase is not yet fully understood. Here, we showed that the TG molecular composition in the liver was significantly altered during liver regeneration following carbon tetrachloride (CCl4)-induced liver injury. Lipid accumulation in livers was observed as early as 12 hours after CCl4 treatment, with transient regeneration-associated steatosis (TRAS) lasting until 24 hours. Hepatocyte proliferation began only after liver lipid levels returned to baseline at 48 hours. Furthermore, the profile of TG species changed significantly during liver regeneration. During the TRAS period, the accumulated TGs in the liver were mainly long-chain triglycerides, with most of the fatty acids constituting these triglycerides having fewer than 20 carbon atoms. In the proliferation phase, the fatty acid composition of these triglycerides shifted from long-chain to ultra-long-chain fatty acids. Our results suggest a significant TRAS-related change in the TG lipid profile of the liver during liver regeneration.

Vitamin D receptor attenuates carbon tetrachloride-induced liver fibrosis via downregulation of YAP.

Liver fibrosis is characterized by the excessive accumulation of extracellular matrix proteins, which can lead to cirrhosis and liver cancer. Metabolic dysfunction-associated steatosis liver diseases are common causes of liver fibrosis, sharing a similar pathogenesis with carbon tetrachloride (CCl4) exposure. This process involves the activation of hepatic stellate cells (HSCs) into myofibroblasts. However, the detailed mechanism and effective treatment strategies require further investigation. In this study, we uncovered a negative correlation between VDR expression and YAP within HSCs. Subsequently, we demonstrated that VDR exerted a downregulatory influence on YAP transcriptional activity in HSCs. Intriguingly, activation VDR effectively inhibited the culture induced activation of primary HSCs by suppressing the transcriptional activity of early YAP. Furthermore, in vivo results manifested that hepatic-specific deletion of YAP/TAZ ameliorates CCl4-induced liver fibrosis, and nullified the antifibrotic efficacy of VDR. Importantly, a YAP inhibitor rescued the exacerbation of liver fibrosis induced by hepatic-specific VDR knockout. Moreover, the combined pharmacological of VDR agonist and YAP inhibitor demonstrated a synergistic effect in diminishing CCl4-induced liver fibrosis, primary HSCs activation and hepatic injury in vivo. These effects were underpinned by their collective ability to inhibit HSC activation through AMPK activation, consequently curbing ATP synthesis and HSCs proliferation. In conclusion, our results not only revealed the inhibition of VDR on YAP-activated liver stellate cells but also identified a synergistic effect of VDR agonist and YAP inhibitor in an AMPKα-dependent manner, providing a practical foundation for integration of multi-targeted drugs in the therapy of CCl4-induced hepatic fibrosis.

Novel microwave assisted carboxymethyl-graphene oxide and its hepatoprotective activity.

This study reports a novel, eco-friendly; fast and cost-effective microwave method for synthesizing carboxymethylated graphene oxide (CMGO) from sugarcane residues. Fourier-transform infrared spectroscopy (FTIR) confirmed successful CMGO synthesis through the presence of characteristic peaks at 1567.93 and 1639.29 cm-1 (COONa vibrations) and increased CH2 intensity compared to unmodified graphene oxide (GO). Furthermore, CMGO derived from sugarcane residues demonstrated potential in mitigating the side effects of toxic materials like carbon tetrachloride (CCl4). Treatment with CMGO partially reduced elevated levels of liver enzymes (ALT and AST) and nitrogenous waste products (urea and uric acid) in CCl4-induced liver damage models, suggesting an improvement in liver function despite ongoing cellular damage.This work paves the way for a sustainable and economical approach to produce functionalized graphene oxide with promising biomedical applications in alleviating toxin-induced liver injury.

Cereblon deficiency ameliorates carbon tetrachloride-induced acute hepatotoxicity in HepG2 cells by suppressing MAPK-mediated apoptosis.

The liver is vulnerable to various hepatotoxins, including carbon tetrachloride (CCl4), which induces oxidative stress and apoptosis by producing reactive oxygen species (ROS) and activating the mitogen-activated protein kinase (MAPK) pathway. Cereblon (CRBN), a multifunctional protein implicated in various cellular processes, functions in the pathogenesis of various diseases; however, its function in liver injury remains unknown. We established a CRBN-knockout (KO) HepG2 cell line and examined its effect on CCl4-induced hepatocellular damage. CRBN-KO cells exhibited reduced sensitivity to CCl4-induced cytotoxicity, as evidenced by decreased levels of apoptosis markers, such as cleaved caspase-3, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. CRBN deficiency enhanced antioxidant defense, with increased superoxide dismutase activity and glutathione ratios (GSH/GSSG), as well as reduced pro-inflammatory cytokine expression. Mechanistically, the protective effects of CRBN deficiency appeared to involve the attenuation of the MAPK-mediated pathways, particularly through decreased phosphorylation of JNK and ERK. Overall, these results suggest the crucial role of CRBN in mediating the hepatocellular response to oxidative stress and inflammation triggered by CCl4 exposure, offering potential clinical implications for liver injury in a wide range of liver diseases.

Yes-associated protein inhibition ameliorates carbon tetrachloride-induced acute liver injury in mice by reducing VDR.

Carbon tetrachloride (CCl4) has a wide range of toxic effects, especially causing acute liver injury (ALI), in which rapid compensation for hepatocyte loss ensures liver survival, but proliferation of surviving hepatocytes (known as endoreplication) may imply impaired residual function. Yes-associated protein (YAP) drives hepatocytes to undergo endoreplication and ploidy, the underlying mechanisms of which remain a mystery. In the present study, we uncover during CCl4-mediated ALI accompanied by increased hepatocytes proliferation and YAP activation. Notably, bioinformatics analyses elucidate that hepatic-specific deletion of YAP substantially ameliorated CCl4-induced hepatic proliferation, effectively decreased the vitamin D receptor (VDR) expression. Additionally, a mouse model of acute liver injury substantiated that inhibition of YAP could suppress hepatocytes proliferation via VDR. Furthermore, we also disclosed that the VDR agonist nullifies CCl4-induced ALI alleviated by the YAP inhibitor in vivo. Importantly, hepatocytes were isolated from mice, and it was spotlighted that the anti-proliferative impact of the YAP inhibitor was abolished by the activation of VDR within these hepatocytes. Similarly, primary hepatic stellate cells (HSCs) were isolated and it was manifested that YAP inhibitor suppressed HSC activation via VDR during acute liver injury. Our findings further elucidate the YAP's role in ALI and may provide new avenues for protection against CCl4-drived acute liver injury.

Pituitary-derived small extracellular vesicles promote liver repair by its cargo miR-143-3p.

The small Extracellular vesicles (sEV) has been recognized to be significant for intercellular communication due to their ability to transfer important cellular cargoes like miRNAs through circulation. The pituitary gland has not been clearly known about the role of its secreted sEV under normal physiological conditions. And Liver disease is a global public health burden. The present study is the first to investigate the effect of pituitary sEV on the liver. Sequencing and qRT-PCR revealed miR-143-3p is one of the richest in the pituitary sEV. MiR-143 Knockout (KO) mice resulted in a remarkable decrease in insulin-like growth factor 1 (IGF-1) levels and a significant increase in insulin-like growth factor binding protein 5 (IGFBP5) levels along with a reduction in liver primary cell growth. More importantly, compared with miR-143-KO-sEV, WT-sEV possesses a more robust capacity to improve miR-143 KO mice liver repair through the Wnt/ß-catenin pathway after an acute injury caused by carbon tetrachloride (CCl4). Our results indicate that pituitary-derived sEV promotes hepatocyte proliferation and liver repair by its cargo miR-143-3p and provides new insight into the regulation mechanism of the pituitary-liver axis, and open a new window for endocrine regulation by using sEV.

Microvesicles from quiescent and TGF-ß1 stimulated hepatic stellate cells: Divergent impact on hepatic vascular injury.

BACKGROUND: This study evaluated the effect of microvesicles(MVs) from quiescent and TGF-ß1 stimulated hepatic stellate cells (HSC-MVs, TGF-ß1HSC-MVs) on H2O2-induced human umbilical vein endothelial cells (HUVECs) injury and CCl4-induced rat hepatic vascular injury. METHODS: HUVECs were exposed to hydrogen peroxide (H2O2) to establish a model for vascular endothelial cell injury. HSC-MVs or TGF-ß1HSC-MVs were co-cultured with H2O2-treated HUVECs, respectively. Indicators including cell survival rate, apoptosis rate, oxidative stress, migration, invasion, and angiogenesis were measured. Simultaneously, the expression of proteins such as PI3K, AKT, MEK1+MEK2, ERK1+ERK2, VEGF, eNOS, and CXCR4 was assessed, along with activated caspase-3. SD rats were intraperitoneally injected with CCl4 twice a week for 10 weeks to induce liver injury models. HSC-MVs or TGF-ß1HSC-MVs were injected into the tail vein of rats. Liver and hepatic vascular damage were also detected. RESULTS: In H2O2-treated HUVECs, HSC-MVs increased cell viability, reduced cytotoxicity and apoptosis, improved oxidative stress, migration, and angiogenesis, and upregulated protein expression of PI3K, AKT, MEK1/2, ERK1/2, VEGF, eNOS, and CXCR4. Conversely, TGF-ß1HSC-MVs exhibited opposite effects. CCl4- induced rat hepatic injury model, HSC-MVs reduced the release of ALT and AST, hepatic inflammation, fatty deformation, and liver fibrosis. HSC-MVs also downregulated the protein expression of CD31 and CD34. Conversely, TGF-ß1HSC-MVs demonstrated opposite effects. CONCLUSION: HSC-MVs demonstrated a protective effect on H2O2-treated HUVECs and CCl4-induced rat hepatic injury, while TGF-ß1HSC-MVs had an aggravating effect. The effects of MVs involve PI3K/AKT/VEGF, CXCR4, and MEK/ERK/eNOS pathways.

Bone marrow-derived mesenchymal stem cells reduce CCl4-induced kidney injury and fibrosis in male Wistar rats.

AIM: This study explores the possible therapeutic role of rats and mice bone marrow-derived mesenchymal stem cells (BM-MSCs) on renal damage and toxicity brought on by carbon tetrachloride (CCl4) in Wistar rats. METHODS: Following an intraperitoneal injection of CCl4 (0.5 mL/kg b.w. twice weekly) for eight weeks, male Wistar rats were intravenously treated with rats and mice BM-MSCs (1 × 106 cells in 0.2 mL Dulbecco's Modified Eagle Medium (DMEM)/rat/week) a week for four weeks. Kidney functions were evaluated and kidney samples were examined using hematoxylin and eosin (H&E), Masson's trichrome (MT) staining techniques, and electron microscopy analysis. Kidney cyclooxygenase-2 (COX-2), protein 53 (p53), and tumor necrosis factor-α (TNF-α) were detected by immunohistochemical staining techniques. Additionally, bioindicators of oxidative stress and antioxidant defense systems were identified in kidney tissue. RESULTS: In CCl4-injected rats, serum creatinine, urea, and uric acid levels significantly increased, as did renal lipid peroxidation (LPO), while superoxide dismutase, glutathione peroxidase (GPx), glutathione (GSH) transferase, and GSH levels significantly dropped in the kidneys. Histologically, the kidneys displayed a wide range of structural abnormalities, such as glomerular shrinkage, tubular dilations, inflammatory leukocytic infiltration, fibroblast proliferation, and elevated collagen content. Inflammatory cytokines like COX-2 and TNF-α as well as the pro-apoptotic mediator p53 were considerably upregulated. Treatment of BM-MSCs from mice and rats with CCl4-injected rats considerably reduced the previously noted abnormalities. CONCLUSIONS: By boosting antioxidant defense and reducing apoptosis and inflammation, BM-MSCs from mice and rats were able to enhance kidney function and histological integrity in rats that had received CCl4 injections.

Network pharmacology combined with metabolomics to reveal the anti-fibrotic mechanism of Polygoni Orientalis Fructus in CCl4-induced hepatic fibrosis rats.

Polygoni Orientalis Fructus (POF), a dried ripe fruit of Polygonum orientale L., is commonly used in China for liver disease treatment. However, its therapeutic mechanism remains unclear. The aim of this study was to elucidate the effects of POF on the regulation of endogenous metabolites and identify its key therapeutic targets in hepatic fibrosis (HF) rats by integrating network pharmacology and metabolomics approaches. First, serum liver indices and histopathological analyses were used to evaluate the therapeutic effects of POF on carbon tetrachloride (CCl4)-induced HF. Subsequently, differential metabolites and potential therapeutic targets of POF were screened using plasma metabolomics and network pharmacology, respectively. The key targets of POF were identified by overlapping differential metabolite-associated targets with the potential targets and validated by molecular docking and ELISA experiments. The results showed that POF effectively alleviated HF in rats. A total of 51 metabolites related to HF were screened, and 24 were associated with POF. 232 potential therapeutic targets were identified by network pharmacology analysis. Finally, six key targets were identified through a combined analysis. Furthermore, molecular docking and ELISA validation revealed that AGXT, PAH, and NOS3 are targets of POF action, while CBS, ALDH2, and ARG1 were identified as potential targets. SIGNIFICANCE: POF is now commonly used in the treatment of liver disease, but its mechanism of action remains unclear. Current studies on metabolomics of liver disease primarily focuse on the interpretation of differential metabolites and related metabolic pathways. This research delves into the intricate details of metabolomics findings via network pharmacology to uncover the targets and pathways of drug action.

Estrogen alleviates liver fibrosis and restores metabolic homeostasis in ovariectomy-induced liver injury and carbon tetrachloride (CCl4) exposure.

AIM: Given estrogen's recognized regulatory influence on diverse metabolic and immune functions, this study sought to explore its potential impact on fibrosis and elucidate the underlying metabolic regulations. METHODS: Female mice underwent ovary removal surgery, followed by carbon tetrachloride (CCl4) administration to induce liver injury. Biochemical index analysis and histopathological examination were then conducted. The expression levels of alpha-smooth muscle actin (α-SMA), transforming growth factor-ß (TGF-ß), and collagen type 1 alpha 1 chain (COL1A1) were assessed using western blotting to further elucidate the extent of liver injury. Finally, metabolite extraction and metabolomic analysis were performed to evaluate metabolic changes. RESULTS: Ovary removal exacerbated CCl4-induced liver damage, while estrogen supplementation provided protection against hepatic changes resulting from OVX. Furthermore, estrogen mitigated liver injury induced by CCl4 treatment in vivo. Estrogen supplementation significantly restored liver damage induced by OVX and CCl4. Comparative analysis revealed significant alterations in pathways including aminoacyl-tRNA biosynthesis, glycine, serine, and threonine metabolism, lysine degradation, and taurine and hypotaurine metabolism in estrogen treatment. CONCLUSION: Estrogen supplementation alleviates liver injury induced by OVX and CCl4, highlighting its protective effects against fibrosis and associated metabolic alterations.