Pathological Changes Of Liver Tissues Research Articles

The Antidiabetic Effect and Mechanism of JinXiaoXiaoKe Decoction in Type 2 Diabetic Goto–Kakizaki Rats

Modern research and clinical practice have proved that Traditional Chinese Medicine (TCM) has unique advantages in the treatment of diabetes. JinXiaoXiaoKe decoction (JXXKD) is a prescription for treating diabetes used in ancient China and still has a good clinical effect today. However, the mechanism of JXXKD against T2DM is unclear. The purpose of this study is to screen the targets through network pharmacology, and to explore the therapeutic effect and mechanism of JXXKD on diabetic rats. The JXXKD's active components, related targets and T2DM targets were obtained from the public database, and Venny 2.1 was used to determine the common targets of JXXKD in the treatment of T2DM, and PPI, GO and KEGG analysis were performed. The core components and targets are verified by molecular docking. The diabetic Goto–Kakizaki rat model was established by high-sugar and high-fat diet, Wistar rats were used as a blank control group. The diabetic rats were randomized into three groups and administered saline (MO), metformin (MET), or JXXKD once a day for 4 weeks. Fasting blood glucose (FBG), Fasting serum insulin (FINS) , and HOMA-IR were detected, hematoxylin-eosin (HE) staining was used to observe the pathological changes of liver tissue; and qPCR was used to detect the expression of relevant genes screened by network pharmacology. 93 active components and 296 targets of JXXKD were identified, of which 156 overlapped with T2DM-related targets. PPI network showed that APP, AKT1, ANXA1, RXRA, C3, EGFR, ESR1, RELA, IL-6 , and MAPK8 were the top 10 relevant targets. GO analysis showed the common targets are mainly involved in oxidative stress, lipid metabolism, and nutrient levels, while KEGG analysis showed these targets may regulate lipids and atherosclerosis, AGE-RAGE signaling pathway, and TNF signaling pathway. Molecular docking suggested a satisfactory potential for key components to bind to these significant targets. The animal experiments showed that JXXKD significantly improved the symptoms of polyuria, decreased the levels of FBG and HOMA-IR, improved liver fat deposition, and decreased the expressions of foxO1, PPARγ and Akt in diabetic rats. The mechanism of JXXKD treat T2DM may be achieved by modulating the expression of foxO1, PPARγ, and Akt, regulating the glucose and lipids metabolism.

The Metabolites and Mechanism Analysis of Genistin against Hyperlipidemia via the UHPLC-Q-Exactive Orbitrap Mass Spectrometer and Metabolomics.

Genistin, an isoflavone, has been reported to have multiple activities. However, its improvement of hyperlipidemia is still unclear, and the same is true with regard to its mechanism. In this study, a high-fat diet (HFD) was used to induce a hyperlipidemic rat model. The metabolites of genistin in normal and hyperlipidemic rats were first identified to cause metabolic differences with Ultra-High-Performance Liquid Chromatography Quadrupole Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS). The relevant factors were determined via ELISA, and the pathological changes of liver tissue were examined via H&E staining and Oil red O staining, which evaluated the functions of genistin. The related mechanism was elucidated through metabolomics and Spearman correlation analysis. The results showed that 13 metabolites of genistin were identified in plasma from normal and hyperlipidemic rats. Of those metabolites, seven were found in normal rat, and three existed in two models, with those metabolites being involved in the reactions of decarbonylation, arabinosylation, hydroxylation, and methylation. Three metabolites, including the product of dehydroxymethylation, decarbonylation, and carbonyl hydrogenation, were identified in hyperlipidemic rats for the first time. Accordingly, the pharmacodynamic results first revealed that genistin could significantly reduce the level of lipid factors (p < 0.05), inhibited lipid accumulation in the liver, and reversed the liver function abnormalities caused by lipid peroxidation. For metabolomics results, HFD could significantly alter the levels of 15 endogenous metabolites, and genistin could reverse them. Creatine might be a beneficial biomarker for the activity of genistin against hyperlipidemia, as revealed via multivariate correlation analysis. These results, which have not been reported in the previous literature, may provide the foundation for genistin as a new lipid-lowering agent.

Aspartate Reduces Liver Inflammation and Fibrosis by Suppressing the NLRP3 Inflammasome Pathway via Upregulating NS3TP1 Expression.

Aspartate (Asp) can act on liver Kupffer cells, inhibit NOD-like receptor-P 3 (NLRP3) inflammatory bodies, and improve liver inflammation in acute hepatitis. However, the effect of Asp on the role of hepatic stellate cells (HSCs) in the pathogenesis of liver fibrosis in chronic liver injury remains unexplored. This study aimed to investigate the effects of Asp on CCl4-induced liver fibrosis in mice and HSCs via the NF-κB/NLRP3 signaling pathway. Liver fibrosis was induced in C57BL/6J mice by intraperitoneally (IP) injecting 0.5 mL/kg 2% CCl4 three times weekly for 8 weeks. Asp was administered to mice by gavage once every morning for 4 weeks. Masson's trichrome staining, Sirius red staining and hematoxylin and eosin staining were used to detect and analyze the pathological changes in liver tissues. Western blot analysis and immunohistochemistry were applied to determine the protein expression levels of α-smooth muscle actin (α-SMA), collagen Ⅲ (COL III), NLRP3, and IL-1β. In addition, reverse transcription-quantitative PCR was performed to detect the mRNA expression levels. In the liver fibrosis model, the pathological changes in liver tissues improved following treatment with Asp. A marked decrease was observed in protein and mRNA expression levels of α-SMA, COL III, NLRP3, and IL-1β. In addition, HSCs were treated with Asp. The expression levels of α-SMA, COL III, NLRP3, and IL-1β reduced in dose- and time-dependent manners. Furthermore, Asp upregulated the expression of NS3TP1 in vivo and in vitro, and NS3TP1 had a significant inhibitory effect on liver fibrosis. Asp attenuated liver fibrosis and reduced collagen production by suppressing the NF-κB/NLRP3 signaling pathway via upregulating the expression of NS3TP1.

ADSCs-exo attenuates hepatic ischemia-reperfusion injury after hepatectomy by inhibiting endoplasmic reticulum stress and inflammation.

Hepatic ischemia-reperfusion (I/R) injury commonly occurs during liver surgery. Exosomes from adipose-derived stem cells (ADSCs-exo) induce a hepatoprotective effect during hepatic I/R injury. This study aimed to investigate the possible mechanism by which ADSCs-exo attenuates hepatic I/R injury in rats. Rats were randomly divided into four groups: Sham, I30R + PH, ADSCs, and ADSCs-exo groups. Liver tissues were collected immediately after 24 h of reperfusion for further analyses. The content of inflammatory factors in liver tissue was detected using enzyme-linked immunosorbent assay. The pathological changes in liver tissue were analyzed using HE staining. Transmission electron microscopy was used to visualize the ultrastructural changes of hepatocytes. Real-time quantitative polymerase chain reaction(RT-qPCR) and western blot analysis were used to detect the expression of endoplasmic reticulum stress (ERS)-related genes and proteins. Liver histomorphology and hepatocyte ultrastructure changes improved after ADSCs-exo treatment. Moreover, ADSCs-exo treatment significantly downregulated tumor necrosis factor-α,interleukin-1β(IL-1β), and IL-6 levels while upregulating IL-10 levels. Western blot analysis suggested that the protein expressions of GRP78, p-PERK, p-eIF2α, p-IRE1α, XBP1s, ATF-6, ATF-4, CHOP, p-JNK, cleaved-Caspase-3, cleaved Caspase-9, and cleaved Caspase-12 significantly decreased after ADSCs-exo treatment. RT-qPCR results demonstrated that mRNA expression of GRP78, IRE1α, XBP1, ATF-6, ATF-4, CHOP, JNK, Caspase-3, Caspase-9, and Caspase-12 markedly reduced after ADSCs-exo treatment. In conclusion,ADSCs-exo protects against hepatic I/R injury after hepatectomy by inhibiting ERS and inflammation. Therefore, ADSCs-exo can be considered as a viable option for the treatment of hepatic I/R injury.

Upregulated microRNA-450b-5p represses the development of acute liver failure via modulation of liver function, inflammatory response, and hepatocyte apoptosis.

It has been evidenced that microRNAs (miRs) exert crucial effects on acute liver failure (ALF), while the detailed function of miR-450b-5p in ALF progression remained obscure. The purpose of this research was to unravel the regulatory mechanism of miR-450b-5p in ALF via modulating Mouse Double Minute 2 protein (MDM2). ALF was induced in mice by intraperitoneal injection of d-galactosamine ( d-GalN) and lipopolysaccharide (LPS). Adenoviruses containing overexpressed miR-450b-5p, MDM2 shRNA, and overexpressed MDM2 sequences were utilized to manipulate miR-450b-5p and MDM2 expression in the liver before the mice were treated with d-GalN/LPS-induced ALF. Subsequently, miR-450b-5p and MDM2 expression levels in liver tissues of ALF mice were examined. Serum biochemical parameters of liver function were tested, serum inflammatory factors were assessed, and the histopathological changes and hepatocyte apoptosis in liver tissues were observed. The relation between miR-450b-5p and MDM2 was verified. In ALF mice, miR-450b-5p was low-expressed while MDM2 was high-expressed. The upregulation of miR-450b-5p or downregulation of MDM2 could alleviate liver function, mitigate the serum inflammatory response and pathological changes in liver tissues, as well as inhibit the apoptosis of hepatocytes. MiR-450b-5p targeted MDM2. MDM2 overexpression reversed the repressive effects of elevated miR-450b-5p on ALF. The upregulated miR-450b-5p blocks the progression of ALF via targeting MDM2. This study contributes to affording novel therapeutic targets for ALF treatment.

Antioxidant Activity of Aqueous Extract of Spatholobi Caulis and Its Ameliorative Effect on CCl4 Induced Acute Liver Injury in Mice

The acute liver injury model of mice induced by carbon tetrachloride (CCl₄) was used to explore the protective effect of aqueous extract of Spatholobi Caulis on acute liver injury. Methods: The contents of total phenols and total flavonoids in the aqueous and ethanol extracts of Caulis spatholobi were determined, and the antioxidant activity was evaluated by DPPH, ABTS free radical scavenging rate and FRAP total reducing capacity. 30 C57BL/6J male mice were randomly divided into model group, bifendate group, the low-dose, medium-dose and high-dose groups of aqueous extract of Spatholobi Caulis (n = 6). The animal model of acute liver injury was established by intraperitoneal injection of CCl₄ after 7 days with corresponding drugs. The pathological changes of liver tissue were observed by H&E staining. Serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor α (TNF-α) were determined by ELISA. The indexes of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and antioxidant effects of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were determined. Results: Liver histomorphology observation showed that aqueous extract of Spatholobi Caulis could significantly improve liver cell injury and inflammatory cell infiltration, and significantly increased the activities of CAT, SOD and GSH-Px antioxidant enzymes in liver, decreased the contents of MDA and ALT and AST in serum, significantly down-regulated the levels of IL-1β, IL-6 and TNF-α in serum, and up-regulated the levels of IL-10 and other inflammatory factors. Conclusion: The aqueous extract of Spatholobi Caulis can improve acute liver injury induced by CCl₄ in mice through improving antioxidant and anti-inflammatory effects.

Anlotinib improves bile duct ligature-induced liver fibrosis in rats via antiangiogenesis regulated by VEGFR2/mTOR pathway.

Cholestasis is a main clinical feature of biliary atresia (BA), which leads to liver fibrosis (LF). The focus of BA treatment is preventing and slowing the progress of LF. This study reports the improvement effect of anlotinib on common bile duct ligature (BDL)-induced LF in young rats. The BDL young rats were treated with anlotinib and the serum levels of aspartate aminotransferase, alanine aminotransferase, albumin, and total bilirubin were determined. Histological staining was performed and pathological changes in liver tissue were observed. The expression levels of α-SMA, collagen I, CD31, TGF-β1, phospho-VEGFR2, phospho-4E/BP1, and phospho-S6K1 were determined. The results showed that anlotinib significantly improved the liver function and histopathological injury of BDL rats, inhibited the deposition of collagen and hepatocyte apoptosis, and downregulated the protein expression of α-SMA and collagen I. Furthermore, anlotinib treatment significantly inhibited microvascular formation in the liver and downregulated the expression level of phospho-VEGFR2, thereby suggesting that the antifibrosis effect of anlotinib may be achieved by antiangiogenesis. In addition, anlotinib downregulated the expression of phospho-S6K1 and upregulated the expression of phospho-4E/BP1, two downstream proteins of the mammalian target of rapamycin (mTOR) pathway. MHY1485, an agonist of mTOR, significantly reversed the inhibitory effect of anlotinib on angiogenesis and LFbut did not influence the effect of anlotinib on the downregulation of phospho-VEGFR2 expression. Together, the above-mentioned results suggest that the effect of anlotinib on BDL-induced LF involves at least antiangiogenesis regulated by the VEGFR2/mTOR signaling pathway.

Expression changes of Tim-3 as one of supplementary indicators for monitoring prognosis of liver pathological changes in chronic HBV infection

PurposeThis study was designed to analyze the liver tissue changes among the CHB patients who received treatment for at least 6 months and follow-up for at least 1 year, together with the correlation between the different disease condition and serum markers.MethodsOne-hundred and eighty-five CHB patients underwent antiviral therapy for at least 6 months were enrolled. In the 12-month follow-up, ultrasonography-guided biopsy was performed. The patients were grouped based on the serum markers and pathological changes in liver tissues. Then we determined the serum markers, virological tests and Tim-3 expression among these groups.ResultsAntiviral therapy significantly reduced liver inflammation indicators and serum Tim-3 level. However, the fibrosis process of liver tissue was not changed, and there are still disputes on the serum marker and hepatic lesion outcomes. Under normal liver function or negative hepatitis B e antigen (HBeAg) of CHB patients, there might be consensus between Tim-3 change and liver pathological outcome. According to the liver tissue inflammation and fibrosis conditions, Tim-3 was positively correlated with liver function indices. Besides, it was also related to fibrosis stage and inflammation grade.ConclusionThere were inconsistent changes between serum markers and liver tissue conditions after anti-viral therapy. Tim-3 expression was more suitable to indicate the changes of liver inflammatory and fibrosis response to some extent than ALT and AST. It may serve as a certain indicator to predict the CHB prognosis, which could be used as one of the monitoring indicators in liver pathological changes of chronic HBV infection, especially in monitoring liver tissue inflammation.

Effects of curcumin on liver injury induced by chronic alcohol addiction

Objective: To investigate the intervention effects of curcumin (Curc) on liver injury induced by chronic alcohol addiction in mice. Methods: Thirty Balb/c mice were randomly divided into normal control group (Control), model group (Model), low-dose Curc group (5 mg/kg, Curc-L), medium dose Curc group (10 mg/kg, Curc-M) and high-dose Curc group (15 mg/kg, Curc-H), with 6 mice in each group. The chronic alcohol addiction liver injury model was prepared with 20% liquor. The mice in control group were given 2 ml of normal saline every day. The mice in model group were given 5 ml/kg of 20% liquor every day, and the mice in Curc treatment group were treated with Curc at the doses of 5, 10, 15 mg/kg in 2 ml saline every day for 35 days. The weight of liver was measured and the health status of mice was observed. Serum ALT, AST, ALP and liver TG, TC, HDL-C, LDL-C, MDA, SOD, GSH-Px and NO were measured. The pathological changes of liver tissues stained with hematoxylin and eosin were observed. Results: Compared with the control group, the liver mass and serum levels of ALT, AST, ALP, MDA, NO, TC, TG, HDL-C, LDL-C in the model group were increased significantly (P＜0.05, P＜0.01), the activities of SOD and GSH-Px were decreased significantly (P＜0.05, P＜0.01), the liver cells were vacuolated and infiltrated with inflammatory cells, and the expression levels of NF-κB and MAPK protein in liver tissues were increased significantly (P＜0.01). Compared with the model group, the levels of ALT, AST, ALP, MDA, NO, TC, TG, HDL-C, LDL-C in Curc group were decreased significantly nd the activities of SOD and GSH-Px were increased significantly (P＜0.05, P＜0.01). Conclusion: Curc can effectively reduce liver tissue damage by regulating NF-κB/MAPK signal pathway.

Expanding antiviral therapy indications for HBeAg-negative chronic hepatitis B patients with normal ALT and positive HBV DNA.

With the improved efficacy and accessibility of antiviral agents as well as the concerns about disease progression, there is a hot discussion on whether HBeAg-negative chronic hepatitis B (CHB) patients with normal alanine aminotransferase (ALT) and positive HBV DNA should be treated. According to the international guidelines on the stages of the natural history of HBV infection, HBeAg-negative CHB patients with normal ALT and positive HBV DNA can be divided into two groups: one is the well-known "inactive carrier phase", which is defined as serum HBV DNA<2000 IU/ml and no significant liver inflammation; and the other is the "indeterminate phase", which is defined as serum HBV DNA≥2000 IU/mL regardless of the pathological changes in liver tissue, or HBV DNA<2000 IU/mL but accompanied by significant pathological changes in the liver. In this minireview, we will expound the disease characteristics, disease progression, and clinical management status of these two groups. Based on the analysis, we propose that HBeAg-negative patients with normal ALT but detectable serum HBV DNA should be treated, regardless of their age, family history of hepatocellular carcinoma (HCC) or the severity of liver necroinflammation. Expanding the indications of antiviral therapy will help improve the survival and quality of life of patients by preventing disease progression, and consequently reduce the risk of HCC development.

Exploring Anti-Type 2 Diabetes Mellitus Mechanism of Gegen Qinlian Decoction by Network Pharmacology and Experimental Validation.

Purpose Gegen Qinlian Decoction (GGQL) has been employed to treat type 2 diabetes mellitus (T2DM) in the clinical practice of traditional Chinese medicine. However, the underlying mechanism of GGQL in the treatment of T2DM remains unknown. This study was aimed at exploring the pharmacological mechanisms of GGQL against T2DM via network pharmacology analysis combined with experimental validation. Methods The effective components of GGQL were screened, and the target was predicted by using traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). The candidate targets of GGQL were predicted by network pharmacological analysis, and crucial targets were chosen by the protein-protein interaction (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed to predict the core targets and pathways of GGQL against T2DM. Then, T2DM mice were induced by a high-fat diet combined with streptozotocin. The model and GGQL groups were given normal saline and GGQL aqueous solution (10 and 20 g/kg/d) intragastric administration, respectively, for 8 weeks. The mice in the GGQLT groups were administered with GGQLT at 10 and 20 g/kg/d, respectively. The pathological changes in liver tissues were observed by hematoxylin-eosin staining. The protein expression of TNF-α and NF-κB was verified by western blotting. Results A total of 204 common targets of GGQL for the treatment of T2DM were obtained from 140 active ingredients and 212 potential targets of T2DM. GO and KEGG enrichment analysis involved 119 signaling pathways, mainly in inflammatory TNF signaling pathways. Animal experiments showed that GGQL significantly reduced the serum levels of body mass, fasting blood glucose, fasting insulin, HOMA-IR, TNF-α, and IL-17. The liver pathological section showed that GGQL could improve the vacuolar degeneration and lipid deposition in the liver of T2DM mice. Mechanistically, GGQL downregulated the mRNA expression of TNF-α and NF-κB. Conclusions This study demonstrated that GGQL may exert antidiabetic effects against T2DM by suppressing TNF-α signaling pathway activation, thus providing a basis for its potential use in clinical practice and further study in treating T2DM.

ARRDC3 inhibits liver fibrosis and epithelial-to-mesenchymal transition via the ITGB4/PI3K/Akt signaling pathway

Objective The effect of ARRDC3 has not been reported in liver fibrosis. Our study aimed to explore the molecular mechanisms by which ARRDC3 attenuates liver fibrosis. Methods The vectors pcDNA-ARRDC3 (which promotes ARRDC3 expression) and si-ITGB4 (which blocks IGTB4 expression) and their negative controls were constructed. The rat liver fibrosis model was established by intraperitoneal injection of CCl4 with or without intraperitoneal injection of pcDNA-ARRDC3. ELISA was used to detect the concentrations of γ-GGT, ALT, AST, and ALP in serum. HE, Masson’s trichome, and Sirius red staining were used to observe the pathological changes in liver tissue. LX-2 cells were treated with TGF-β, and pcDNA-ARRDC3 or si-ITGB4RNA was transfected to promote ARRDC3 expression or knock down ITGB4 expression. Western blotting was used to detect the expression levels of proteins. Results ARRDC3 effectively reduced liver injury, improved liver function, and decreased collagen production and deposition in the CCl4-induced rat fibrosis model. The studies showed that overexpressed ARRDC3 remarkably reduced the expression of E-cadherin and collagen-related protein and increased the expression of mesenchymal markers and EMT-related transcription factors, consequently inhibiting the activity of the ITGB4/PI3K/Akt signaling pathway. Conclusion Our study shows that ARRDC3 could ameliorate CCl4-induced liver fibrosis and EMT progression via the ITGB4/PI3K/Akt signaling pathway, which provides a meaningful reference for the clinical targeted treatment of liver fibrosis.

Anti-fibrotic effect of extracellular vesicles derived from tea leaves in hepatic stellate cells and liver fibrosis mice.

BackgroundActivation of hepatic stellate cells (HSCs) is essential for the pathogenesis of liver fibrosis, there is no effective drug used to prevent or reverse the fibrotic process.MethodsWith human hepatic stellate cell line LX-2 and mouse model of CCl4-induced liver fibrosis, we investigated the anti-fibrotic effect to liver fibrosis of extracellular vesicles (EVs) extracted from tea leaves through cytological tests such as cell proliferation, cell migration, and cell fibrotic marker.ResultsIt was found that tea-derived EVs (TEVs) inhibited HSCs activation. In CCl4-induced liver fibrosis model, TEVs treatment can significantly improve the pathological changes of liver tissue, inhibit collagen deposition, reduce the number of lipid droplets in liver tissue, and reduce serum AST and ALT levels. In addition, TEVs inhibited TGF-β1 signaling and miR-44 in TEVs had the potential inhibitory effect on liver fibrosis.ConclusionsTaken together, our work suggesting that TEVs are novel therapeutic potential for liver fibrosis.

Antihepatofibrotic effect of Guizhifuling pill on carbon tetrachloride-induced liver fibrosis in mice.

To evaluate the protective effects and the underlying mechanism of Guizhifuling pill (, GZFL) on carbon tetrachloride (CCl)-induced hepatic fibrosis in mice. Male ICR mice by intraperitoneally administered with 20% CCl (mixed 1∶4 in soybean oil) to induce liver fibrosis. Mice that underwent CCl were orally with GZFL. Using hematoxylin and eosin and Masson staining to examine the pathological changes in liver tissue. Serum biochemical parameters, antioxidant enzyme activity and proinflammatory cytokines was assessed. Nuclear factor-kappaB (NF-κB) pathway and nuclear factor-erythroid 2-related factor 2 (Nrf2) family members were evaluated by Western blotting. Our findings indicated that GZFL could effectively suppress the progression of liver fibrosis in mice, which was determined based on the improvement in liver function and reduction of collagen deposition. GZFL treatment also decreased the level of cytokines and increased the activity of antioxidant enzymes in liver tissue. Moreover, GZFL exerted anti-inflammatory and antioxidant effects through regulating the Nrf2-mediated antioxidant system and inhibiting the NF-κB pathway. GZFL may prevent the progression of liver fibrosis by regulating the Nrf2/ heme oxygenase-1 and NF-κB signaling pathways, thereby highlighting its role in the management of liver fibrosis.

α-lipoic acid ameliorates liver injury in rats with type 2 diabetes mellitus via activating AMPK/mTOR pathway

To investigate the effect of α-lipoic acid in ameliorating liver injury in rats with type 2 diabetes mellitus via activating adenosine 5'-monophosphate-activate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. The T2DM rat models were established by feeding with high-fat, high-sucrose diet and intraperitoneal injection of 27.5 mg/(kg·d) streptozotocin. The 32 rats with T2DM were randomly divided into 4 groups: T2DM group, α-lipoic acid group (LA), Compound C group (Comp C, an inhibitor of AMPK) and LA+Comp C group, with 8 rats in each group. Additionally, 8 Sprague-Dawlay (SD) rats without diabetes were set as normal control. The rats received α-lipoic acid at a dosage of 100 mg/(kg·d) or Compound C at a dosage of 20 mg/(kg·d) by intraperitoneal injection for 8 weeks as needed. The levels of relevant biochemical indexes were detected. The weight of liver was recorded to calculate liver weight index (LWI), and the pathological changes of liver tissues were detected by light and electron microscopy. The levels of AMPK, p-AMPK, mTOR, p-mTOR in rat liver were detected by Western blot. Compared with control group, the levels of LWI, homeostasis model assessment of insulin resistance, fasting blood glucose, alanine transaminase, aspartate transaminase, gamma glutamyl transferase and triglyceride in T2DM group were increased significantly (all P＜0.05). The liver tissue lesions were more serious and hepatic steatosis grade was higher. The expression of p-AMPK was decreased (P＜0.05) and the expression of p-mTOR was increased significantly(P＜0.05). α-lipoic acid could reverse the above-mentioned changes, ameliorate insulin resistance (all P＜0.05), protect the structure and function of liver, and activate the AMPK/mTOR pathway (P＜0.05). The protection of α-lipoic acid was weakened by the inhibition of AMPK with Compound C (P＜0.05). α-lipoic acid could protect the liver of rats with T2DM by activating AMPK/mTOR pathway.

Xiao-Yao-San protects against anti-tuberculosis drug-induced liver injury by regulating Grsf1 in the mitochondrial oxidative stress pathway.

Background: Xiao-Yao-San (XYS) is a traditional Chinese prescription that regulates gastrointestinal function, improves mental and psychological abnormalities, and enhances liver function. However, the underlying mechanism of XYS for relieving anti-tuberculosis (AT) drug-induced liver injury is not clear. Objective: The current study examined whether XYS alleviated the symptoms of AT drug-induced liver injury in mice via the mitochondrial oxidative stress pathway. Methods: BALB/c male mice were randomly divided into four groups of 12 animals, including a control group, a model group, a 0.32 g/kg XYS group, and a 0.64 g/kg XYS group. The effect of XYS on the degree of liver injury was observed using haematoxylin and eosin staining (HE) and oil red O staining of pathological sections, biochemical parameters, and reactive oxygen species (ROS) levels. The protein expression of mitochondrial synthesis-related proteins and ferroptosis-related proteins was examined using Western blotting. Results: XYS improved the pathological changes in liver tissue and reduced the level of oxidative stress in liver-injured mice. XYS increased the expression of mitochondrial synthesis-related proteins and reversed the expression of ferroptosis-related proteins. Knockdown of G-rich RNA sequence binding factor 1 (Grsf1) expression with Grsf1 shRNA blocked the protective effects of XYS in liver injury. Conclusion: Our findings suggest that XYS alleviates AT drug-induced liver injury by mediating Grsf1 in the mitochondrial oxidative stress pathway.

Protective effect of adipose-derived stromal cell-secretome attenuate autophagy induced by liver ischemia–reperfusion and partial hepatectomy

BackgroundThe therapeutic effects of adipose-derived mesenchymal stromal cells (ADSCs) may be mainly mediated by their paracrine effects. The ADSC-secretome can ameliorate hepatic ischemia–reperfusion injury (IRI). We explored the therapeutic effect of the ADSC-secretome from the perspective of excessive hepatocyte autophagy induced by hepatic IRI.MethodsWe established a miniature pig model of hepatic ischemia–reperfusion (I/R) and hepatectomy using a laparoscopic technique and transplanted ADSCs and the ADSC-secretome into the liver parenchyma immediately after surgery. Liver injury and hepatocyte autophagy were evaluated by histopathological examination and assessment of relevant cytokines and other factors.ResultsThe results showed that the ADSC-secretome alleviated the pathological changes of liver tissue and the microstructural damage of hepatocytes after IRI. Moreover, the expression levels of autophagy-related markers including Beclin-1, ATG5, ATG12, and LC3II/LC3I decreased, whereas those of p62 increased during phagophore expansion. Furthermore, the expression levels of markers related to the autophagy inhibition pathway phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR), including PI3K, Akt, and mTOR, increased.ConclusionThe ADSC-secretome attenuates hepatic I/R and hepatectomy-induced liver damage by inhibiting autophagy, which is possibly mediated by activation of the PI3K/Akt/mTOR signaling pathway. In addition, there was no significant difference between ADSCs and the ADSC-secretome in the regulation of hepatocyte autophagy. Therefore, ADSCs may improve the excessive autophagy-induced injury of hepatocytes in hepatic I/R and hepatectomy through paracrine effect. Our findings provide new insight into the therapeutic potential of cell-free products, which could replace cell therapy in liver diseases.

Integrated spatially resolved metabolomics and network toxicology to investigate the hepatotoxicity mechanisms of component D of Polygonum multiflorum Thunb

Ethnopharmacological relevanceThe liver toxicity of Reynoutria multiflora (Thunb.) Moldenke. (Polygonaceae) (Polygonum multiflorum Thunb, PM) has always attracted much attention, but the related toxicity materials and mechanisms have not been elucidated due to multi-component and multi-target characteristics. In previous hepatotoxicity screening, different components of PM were first evaluated and the hepatotoxicity of component D [95% ethanol (EtOH) elution] in a 70% EtOH extract of PM (PM-D) showed the highest hepatotoxicity. Furthermore, the main components of PM-D were identified and their hepatotoxicity was evaluated based on a zebrafish embryo model. However, the hepatotoxicity mechanism of PM-D is unknown. Aim of the studyThis work is to explore the hepatotoxicity mechanisms of PM-D by integrating network toxicology and spatially resolved metabolomics strategy. Materials and methodsA hepatotoxicity interaction network of PM-D was constructed based on toxicity target prediction for eight key toxic ingredients and a hepatotoxicity target collection. Then the key signaling pathways were enriched, and molecular docking verification was implemented to evaluate the ability of toxic ingredients to bind to the core targets. The pathological changes of liver tissues and serum biochemical assays of mice were used to evaluate the liver injury effect of mice with oral administration of PM-D. Furthermore, spatially resolved metabolomics was used to visualize significant differences in metabolic profiles in mice after drug administration, to screen hepatotoxicity-related biomarkers and analyze metabolic pathways. ResultsThe contents of four key toxic compounds in PM-D were detected. Network toxicology identified 30 potential targets of liver toxicity of PM-D. GO and KEGG enrichment analyses indicated that the hepatotoxicity of PM-D involved multiple biological activities, including cellular response to endogenous stimulus, organonitrogen compound metabolic process, regulation of the apoptotic process, regulation of kinase, regulation of reactive oxygen species metabolic process and signaling pathways including PI3K-Akt, AMPK, MAPK, mTOR, Ras and HIF-1. The molecular docking confirmed the high binding activity of 8 key toxic ingredients with 10 core targets, including mTOR, PIK3CA, AKT1, and EGFR. The high distribution of metabolites of PM-D in the liver of administrated mice was recognized by mass spectrometry imaging. Spatially resolved metabolomics results revealed significant changes in metabolic profiles after PM-D administration, and metabolites such as taurine, taurocholic acid, adenosine, and acyl-carnitines were associated with PM-D-induced liver injury. Enrichment analyses of metabolic pathways revealed tht linolenic acid and linoleic acid metabolism, carnitine synthesis, oxidation of branched-chain fatty acids, and six other metabolic pathways were significantly changed. Comprehensive analysis revealed that the hepatotoxicity caused by PM-D was closely related to cholestasis, mitochondrial damage, oxidative stress and energy metabolism, and lipid metabolism disorders. ConclusionsIn this study, the hepatotoxicity mechanisms of PM-D were comprehensively identified through an integrated spatially resolved metabolomics and network toxicology strategy, providing a theoretical foundation for the toxicity mechanisms of PM and its safe clinical application.

Spleen deficiency and phlegm dampness syndrome model in rats treated by Citri Reticulatae Pericarpium based on metabolomics

The present study analyzed the effect of Citri Reticulatae Pericarpium on endogenous metabolites in spleen deficiency and phlegm dampness syndrome by metabolomics, and explored the underlying mechanism of Citri Reticulatae Pericarpium in the treatment of spleen deficiency and phlegm dampness syndrome.The model of spleen deficiency and phlegm dampness syndrome was induced in rats by the multi-factor modeling method.The intervention effects of Citri Reticulatae Pericarpium on rats with spleen deficiency and phlegm dampness syndrome were preliminarily evaluated by observing the pathological changes of rat liver tissues and measuring the plasma content of pathological and biochemical indexes such as triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C).Immunohistochemistry was used to detect the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland, and the effect of Citri Reticulatae Pericarpium on aquaporin expression in rats with spleen deficiency and phlegm dampness syndrome was evaluated.Furthermore, UHPLC-ESI-MS/MS was used to analyze the metabolic profiles of rat plasma samples.Multiple methods, such as principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were used for pattern recognition.Differential metabolites were screened out by t-test and variable importance in projection(VIP), followed by pathway analysis based on MetaboAnalyst 5.0.As revealed by experimental results, Citri Reticulatae Pericarpium could improve the pathological changes of liver tissues, increase the levels of HDL-C in the plasma, reduce the levels of TC, TG, and LDL-C, and enhance the expression of AQP2 in the kidney, AQP3 in the colon, and AQP5 in the submandibular gland of rats with spleen deficiency and phlegm dampness syndrome.In addition, 87 differential metabolites of spleen deficiency and phlegm dampness syndrome were screened out by UHPLC-ESI-MS/MS(the levels of 39 metabolites increased significantly and the levels of 48 metabolites decreased significantly), with the representatives of glycine, L-isoleucine, N-acetyl-L-tyrosine, xanthine, hypoxanthine, and trigonelline.The differential metabolites were mainly enriched in the pathways of steroid hormone biosynthesis, linoleic acid metabolism, and purine metabolism.This study distinguished and revealed the characteristic metabolic pattern of spleen deficiency and phlegm dampness syndrome by metabolomics.The preliminary construction of the OPLS-DA model provides an objective basis for the differentiation of spleen deficiency and phlegm dampness syndrome in traditional Chinese medi-cine(TCM), as well as ideas and methods for exploring the biological basis of TCM syndrome from the molecular level and the overall level.

Qingluo Tongbi Formula Alleviates Hepatotoxicity Induced by Tripterygium wilfordii Hook. F. by Regulating Excessive Mitophagy Through the PERK-ATF4 Pathway.

Qingluo Tongbi Formula (QTF) is an empirical formula of Chinese medicine master Zhongying Zhou for the treatment of rheumatoid arthritis. Although including Tripterygium wilfordii Hook. F. (TW), it has not shown liver toxicity in clinical application for many years. Our previous studies have shown that QTF can significantly reduce TW-caused hepatotoxicity, but the mechanism is still unclear. This study aimed to explore the important roles of mitophagy and endoplasmic reticulum stress (ERS) and the relationship between them in QTF in alleviating TW-induced hepatotoxicity. In vivo, C57BL/6J female mice were used to build a model of TW-induced liver toxicity; Then mice were randomly divided into control, TW, TW + RG, TW + PN, TW + SA, TW + BM, and QTF groups. After intragastric administration for 7 days, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in serum were detected; H and E staining, Oil Red O staining, transmission electron microscopy, Western blotting, and RT-qPCR were used to detect the pathological changes in liver tissue, the levels of ERS and mitophagy related proteins and genes, including GRP78, PERK, DRP1, LC3, etc., In vitro, triptolide (TP), catalpol (CAT), and panax notoginseng saponins (PNS), the main active ingredients of QTF, were selected. The mitophagy inhibitor, ERS inhibitor, and PERK inhibitor were used to further study the relationship between TW-induced ERS and mitophagy in HepaRG cells. The results showed that, QTF reduced excessive mitophagy and ERS in TW-induced hepatotoxicity in C57BL/6J mice, and the attenuating effects of RG and PN in QTF were most obvious, and they also significantly restrained the TW-induced ERS and mitophagy by the PERK-ATF4 pathway. Furthermore, PNS was superior to CAT in inhibiting the expression levels of GRP78, PERK, and ATF4, while CAT was superior to PNS in reversing the expression levels of DRP1, P62, and LC3. The combination of CAT and PNS had the best attenuating effect and the most significant regulation on ERS and mitophagy. In conclusion, QTF can alleviate TW-induced hepatotoxicity by differentially downregulating the PERK-ATF4 pathway and excessive mitophagy by different components.