calpain and Liver-Cirrhosis

Topics: Animals; Calpain; Cytokines; Fibrosis; Humans; Ion Channels; Liver Cirrhosis; Macrophages; Mice

This study is aimed to investigate whether calpain 2 (CAPN2) serves as an indicator of the hepatitis B virus (HBV) to induce hepatic fibrosis. Differentially-expressed genes (DEGs) in HBV-induced hepatic fibrosis and normal liver tissues were analyzed, and signal pathway which was analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using DEGs. Next, the gene-related network map was constructed using the Search Tool for the Retrieval of Interacting Genes. Moreover, CAPN2 protein expression, level of hepatic fibrosis, CAPN2 messenger RNA level, and protein levels of CAPN2, a-SAM, COL3A1, COL1A1, and MAPK1 were determined using Immunohistochemistry (IHC), hematoxylin and eosin, RT-qPCR, and western blot (WB), respectively. There were 420 DEGs screened in HBV-induced hepatic fibrosis and normal liver tissues, among which, 373 were significantly upregulated and 47 were obviously downregulated. KEGG analysis showed that the upregulated DEGs were mainly concentrated in extracellular matrix-receptor interaction, protein digestion, and absorption signaling pathways. The network diagram analysis showed that the DEGs, such as CAPN2, ITGAV, and CCR2, play the key role in the DEG network map, and CAPN2 related to hepatic fibrosis via MAPK1. The increased CAPN2 expression and obvious hepatic fibrosis was displayed in the HBV-induced hepatic fibrosis tissues. In addition, HBV could induce CAPN2 expression, and the interference of CAPN2 could inhibit the expression of hepatic fibrosis markers, including a-SAM, COL3A1, COL1A1, and MAPK1. CAPN2 is regarded as a biomarker of hepatic fibrosis induced by HBV.

Topics: Biomarkers; Calpain; Computational Biology; Gene Expression Profiling; Hepatitis B; Hepatitis B virus; Humans; Liver Cirrhosis; Prognosis; Protein Interaction Maps

Fibrosis is a common pathologic outcome of chronic disease resulting in the replacement of normal tissue parenchyma with a collagen-rich extracellular matrix produced by myofibroblasts. Although the progenitor cell types and cellular programs giving rise to myofibroblasts through mesenchymal transition can vary between tissues and diseases, their contribution to fibrosis initiation, maintenance, and progression is thought to be pervasive. Here, we showed that the ability of transforming growth factor-β (TGFβ) to efficiently induce myofibroblast differentiation of cultured epithelial cells, endothelial cells, or quiescent fibroblasts is dependent on the induced expression and activity of dimeric calpains, a family of non-lysosomal cysteine proteases that regulate a variety of cellular events through posttranslational modification of diverse substrates. siRNA-based gene silencing demonstrated that TGFβ-induced mesenchymal transition of a murine breast epithelial cell line was dependent on induction of expression of calpain 9 (CAPN9), an isoform previously thought to be restricted to the gastrointestinal tract. Mice lacking functional CAPN9 owing to biallelic targeting of

Topics: Angiotensin II; Animals; Bleomycin; Calcium-Binding Proteins; Calpain; Carbon Tetrachloride; Cell Line; Dogs; Epithelial-Mesenchymal Transition; Fibrosis; Humans; Isoenzymes; Liver Cirrhosis; Male; Mice, Inbred C57BL; Molecular Targeted Therapy; Myocardium; Protein Biosynthesis; Protein Multimerization; RNA Stability; Signal Transduction; Transforming Growth Factor beta

The activation of hepatic stellate cells (HSCs) in response to liver injury is critical to the development of liver fibrosis, thus, the blockage of the activation of HSCs is considered as a rational approach for anti-fibrotic treatment. In this report, we investigated the effects and the underlying mechanisms of gallic acid (GA) in interfering with the activation of HSCs.. The primary cultured rat HSCs were treated with various doses of GA for different time intervals. The morphology, viability, caspase activity, calcium ion flux, calpain I activity, reactive oxygen species generation and lysosomal functions were then investigated.. GA selectively killed HSCs in both dose- and time-dependent manners, while remained no harm to hepatocytes. Besides, caspases were not involved in GA-induced cell death of HSCs. Further results showed that GA toxicity was associated with a rapid burst of reactive oxygen species (ROS) and a subsequent increase of intracellular Ca(2+) and calpain activity. Addition of calpain I but not calpain II inhibitor rescued HSCs from GA-induced death. In parallel, pretreatment with antioxidants or an intracellular Ca(2+) chelator eradicated GA responses, implying that GA-mediated cytotoxicity was dependent on its pro-oxidative properties and its effect on Ca(2+) flux. Furthermore, application of ROS scavengers also reversed Ca(2+) release and the disruption of lysosomal membranes in GA-treated HSCs.. These results provide evidence for the first time that GA causes selective HSC death through a Ca(2+)/calpain I-mediated necrosis cascade, suggesting that GA may represent a potential therapeutic agent to combat liver fibrosis.

Topics: Animals; Antioxidants; Calcium; Calpain; Dose-Response Relationship, Drug; Gallic Acid; Hepatic Stellate Cells; Hepatocytes; Liver Cirrhosis; Male; Necrosis; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Time Factors

Recent reports considered that it was the disturbance of calcium homeostasis and the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that activated hepatic stellate cells (HSCs) apoptosis and promoted fibrosis resolution. However, the signal-transducing events that are activated by ER stress after HSCs activation were incompletely understood. In this study, we induced ER stress with thapsigargin (TG), and determined the activation of calpain and the cleavage of caspase by analyzing the protein levels and the correspondingly increased intracellular calcium levels and the induction of the proapoptotic transcription factor CHOP. Moreover, the phosphorylation of JNK and p38 MAPK were followed by the activation of the executioner caspases, caspase-3. As expected, preventing an increase in intracellular calcium levels using intracellular calcium chelators, EGTA, and BAPTA/AM, could substantially inhibit the phosphorylation of JNK and p38 MAPK, abolish the activation of calpains, namely caspase-12, caspase-9, and caspase-3, and provide significant protection for TG-treated activated HSCs. Interestingly, pretreatment with p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, the pan-caspase inhibitor z-VAD-FMK, or calpain inhibitors calpeptin, significantly reduced the cell apoptosis and the cleavage of caspase-12 and caspase-3. However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain. Additionally, pretreatment with SB202190 and SP600125 also decreased the expression of CHOP. Importantly, PDGF-induced collagen Col1α1 and α-smooth muscle actin (α-SMA), markers for the perpetuation phase of HSCs activation, were inhibited in TG-treated activated HSCs. These findings showed that the Calpain/Caspase-12 activation induced by ER stress and the JNK/p38 MAPK phosphorylation induced by the increase of intracellular calcium concentration releasing from ER are the novel signaling pathway underlying the molecular mechanism of fibrosis recovery.

Topics: Actins; Animals; Apoptosis; Calcium Chelating Agents; Calcium Signaling; Calpain; Caspase 12; Caspase Inhibitors; Cell Line; Collagen Type I; Collagen Type I, alpha 1 Chain; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Hepatic Stellate Cells; JNK Mitogen-Activated Protein Kinases; Liver Cirrhosis; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Rats; RNA Interference; Thapsigargin; Transcription Factor CHOP; Transfection