Anti-inflammation treatment for protection of hepatocytes and amelioration of hepatic fibrosis in rats.

Chronic inflammation is considered as an important pathophysiologic mechanism of hepatic cirrhosis, which induces hepatocyte injury and activates hepatic stellate cells (HSCs), thus resulting in hepatic fibrosis. Previous studies have reported that cyclooxygenase-2 (COX-2) inhibitor can effectively treat liver fibrosis, while somatostatin (SST) analogues inhibit the activation of HSCs. The present study aimed to investigate the effects of a COX-2 inhibitor, celecoxib, combined with a SST analogue, octreotide, for protection of hepatocytes and prevention of fibrosis in a rat model of hepatic fibrosis. Therefore, a hepatic fibrosis rat model was established following peritoneal injection of thioacetamide (TAA), and the rats were then treated with a combination of celecoxib and octreotide (TAA + C). Immunohistochemistry and western blotting assays were used to assess the expression levels of proteins associated with inflammation, epithelial-mesenchymal transition (EMT), proliferation, apoptosis and autophagy. H&E staining, transmission electron microscopy and scanning electron microscopy were used to evaluate the destruction of hepatocytes. Masson's Trichrome and Sirius Red were used to measure the degree of liver fibrosis. The results demonstrated that, compared with those of the control group, the degree of liver fibrosis and the expression of the intrahepatic inflammation factors were aggravated in the TAA group. Furthermore, the apoptosis rate, EMT and autophagy of hepatocytes were also increased in the TAA group. However, treatment with TAA + C restored the aforementioned increased levels compared with the TAA group. In conclusion, treatment of rats with the combination of celecoxib and octreotide could attenuate the progress of hepatic fibrosis via protection of hepatocytes by reducing apoptosis, EMT and autophagy in hepatocytes.

Celecoxib attenuates hepatocyte apoptosis by inhibiting endoplasmic reticulum stress in thioacetamide-induced cirrhotic rats.

BACKGROUND: Endoplasmic reticulum (ER) stress is an important mechanism in the progression of chronic and acute liver diseases, especially in the progression and recovery of liver fibrosis. Excessive and long-term ER stress induces apoptosis. ER stress-induced apoptosis is considered to be an important pathway in the development of liver fibrosis. Cyclooxygenase-2 (COX-2) induction is also closely related to ER stress. In our previous studies, we showed that celecoxib, a COX-2 inhibitor, improves liver fibrosis and portal hypertension. However, the role and mechanism of celecoxib in alleviating liver fibrosis remain unclear. AIM: To investigate whether celecoxib alleviates liver fibrosis by inhibiting hepatocyte apoptosis via the ER stress response. METHODS: Cirrhosis was induced by intraperitoneal injections of thioacetamide (TAA) for 16 wk (injection dose is 200 mg/kg per 3 d for the first 8 wk and 100 mg /kg per 3 d after 8 wk). Thirty-six male Sprague-Dawley rats were randomly divided into three groups, namely, control group, TAA group, and TAA + celecoxib group. In the last 8 wk, TAA-induced cirrhotic rats received celecoxib (20 mg/kg/day) or the vehicle by gastric gavage. After 16 wk, the rats were sacrificed, and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB) were detected. The hepatic fibrosis areas were evaluated by Sirius red staining and the degree of fibrosis was assessed by measuring the level of hydroxyproline. ER stress levels were evaluated by detecting the marker proteins glucose-regulated protein 78 (GRP78), CCAAT/enhancer binding protein homologous protein (CHOP), PKR-like ER protein kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 alpha (IRE1α). Apoptosis levels were evaluated by detecting caspase-12 and caspase-3. RESULTS: The serum ALT and AST levels in the liver were significantly reduced by celecoxib; however, the serum ALB had no significant changes. Celecoxib significantly reduced the degree of liver fibrosis and the levels of hydroxyproline (-38% and -25.7%, respectively, P < 0.01). Celecoxib ameliorated ER stress by reducing the level of GRP78 compared to the TAA group (P < 0.05). Consistently, after celecoxib administration, the upregulation of TAA-induced hepatic apoptosis markers (caspase-12 and caspase-3) and CHOP were significantly inhibited. In addition, after celecoxib treatment, the expression of key molecules associated with ER stress (PERK, ATF6, and IRE1) was decreased (P < 0.05). CONCLUSION: Therapeutic administration of celecoxib effectively reduces hepatic apoptosis in TAA-induced cirrhotic rats. The mechanism of action may be attributed to the suppression of CHOP expression, which subsequently inhibits ER stress.

Expression of cyclooxygenase-2 is correlated with lncRNA-COX-2 in cirrhotic mice induced by carbon tetrachloride.

Multiple long non-coding RNAs (lncRNAs) have been demonstrated to be involved in liver disease. Increased cyclooxygenase-2 (COX­2) levels have also been reported to be involved in the progression of liver cirrhosis. In the present study, the correlations between lncRNA­COX­2 RNA expression levels, COX­2 mRNA expression levels and liver fibrosis were examined. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4) in mice for 2 months (CCl4­2M) or 3 months (CCl4­3M). Liver histopathological evaluation was conducted using hematoxylin and eosin and Masson trichrome staining. Hepatic expression of COX­2 and lncRNA­COX­2 was evaluated by reverse transcription­quantitative polymerase chain reaction and immunohistochemical staining. Compared with the control group, fibrotic areas were increased four and nine times in the CCl4­2M group and the CCl4­3M group, respectively. LncRNA-COX-2 and COX­2 upregulation were observed in the cirrhotic liver. COX­2 mRNA expression levels and lncRNA-COX-2 RNA expression levels were significantly positively correlated with the fibrotic area. In addition, COX­2 mRNA expression was significantly positively correlated with lncRNA­COX­2 expression. These results suggest that expression of COX­2 and lncRNA­COX­2 increased with the progression of liver fibrosis. LncRNA-COX-2 may potentially be considered as a novel therapeutic target for liver fibrosis.

Correlations between VEGF-A expression and prognosis in patients with gastric adenocarcinoma.

Angiogenesis induced by vascular endothelial growth factor A (VEGF-A) plays a critical role in tumor growth and metastasis. The study aimed to evaluate the expression of VEGF-A in gastric adenocarcinoma and investigate its correlations with tumor clinicopathological features and prognostic significance. VEGF-A expression was detected by immunohistochemistry on a tissue microarray containing 90 pairs of human gastric adenocarcinoma and paracancerous tissues. Levels of VEGF-A in gastric adenocarcinoma were significantly higher than those in paracancerous tissues (P=0.018). Furthermore, the result was coincident with that of human gastric adenocarcinoma xenografts in nude-mice (P<0.01). In addition, the VEGF-A expression was positive correlation with TNM stage (P=0.047), tumor size (P=0.028), positive lymph nodes (P=0.002) and lymphovascular invasion (P=0.001). Finally, Kaplan-Meier survival analysis showed that VEGF-A up-regulation indicated a poor prognosis for overall survival (P=0.039). In conclusions, VEGF-A may be used as a biomarker for evaluating both the biological behavior of tumor and the prognosis in patients with gastric adenocarcinoma.

Collapsed Reticular Network and its Possible Mechanism during the Initiation and/or Progression of Hepatic Fibrosis.

Among the researches on hepatic fibrosis, great attention was paid to both hepatocytes and extracellular matrix (ECM). However, little focus was drawn on reticular fibrous network, which is important for demarcation and support of hepatocytes. The aim of this study was to investigate the change pattern of reticular fibers in hepatic fibrosis/cirrhosis and its underlying mechanism. In this study, thioacetamide (TAA) and bile duct ligation (BDL) were utilized to induce rat hepatic fibrosis respectively, and Human liver cirrhotic microassay was analyzed with IHC to confirm the results in animal experiment and to detect the metalloproteinases (MMPs) expressions. As a result, the reticular fibers decreased markedly after 1 week in TAA and 1 day in BDL treated rats. Multiple representative regulators of MMPs and MMPs increased significantly in their expressions and activities. Further more, in human liver cirrhotic microassay, MMPs expressions also showed similar patterns as that of animal experiment. In Conclusions: Degradation or collapse of reticular fibers in hepatic sinusoid can be considered as a pathological feature during the initiation and/or progression of hepatic fibrosis. Moreover, such degradation is associated with and probably caused by the over/dysregulated expression of MMPs.

Celecoxib and octreotide synergistically ameliorate portal hypertension via inhibition of angiogenesis in cirrhotic rats.

Abnormal angiogenesis is critical for portal hypertension in cirrhosis. Except for etiological treatment, no efficient medication or regime has been explored to treat the early stage of cirrhosis when angiogenesis is initiated or overwhelming. In this study, we explored an anti-angiogenesis effort through non-cytotoxic drugs octreotide and celecoxib to treat early stage of cirrhotic portal hypertension in an animal model. Peritoneal injection of thioacetamide (TAA) was employed to induce liver cirrhosis in rats. A combination treatment of celecoxib and octreotide was found to relieve liver fibrosis, portal venous pressure, micro-hepatic arterioportal fistulas, intrahepatic and splanchnic angiogenesis. Celecoxib and octreotide exerted their anti-angiogenesis effect via an axis of cyclooxygenase-2/prostaglandin E2/EP-2/somatostatin receptor-2, which consequently down-regulated phosphorylation of extracellular signal-regulated kinase (p-ERK)-hypoxia-inducible factor-1α (HIF-1α)-vascular endothelial growth factor (VEGF) integrated signaling pathways. In conclusions, combination of celecoxib and octreotide synergistically ameliorated liver fibrosis and portal hypertension of the cirrhotic rats induced by TAA via the inhibition of intrahepatic and extrahepatic angiogenesis. The potential mechanisms behind the regimen may due to the inactivation of p-ERK-HIF-1α-VEGF signaling pathway.

Inhibition of cyclooxygenase-2 alleviates liver cirrhosis via improvement of the dysfunctional gut-liver axis in rats.

Inflammatory transport through the gut-liver axis may facilitate liver cirrhosis. Cyclooxygenase-2 (COX-2) has been considered as one of the important molecules that regulates intestinal epithelial barrier function. This study was aimed to test the hypothesis that inhibition of COX-2 by celecoxib might alleviate liver cirrhosis via reduction of intestinal inflammatory transport in thiacetamide (TAA) rat model. COX-2/prostaglandin E2 (PGE2)/EP-2/p-ERK integrated signal pathways regulated the expressions of intestinal zonula occludens-1 (ZO-1) and E-cadherin, which maintain the function of intestinal epithelial barrier. Celecoxib not only decreased the intestinal permeability to a 4-kDa FITC-dextran but also significantly increased expressions of ZO-1 and E-cadherin. When celecoxib greatly decreased intestinal levels of LPS, TNF-α, and IL-6, it significantly enhanced T cell subsets reduced by TAA. As a result, liver fibrosis induced by TAA was significantly alleviated in the celecoxib group. These data indicated that celecoxib improved the integrity of intestinal epithelial barrier, blocked inflammatory transport through the dysfunctional gut-liver axis, and ameliorated the progress of liver cirrhosis.