Rosuvastatin improves hepatopulmonary syndrome through inhibition of inflammatory angiogenesis of lung.

The hepatopulmonary syndrome (HPS) is characterized by hypoxia and increased intrapulmonary shunts in cirrhotic patients. Emerging evidence showed promising results of treating HPS by abolishment of intrapulmonary inflammation and angiogenesis. Rosuvastatin is a kind of 3-hydroxy-methyl-3-glutamyl coenzyme A reductase inhibitor. In addition to lipid-lowering effects, it has anti-inflammation and anti-angiogenesis properties. We postulated that rosuvastatin treatment can ameliorate HPS. Common bile duct ligation (CBDL) was applied in an experimental HPS animal model. CBDL rats received 2-week rosuvastatin (20 mg/kg/day) treatments from the fifteenth day after operation. The haemodynamic data, blood gas analysis, liver biochemistries, tumour necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) were examined after rosuvastatin treatment. The liver and lung tissues were dissected for histopathological studies and protein analyses. In the parallel groups, intrapulmonary shunts were determined. The haemodynamic and liver biochemistries were not changed after rosuvastatin treatment in CBDL rats, but the alveolar-arterial oxygen pressure gradient was significantly decreased, implying that HPS-induced hypoxia was reversed after rosuvastatin treatment. In addition, rosuvastatin treatment reduced intrapulmonary shunts and plasma levels of VEGF and TNF-α. Besides, the intrapulmonary protein expression of nuclear factor kappa B (NF-κB), VEGF receptor (VEGFR)-1,2 and Rho-associated A kinase were significantly down-regulated and the intrapulmonary angiogenesis was ameliorated. We concluded that rosuvastatin alleviates experimental HPS through blockade of pulmonary inflammatory angiogenesis via TNF-α/NF-κB and VEGF/Rho-associated A kinase pathways down-regulation.

Lipopolysaccharide enhanced renal vascular response to endothelin-1 through ETA overexpression in portal hypertensive rats.

BACKGROUND AND AIM: Hypo-perfusion resulting from intense renal vasoconstriction is traditionally contributed to renal dysfunction in advanced liver disease, although cumulative studies demonstrated renal vasodilatation with impaired vascular contractility to endogenous vasoconstrictors in portal hypertension and compensated liver cirrhosis. The pathophysiology of altered renal hemodynamics remains unclear. This study, using a rat model of portal hypertension with superimposed endotoxemia, was designed to delineate the evolution of renal vascular reactivity and vaso-regulatory gene expression during liver disease progression. METHODS: Rats were randomized into sham surgery (SHAM) or partial portal vein ligation (PVL). Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS) on the seventh day following surgery. Isolated kidney perfusion was performed at 0.5 h or 5 h after LPS to evaluate renal vascular response to endothelin-1. RESULTS: In contrast to impaired vascular contractility of SHAM rats, PVL rats displayed enhanced renal vascular reactivity to endothelin-1 at 5 h following endotoxemia. There were extensive upregulations of inducible nitric oxide synthase in kidney tissues of endotoxemic rats. The changes of renal endothelin receptor type A (ETA ) level paralleled with the changes of renal vascular reactivity in LPS-treated rats. Compared with SHAM rats, PVL rats showed increased renal ETA and phosphorylated extracellular-signal-regulated kinases 1/2 (p-ERK1/2) at 5 h after LPS. CONCLUSION: LPS-induced systemic hypotension induces a paradoxical change of renal vascular response to endothelin-1 between SHAM and PVL rats. LPS-induced renal vascular hyperreactivity in PVL rats was associated with upregulation of renal ETA and subsequent activation of ERK1/2 signaling.

Cannabinoid receptor 2 agonist ameliorates mesenteric angiogenesis and portosystemic collaterals in cirrhotic rats.

UNLABELLED: Angiogenesis in liver cirrhosis leads to splanchnic hyperemia, increased portal inflow, and portosystemic collaterals formation, which may induce lethal complications, such as gastroesophageal variceal hemorrhage and hepatic encephalopathy. Cannabinoids (CBs) inhibit angiogenesis, but the relevant influences in cirrhosis are unknown. In this study, Spraque-Dawley rats received common bile duct ligation (BDL) to induce cirrhosis. BDL rats received vehicle, arachidonyl-2-chloroethylamide (cannabinoid receptor type 1 [CB(1) ] agonist), JWH-015 (cannabinoid receptor type 2 [CB(2) ] agonist), and AM630 (CB(2) antagonist) from days 35 to 42 days after BDL. On the 43rd day, hemodynamics, presence of CB receptors, severity of portosystemic shunting, mesenteric vascular density, vascular endothelial growth factor (VEGF), VEGFR-1, VEGFR-2, phospho-VEGFR-2, cyclooxygenase (COX)-1, COX-2, and endothelial nitric oxide synthase (eNOS) expressions as well as plasma VEGF levels were evaluated. Results showed that CB(1) and CB(2) receptors were present in left adrenal veins of sham rats, splenorenal shunts (the most prominent intra-abdominal shunts) of BDL rats, and mesentery of sham and BDL rats. CB(2) receptor was up-regulated in splenorenal shunts of BDL rats. Both acute and chronic JWH-015 treatment reduced portal pressure and superior mesenteric arterial blood flow. Compared with vehicle, JWH-015 significantly alleviated portosystemic shunting and mesenteric vascular density in BDL rats, but not in sham rats. The concomitant use of JWH-015 and AM630 abolished JWH-015 effects. JWH-133, another CB(2) agonist, mimicked the JWH-015 effects. JWH-015 decreased mesenteric COX-1, COX-2 messenger RNA expressions, and COX-1, COX-2, eNOS protein expressions. Furthermore, JWH-015 decreased intrahepatic angiogenesis and fibrosis. CONCLUSIONS: CB(2) agonist alleviates portal hypertension (PH), severity of portosystemic collaterals and mesenteric angiogenesis, intrahepatic angiogenesis, and fibrosis in cirrhotic rats. The mechanism is, at least partly, through COX and NOS down-regulation. CBs may be targeted in the control of PH and portosystemic collaterals.

Sorafenib treatment improves hepatopulmonary syndrome in rats with biliary cirrhosis.

HPS (hepatopulmonary syndrome) is characterized by oxygen desaturation in patients with chronic liver disease. The initiation of HPS comes from abnormal pulmonary vasodilatation and/or angiogenesis. In the present study, we evaluated anti-angiogenesis therapy using sorafenib in experimental HPS animals. HPS was induced by CBDL (common bile duct ligation) in rats. A 2-week 10 mg·(kg of body weight)-1·day-1 treatment regimen of sorafenib or distilled water (control) was initiated 2 weeks after the surgical procedure. Haemodynamics, liver biochemistry, plasma VEGF (vascular endothelial growth factor) measurements and blood gas analysis of the CBDL rats were performed. The livers of the CBDL rats were dissected for histopathology examination, and the lungs were examined by immunohistochemical staining, real-time PCR and Western blot analysis. In another two parallel groups, intrapulmonary shunts were determined. The AaPO2 (alveolar-arterial O2 gradient) and plasma VEGF levels were reduced after sorafenib treatment [AaPO2, 7.2±3.4 mmHg in sorafenib-treated rats compared with 15.3±4.2 mmHg in controls (P=0.004); VEGF, 45.3±2.7 pg/ml in sorafenib-treated rats compared with 54.4±7.7 pg/ml in controls (P=0.021)]. Sorafenib attenuated pulmonary VEGF mRNA and VEGF, VEGFR-2 (VEGF receptor 2), phospho-VEGFR-2 and Akt protein expression. In addition, sorafenib significantly attenuated intrapulmonary angiogenesis and decreased the degree of intrapulmonary shunting by 33.7% (11.2±5.7% in sorafenib-treated rats compared with 16.9±5.9% in controls; P=0.003). Our findings suggest that sorafenib attenuates intrapulmonary shunting and decreases the AaPO2 in CBDL rats, implicating the improvement of HPS in this experimental animal model. The beneficial effect may be attributed to the reduction in intrapulmonary angiogenesis through inhibition of the VEGF/VEGFR-2/Akt pathway.

Selective cyclooxygenase inhibition by SC-560 improves hepatopulmonary syndrome in cirrhotic rats.

OBJECTIVE: Hepatopulmonary syndrome (HPS) is characterized by hypoxia in patients with chronic liver disease. The mechanism of HPS includes pulmonary vasodilatation, inflammation, and angiogenesis. Prostaglandins synthesized by cyclooxygenases (COX) participate in vascular responsiveness, inflammation and angiogenesis, which can be modulated by COX inhibitors. We therefore evaluated the impact of COX inhibition in rats with common bile duct ligation (CBDL)-induced liver cirrhosis and HPS. METHODS: Cirrhotic rats were randomly allocated to receive non-selective COX inhibitor (indomethacin), selective COX-1 inhibitor (SC-560), or COX-2 inhibitor (celecoxib) for 14 days. After that, hemodynamic parameters, severity of hypoxia and intrapulmonary shunts, liver and renal biochemistry parameters, histological finding and protein expressions were evaluated. RESULTS: Non-selective COX inhibition by indomethacin improved hepatic fibrosis and pulmonary inflammation in cirrhotic rats with HPS. It also decreased mean arterial blood pressure, portal pressure, and alleviated hypoxia and intrapulmonary shunts. However, indomethacin increased mortality rate. In contrast, selective COX inhibitors neither affected hemodynamics nor increased mortality rate. Hypoxia was improved by SC-560 and celecoxib. In addition, SC-560 decreased intrapulmonary shunts, attenuated pulmonary inflammation and angiogenesis through down-regulating COX-, NFκB- and VEGF-mediated pathways. CONCLUSION: Selective COX-1 inhibitor ameliorated HPS by mitigating hypoxia and intrapulmonary shunts, which are related to anti-inflammation and anti-angiogenesis.

Kinetics of cytokine expression in cirrhotic rats.

BACKGROUND: Cytokines are involved in liver injury and cirrhosis and systemic and hepatic cytokine levels may help predict cirrhosis evolution. However, the relevant survey has not been performed. METHODS: Male Sprague-Dawley rats (240-270 g) received either common bile duct ligation (BDL, animal model of cholestatic liver injury) or sham operation (control). Five rats were sacrificed and liver and serum were collected from each in weeks 1, 2, 4, 6, 8 and 10 after surgery. Hepatic expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) were analyzed by immunohistochemial staining. The corresponding serum levels were measured by ELISA. RESULTS: Compared to the corresponding sham groups, hepatic expression of these cytokines in BDL rats was significantly and progressively enhanced during cirrhosis development. However, serum IFN-γ levels of BDL rats did not change significantly. Serum TNF-α of BDL rats increased gradually and reached a peak in week 6. Serum TGF-ß level was elevated up to week 8, whereas IL-10 level decreased progressively until week 6. CONCLUSION: Cirrhosis development in BDL rats is associated with progressively enhanced expression of hepatic pro-inflammatory and anti-inflammatory cytokines, which is not in accord with the corresponding serum concentration. The circulating cytokine concentration may not totally reflect the hepatic expression level throughout the development of cirrhosis.

Interleukin-17 induces src/MAPK cascades activation in human renal epithelial cells.

Interleukin-17 (IL-17) is a T cell derived pro-inflammatory cytokine exhibiting multiple biological activities in a variety of cells. In our previous study, we found that IL-17 expressed early on borderline change of renal allograft rejection by Banff classification both in rat renal allograft model and human renal specimens. Renal epithelial cells (RECs) are the important targets in renal allograft rejection. The purpose of this study was to explore the signalling pathways by which human interleukin-17 (hIL-17) contributes to renal allograft rejection by inducing IL-6, IL-8 and MCP-1 expression in human renal epithelial cells (hRECs). Using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoprecipitation and western blot analysis, we report that the early signalling events triggered by the hIL-17 involved tyrosyl phosphorylation of proteins and increased the levels of IL-6, IL-8 and MCP-1 in a dose-dependent manner. Tyrosyl phosphorylation of proteins was induced by IL-17 in 1 min and peaked in 5 min. Further, IL-17 induced the phosphorylation of src kinase and mitogen-activated protein (MAP) kinase. Using a specific src kinase inhibitor, PP2, to treat the hRECs before hIL-17 stimulation, we found that PP2 not only inhibited the phosphorylation of src kinase but also inhibited IL-6, IL-8 and MCP-1 mRNA expression, in a dose-dependent manner. These findings provide the first evidence that the mechanism of IL-17 signalling involves src/MAPK cascades activation.

Evidence for the early involvement of interleukin 17 in human and experimental renal allograft rejection.

Inflammatory processes can stimulate renal epithelial cells to release cytokines, chemoattractants and matrix proteins into the interstitium, thus contributing to interstitial injury during acute allograft rejection. To test the role of interleukin 17 (IL-17) in this process, cultured human renal epithelial cells (hRECs) were first established and treated with or without human IL-17 (hIL-17) for 2, 4, 8 and 10 h in vitro. Significant elevations of IL-6 and IL-8 levels were noted in the supernatants in a dose-dependent and time-dependent manner, as also for IL-6 mRNA expression. Secondly, using a rat acute allograft rejection model, the correlation between IL-17 expression and histopathological changes was serially studied. The results demonstrated that increased expression of IL-17 protein on infiltrating mononuclear cells (MNCs) was detectable on day 2. This corresponds to the borderline change of acute rejection according to the Banff classification, and it increased progressively to day 5. Serial study of IL-6, IL-8 and IL-17 mRNA expression of the renal allograft confirmed IL-17 mRNA expression in the allograft early on post-transplant day 2, whereas IL-6 and IL-8 expression started on day 3. Thirdly, IL-17 expression was observed in human renal allograft and urinary sediment. IL-17 protein expression was found in human subclinical (borderline) rejection renal allograft biopsy tissue and none in biopsy tissue not showing any evidence of rejection. There was also a 100% detectable rate of IL-17 mRNA expression in the MNCs of urinary sediment of patients with subclinical borderline rejection. These results demonstrate that hRECs exposed to IL-17 can produce inflammatory mediators with the potential to stimulate early alloimmune responses, which may also serve to give warning of acute renal allograft rejection.

H1-A extracted from Cordyceps sinensis suppresses the proliferation of human mesangial cells and promotes apoptosis, probably by inhibiting the tyrosine phosphorylation of Bcl-2 and Bcl-XL.

H1-A, a pure compound used in traditional Chinese medicine, is effective in the treatment of autoimmune disorders of MRL lpr/lpr mice. We have previously reported that after 8 weeks of oral therapy with H1-A, 40 microg/kg/day, MRL lpr/lpr mice demonstrated significantly less proteinuria, lower serum creatinine levels, and less renal mesangial proliferation than mice in an untreated group. To clarify the pharmacologic properties of H1-A, we studied its cellular and subcellular effects in cultured human mesangial cells. Our results show that H1-A inhibits cell proliferation and promotes the apoptosis of interleukin (IL)-1- and platelet-derived growth factor (PDGF)-BB-activated human mesangial cells in vitro. Uptake of tritiated thymidine was nearly totally suppressed by the addition of 12.5 micromol/L H1-A (counts per minute decreased from 3905 +/- 70 to 141 +/- 5). The population of S-phase cells decreased from 15.5% +/- 1.7% to 10.0% +/- 0.3%, and G0 + G1 phase cells increased from 68.8% +/- 0.07% to 74.6% +/- 0.05%. This suppression was not a result of cytotoxicity. Apoptosis of human mesangial cells was detectable after treatment with 12.5 or 25 micromol/L H1-A. Using immunoprecipitation and immunoblotting, we found that H1-A inhibits tyrosine phosphorylation of human mesangial proteins and that Bcl-2 and Bcl-XL were probably among these proteins. These findings suggest that H1-A modulates some subcellular signal-transduction pathways and changes the balance between proliferation and apoptosis of mesangial cells in vitro or in vivo. H1-A may be effective in the management of autoimmune disorders, and the modulation of the signal transduction proteins Bcl-2 and Bcl-XL may represent a target for future pharmacologic interventions.