Hydrogen sulfide attenuated tumor necrosis factor-α-induced inflammatory signaling and dysfunction in vascular endothelial cells

Background

Hydrogen sulfide (H₂S), the third physiologically relevant gaseous molecule, is recognized increasingly as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the effects and mechanisms of sodium hydrosulfide (NaHS, a H₂S donor) on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.

Methodology and Principal Findings

Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.

Significance

H₂S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1.     

Protective role of hydrogen sulfide against noise-induced cochlear damage: a chronic intracochlear infusion model

Background

A reduction in cochlear blood flow plays an essential role in noise-induced hearing loss (NIHL). The timely regulation of cochlear perfusion determines the progression and prognosis of NIHL. Hydrogen sulfide (H₂S) has attracted increasing interest as a vasodilator in cardiovascular systems. This study identified the role of H₂S in cochlear blood flow regulation and noise protection.

Methodology/Principal Findings

The gene and protein expression of the H₂S synthetase cystathionine-γ-lyase (CSE) in the rat cochlea was examined using immunofluorescence and real-time PCR. Cochlear CSE mRNA levels varied according to the duration of noise exposure. A chronic intracochlear infusion model was built and artificial perilymph (AP), NaHS or DL-propargylglycine (PPG) were locally administered. Local sodium hydrosulfide (NaHS) significantly increased cochlear perfusion post-noise exposure. Cochlear morphological damage and hearing loss were alleviated in the NaHS group as measured by conventional auditory brainstem response (ABR), cochlear scanning electron microscope (SEM) and outer hair cell (OHC) count. The highest percentage of OHC loss occurred in the PPG group.

Conclusions/Significance

Our results suggest that H₂S plays an important role in the regulation of cochlear blood flow and the protection against noise. Further studies may identify a new preventive and therapeutic perspective on NIHL and other blood supply-related inner ear diseases.     

Differential Effects of Cystathionine-γ-lyase-Dependent Vasodilatory H(2)S in Periadventitial Vasoregulation of Rat and Mouse Aortas

Background

Hydrogen sulfide (H₂S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H₂S are not fully understood. We tested the hypotheses that (1) H₂S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K_v) K⁺ channels and (2) that H₂S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway.

Methodology/Principal Findings

Wire myography of rat and mouse aortas was used. NaHS and 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) were used as H₂S donors. KCNQ-type K_v channels were blocked by XE991. 4-Propargylglycine (PPG) and ß-cyano-l-alanine (BCA), or 2-(aminooxy)-acetic acid (AOAA) were used as inhibitors of CSE or cystathionine-ß-synthase (CBS), respectively. NaHS and ADTOH produced strong vasorelaxation in rat and mouse aortas, which were abolished by KCNQ channel inhibition with XE991. Perivascular adipose tissue (PVAT) exerted an anticontractile effect in these arteries. CSE inhibition by PPG and BCA reduced this effect in aortas from rats but not from mice. CBS inhibition with AOAA did not inhibit the anticontractile effects of PVAT. XE991, however, almost completely suppressed the anticontractile effects of PVAT in both species. Exogenous l-cysteine, substrate for the endogenous production of H₂S, induced vasorelaxation only at concentrations >5 mmol/l, an effect unchanged by CSE inhibition.

Conclusions/Signficance

Our results demonstrate potent vasorelaxant effects of H₂S donors in large arteries of both rats and mice, in which XE991-sensitive KCNQ-type channel opening play a pivotal role. CSE-H₂S seems to modulate the effect of adipocyte-derived relaxing factor in rat but not in mouse aorta. The present study provides novel insight into the interaction of CSE-H₂S and perivascular adipose tissue. Furthermore, with additional technical advances, a future clinical approach targeting vascular H₂S/KCNQ pathways to influence states of vascular dysfunction may be possible.     

Down-Regulation of Hydrogen Sulfide Biosynthesis Accompanies Murine Interstitial Cells of Cajal Dysfunction in Partial Ileal Obstruction

Purpose

To investigate the role of endogenous hydrogen sulfide (H₂S) in partial obstruction-induced dysfunction of the interstitial cells of Cajal (ICC) in mice ileum.

Materials and Methods

Partial intestinal obstruction was induced surgically in male imprinting control region (ICR) mice. ICC networks were studied by Immunohistochemistry. Electrical activity was recorded by intracellular recording techniques. The expression of ICC phenotype marker c-kit receptor tyrosine kinase (c-kit), membrane binding stem cell factor (mSCF), the endogenous H₂S biosynthesis enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) was studied by Western blotting. The expression of tumor necrosis factor-α (TNF-α) mRNA was observed by using real-time polymerase chain reaction.

Results

Partial intestinal obstruction resulted in ICC networks were disrupted above obstruction 14 days after the operation. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed and their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized. The expression of c-kit and mSCF was significantly decreased, also suggesting the disruption of the ICC network. The expression of TNF-α was significantly increased in the tunica muscularis of the obstructed intestine. Treatment of cultured intestinal smooth muscle cells with TNF-α caused dramatic down regulation of mSCF. The expression of CBS and CSE was significantly decreased in the tunica muscularis of the obstructed intestine. Intraperitoneal injection (i.p) of DL-propargylglycine, an irreversible inhibitor of CSE, and aminooxyacetic acid, an inhibitor of CBS, elevated the expression of TNF-α mRNA in the tunica muscularis of the ileum. Obstruction-induced over expression of TNF-α was significantly improved by supplementation of NaHS, but not the expressions of mSCF and c-kit.

Conclusions

The down regulation of endogenous H₂S biosynthesis is related to over expression of TNF-α in obstructed small intestine. TNF-α-mediated mSCF down-regulation is not the only reason of partial intestinal obstruction-induced loss of ICC.     

Upregulation of Cystathionine β-Synthetase Expression Contributes to Visceral Hyperalgesia Induced by Heterotypic Intermittent Stress in Rats

Background

Hydrogen sulfide (H₂S) functions as a neuromodulator, but whether it modulates visceral pain is not well known. This study was designed to determine the role for the endogenous H₂S producing enzyme cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CSE) in a validated rat model of visceral hyperalgesia (VH).

Methods

VH was induced by nine-day heterotypic intermittent stress (HIS). Abdominal withdrawal reflex (AWR) scores were determined by measuring the visceromoter responses to colorectal distension (CRD). Dorsal root ganglia (DRG) neurons innervating the colon were labeled by injection of DiI (1,1''-dioleyl-3,3,3'',3-tetramethylindocarbocyanine methanesulfonate) into the colon wall. Patch clamp recording techniques were employed to examine excitability and sodium channel currents of colon specific DRG neurons. Tissues from colon related thoracolumbar DRGs were analyzed for CBS, CSE and sodium channel expression.

Results

HIS significantly increased the visceromotor responses to CRD in association with an upregulated expression of CBS not CSE proteins in colon related DRGs. Administration of O-(Carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, attenuated the AWR scores in HIS-treated rats, in a dose dependent fashion. In contrast, AOAA did not produce any effect on AWR scores in healthy control rats. AOAA reversed the potentiation of sodium channel current densities of colon specific DRG neurons of HIS rats. To further confirm the role for CBS-H₂S signaling, NaHS was used to mimic the production of H₂S by CBS. Application of NaHS significantly enhanced neuronal excitability and potentiated sodium channel current densities of colon DRG neurons from healthy control rats. Furthermore, AOAA reversed the upregulation of Na_V1.7 and Na_V1.8 in colon related DRGs of HIS rats.

Conclusion

Our results suggest that upregulation of CBS expression might play an important role in developing VH via sensitization of sodium channels in peripheral nociceptors, thus identifying a specific neurobiological target for the treatment of VH in functional bowel syndromes.     

Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

Pathological Changes in Pulmonary Circulation in Carbon Tetrachloride (ccl4)-Induced Cirrhotic Mice

Rationale

Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease.

Objective

We investigated the effects of CCl₄-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH.

Methods And Results

Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl₄ 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl₄-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl₄ did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery.

Conclusion

Collectively, our data demonstrate that chronic CCl₄ treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.     

Spironolactone lowers portal hypertension by inhibiting liver fibrosis, ROCK-2 activity and activating NO/PKG pathway in the bile-duct-ligated rat

Objective

Aldosterone, one of the main peptides in renin angiotensin aldosterone system (RAAS), has been suggested to mediate liver fibrosis and portal hypertension. Spironolactone, an aldosterone antagonist, has beneficial effect on hyperdynamic circulation in clinical practice. However, the mechanisms remain unclear. The present study aimed to investigate the role of spionolactone on liver cirrhosis and portal hypertension.

Methods

Liver cirrhosis was induced by bile duct ligation (BDL). Spironolactone was administered orally (20 mg/kg/d) after bile duct ligation was performed. Liver fibrosis was assessed by histology, Masson''s trichrome staining, and the measurement of hydroxyproline and type I collagen content. The activation of HSC was determined by analysis of alpha smooth muscle actin (α-SMA) expression. Protein expressions and protein phosphorylation were determined by immunohistochemical staining and Western blot analysis, Messenger RNA levels by quantitative real time polymerase chain reaction (Q-PCR). Portal pressure and intrahepatic resistance were examined in vivo.

Results

Treatment with spironolactone significantly lowered portal pressure. This was associated with attenuation of liver fibrosis, intrahepatic resistance and inhibition of HSC activation. In BDL rat liver, spironolactone suppressed up-regulation of proinflammatory cytokines (TNFα and IL-6). Additionally, spironolactone significantly decreased ROCK-2 activity without affecting expression of RhoA and Ras. Moreover, spironolactone markedly increased the levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS and the activity of NO effector- protein kinase G (PKG) in the liver.

Conclusion

Spironolactone lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2 activity and activating NO/PKG pathway. Thus, early spironolactone therapy might be the optional therapy in cirrhosis and portal hypertension.     

The dual role of the cystathionine γ-lyase/hydrogen sulfide pathway in CVB3-induced myocarditis in mice

The present study found that serum H₂S level, H₂S production rate, CSE mRNA and CSE protein levels were increased in CVB3-induced myocarditis. dl-proparglygylcine (PAG), an irreversible CSE inhibitor, decreased the infected myocardium titers on postinfection day 4, while NaHS, a H₂S donor, alleviated myocardial injury and necrosis, inflammatory cell infiltration and interstitial edema on postinfection day 10. These data reveal that the CSE/H₂S pathway is upregulated in the heart in a murine model of CVB3-induced myocarditis and that inhibition of endogenous H₂S is beneficial to treatment early in the disease while administration of exogenous H₂S is protective to infected myocardium during the later stage.     

Hydrogen sulfide guards myoblasts from ferroptosis by inhibiting ALOX12 acetylation

Recognized as a novel and important gasotransmitter, hydrogen sulfide (H₂S) is widely present in various tissues and organs. Cystathionine gamma-lyase (CSE)-derived H₂S has been shown to regulate oxidative stress and lipid metabolism. The aim of the present study is to examine the role of H₂S in ferroptosis and lipid peroxidation in mouse myoblasts and skeletal muscles. Ferroptosis agonist RSL3 inhibited the expressions of Gpx4 and reduced CSE/H₂S signaling, which lead to increased oxidative stress, lipid peroxidation, and ferroptotic cell death. In addition, ferroptosis antagonist ferrostatin-1 (Fer-1) up-regulated the expression of CSE, scavenged the generation of reactive oxygen species (ROS) and lipid peroxidation, and improved cell viability. Exogenously applied NaHS was also able to block RSL3-induced ferroptotic cell death. Neither RSL3 nor H₂S affected cell apoptosis. Furthermore, H₂S reversed RSL3-induced Drp1 expression and mitochondrial damage, which lead to abnormal lipid metabolism as evidenced by altered expressions of ACSL4, FAS, ACC and CPT1 as well as higher acetyl-CoA contents in both cytoplasm and mitochondria. RSL3 promoted the protein expression and acetylation of ALOX12, a key protein in initiating membrane phospholipid oxidation, while the addition of NaHS attenuated ALOX12 acetylation and protected from membrane lipid peroxidation. Moreover, we observed that CSE deficiency alters the expressions of ferroptosis and lipid peroxidation-related proteins and enhances global protein acetylation in mouse skeletal muscles under aging or injury conditions. These results indicate that downregulation of CSE/H₂S signaling would contribute to mitochondrial damage, abnormal lipid metabolism, membrane lipid peroxidation, and ferroptotic cell death. CSE/H₂S system can be a target for preventing ferroptosis in skeletal muscle.     

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction

Hydrogen sulfide (H₂S) produced by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H₂S in kidney fibrosis remains to be defined. Here, we investigated the role of H₂S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H₂S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H₂S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H₂S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-β1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H₂S in UO-induced kidney fibrosis, suggesting that H₂S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.     

Dietary Supplementation of Blueberry Juice Enhances Hepatic Expression of Metallothionein and Attenuates Liver Fibrosis in Rats

Aim

To investigate the effect of blueberry juice intake on rat liver fibrosis and its influence on hepatic antioxidant defense.

Methods

Rabbiteye blueberry was used to prepare fresh juice to feed rats by daily gastric gavage. Dan-shao-hua-xian capsule (DSHX) was used as a positive control for liver fibrosis protection. Liver fibrosis was induced in male Sprague-Dawley rats by subcutaneous injection of CCl₄ and feeding a high-lipid/low-protein diet for 8 weeks. Hepatic fibrosis was evaluated by Masson staining. The expression of α-smooth muscle actin (α-SMA) and collagen III (Col III) were determined by immunohistochemical techniques. The activities of superoxide dismutase (SOD) and malondialdehyde (MDA) in liver homogenates were determined. Metallothionein (MT) expression was detected by real-time RT-PCR and immunohistochemical techniques.

Results

Blueberry juice consumption significantly attenuates CCl₄-induced rat hepatic fibrosis, which was associated with elevated expression of metallothionein (MT), increased SOD activity, reduced oxidative stress, and decreased levels of α-SMA and Col III in the liver.

Conclusion

Our study suggests that dietary supplementation of blueberry juice can augment antioxidative capability of the liver presumably via stimulating MT expression and SOD activity, which in turn promotes HSC inactivation and thus decreases extracellular matrix collagen accumulation in the liver, and thereby alleviating hepatic fibrosis.     

H2S Donor,S-Propargyl-Cysteine,Increases CSE in SGC-7901 and Cancer-Induced Mice: Evidence for a Novel Anti-Cancer Effect of Endogenous H2S?

Background

S-propargyl-cysteine (SPRC), an H₂S donor, is a structural analogue of S-allycysteine (SAC). It was investigated for its potential anti-cancer effect on SGC-7901 gastric cancer cells and the possible mechanisms that may be involved.

Methods and Findings

SPRC treatment significantly decreased cell viability, suppressed the proliferation and migration of SPRC-7901 gastric cancer cells, was pro-apoptotic as well as caused cell cycle arrest at the G₁/S phase. In an in vivo study, intra-peritoneal injection of 50 mg/kg and 100 mg/kg of SPRC significantly reduced tumor weights and tumor volumes of gastric cancer implants in nude mice, with a tumor growth inhibition rate of 40–75%. SPRC also induced a pro-apoptotic effect in cancer tissues and elevated the expressions of p53 and Bax in tumors and cells. SPRC treatment also increased protein expression of cystathione-γ-lyase (CSE) in cells and tumors, and elevated H₂S levels in cell culture media, plasma and tumoral CSE activity of gastric cancer-induced nude mice by 2, 2.3 and 1.4 fold, respectively. Most of the anti-cancer functions of SPRC on cells and tumors were significantly suppressed by PAG, an inhibitor of CSE activity.

Conclusions

Taken together, the results of our study provide insights into a novel anti-cancer effect of H₂S as well as of SPRC on gastric cancer through inducing the activity of a new target, CSE.     

Oroxylin A Accelerates Liver Regeneration in CCI4-Induced Acute Liver Injury Mice

Introduction

Based on the previous research that oroxylin A can suppress inflammation, we investigated the hepatoprotective role of oroxylin A against CCl₄-induced liver damage in mice and then studied the possible alteration of the activities of cytokine signaling participating in liver regeneration. Wild type (WT) mice were orally administrated with oroxylin A (60 mg/kg) for 4 days after CCl₄ injection, the anti-inflammatory effects of oroxylin A were assessed directly by hepatic histology and indirectly by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Albumin. Proliferating cell nuclear antigen (PCNA) staining was performed to evaluate the role of oroxylin A in promoting hepatocyte proliferation. Serum IL-1β, TNF-α, IL-6 and IL-1Ra levels were measured by enzyme-linked immunosorbent assay (ELISA) and liver HGF, EGF, TNF-α, IL-6, IL-1Ra and IL-1β gene expression was determined by quantitative real-time PCR. The data indicated that the IL-6 and TNF-α mRNA of oroxylin A administered group significantly increased higher than the control within 12 hours after CCl₄ treatment. Meanwhile, oroxylin A significantly enhanced the expression of IL-1Ra at the early phase, which indicated that oroxylin A could facilitate the initiating events in liver regeneration by increasing IL-1Ra which acts as an Acute-Phase Protein (APP). In addition, a lethal CCl₄-induced acute liver failure model offers a survival benefit in oroxylin A treated WT mice. However, oroxylin A could not significantly improve the percent survival of IL-1RI^−/− mice with a lethal CCl₄-induced acute liver failure.

Conclusions

Our study confirmed that oroxylin A could strongly promote liver structural remodeling and functional recovery through IL-1Ra/IL-1RI signaling pathway. All these results support the possibility of oroxylin A being a therapeutic candidate for acute liver injury.     

A Ribosomal S-6 Kinase–Mediated Signal to C/EBP-β Is Critical for the Development of Liver Fibrosis

Background

In response to liver injury, hepatic stellate cell (HSC) activation causes excessive liver fibrosis. Here we show that activation of RSK and phosphorylation of C/EBPβ on Thr217 in activated HSC is critical for the progression of liver fibrosis.

Methodology/Principal Findings

Chronic treatment with the hepatotoxin CCl₄ induced severe liver fibrosis in C/EBPβ^+/+ mice but not in mice expressing C/EBPβ-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPβ-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. The C/EBPβ-Ala217 peptides directly stimulated caspase 8 activation in a cell-free system. C/EBPβ^+/+ mice with CCl₄-induced severe liver fibrosis, while continuing on CCl₄, were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis. We found a similar activation of RSK and phosphorylation of human C/EBPβ on Thr266 (human phosphoacceptor) in activated HSC in patients with severe liver fibrosis but not in normal livers, suggesting that this pathway may also be relevant in human liver fibrosis.

Conclusions/Significance

These data indicate that the RSK-C/EBPβ phosphorylation pathway is critical for the development of liver fibrosis and suggest a potential therapeutic target.     

Activation of protein serine/threonine phosphatase PP2Cα efficiently prevents liver fibrosis

Background

Over-activation of TGFβ signaling pathway and uncontrolled cell proliferation of hepatic stellate cells (HSCs) play pivotal roles in liver fibrogenesis, while the protein serine/threonine phosphatase PP2Cα was reported to negatively regulate TGFβ signaling pathway and cell cycle. Our study aimed to investigate the role of PP2Cα in liver fibrogenesis.

Methodology/Principal Findings

The effects of PP2Cα activation on liver fibrosis were investigated in human HSCs and primary rat HSCs in vitro using western blotting, real-time PCR, nuclear translocation, cell viability and cell cycle analyses. The antifibrogenic effects in carbon tetrachloride (CCl₄)- and bile duct ligation (BDL)-induced mice in vivo were assessed using biochemical, histological and immunohistochemical analyses. The results demonstrated that activation of PP2Cα by overexpression or the new discovered small molecular activator NPLC0393 terminated TGFβ-Smad3 and TGFβ-p38 signaling pathways, induced cell cycle arrest in HSCs and decreased α-smooth muscle actin (α-SMA) expression, collagen deposition and hepatic hydroxyproline (HYP) level in CCl₄- and BDL-induced mice.

Conclusions/Significance

Our findings suggested that PP2Cα activation might be an attractive new strategy for treating liver fibrosis while the small molecular activator NPLC0393 might represent a lead compound for antifibrogenic drug development. Moreover, our study might provide the first evidence for the role of PP2C family members in the fibrotic disease.     

Expression of cystathionine β-synthase and cystathionine γ-lyase in human pregnant myometrium and their roles in the control of uterine contractility

Background

Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H₂S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H₂S produced locally in the control of uterine contractility during labour is unknown.

Methodology/Principal Findings

Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H₂S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H₂S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10⁻⁷–10⁻² mol/L), a precursor of H₂S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10⁻³ mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.

Conclusion/Significance

H₂S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H₂S synthetic enzymes is down-regulated during labour, suggesting that H₂S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.     

Role of the Sympathetic Nervous System in Carbon Tetrachloride-Induced Hepatotoxicity and Systemic Inflammation

Carbon tetrachloride (CCl₄) is widely used as an animal model of hepatotoxicity and the mechanisms have been arduously studied, however, the contribution of the sympathetic nervous system (SNS) in CCl₄-induced acute hepatotoxicity remains controversial. It is also known that either CCl₄ or SNS can affect systemic inflammatory responses. The aim of this study was to establish the effect of chemical sympathectomy with 6-hydroxydopamine (6-OHDA) in a mouse model of CCl₄-induced acute hepatotoxicity and systemic inflammatory response. Mice exposed to CCl₄ or vehicle were pretreated with 6-OHDA or saline. The serum levels of aminotransferases and alkaline phosphatase in the CCl₄-poisoning mice with sympathetic denervation were significantly lower than those without sympathetic denervation. With sympathetic denervation, hepatocellular necrosis and fat infiltration induced by CCl₄ were greatly decreased. Sympathetic denervation significantly attenuated CCl₄-induced lipid peroxidation in liver and serum. Acute CCl₄ intoxication showed increased expression of inflammatory cytokines/chemokines [eotaxin-2/CCL24, Fas ligand, interleukin (IL)-1α, IL-6, IL-12p40p70, monocyte chemoattractant protein-1 (MCP-1/CCL2), and tumor necrosis factor-α (TNF-α)], as well as decreased expression of granulocyte colony-stimulating factor and keratinocyte-derived chemokine. The overexpressed levels of IL-1α, IL-6, IL-12p40p70, MCP-1/CCL2, and TNF-α were attenuated by sympathetic denervation. Pretreatment with dexamethasone significantly reduced CCl₄-induced hepatic injury. Collectively, this study demonstrates that the SNS plays an important role in CCl₄-induced acute hepatotoxicity and systemic inflammation and the effect may be connected with chemical- or drug-induced hepatotoxicity and circulating immune response.