Better detoxifying effect of ripe forsythiae fructus over green forsythiae fructus and the potential mechanisms involving bile acids metabolism and gut microbiota.

Forsythiae Fructus (FF), the fruit of Forsythia suspensa (Thunb.) Vahl. (Lianqiao), is one of the most fundamental herbs in Traditional Chinese Medicines (TCM), mainly due to its heat-clearing and detoxifying effects. There are two types of FF, the greenish fruits that start to ripen (GF) and the yellow fruits that are fully ripe (RF), called "Qingqiao" and "Laoqiao" referred to the Chinese Pharmacopoeia, respectively. It undergoes a complex series of changes during the maturation of FF. However, the clinical uses and preparation of phytopharmaceuticals of FF have not been distinguished to date. Moreover, there is limited information on the study of the difference in pharmacological activity between RF and GF. In this study, a rat model of bile duct ligation (BDL)-induced cholestasis was used to compare the differences in their effects. RF was found to have better results than GF in addressing toxic bile acids (BAs) accumulation and related pathological conditions caused by BDL. The underlying mechanism may be related to the interventions of gut microbiota. The results of the present study suggest that the better detoxifying effect of RF than GF may be indirectly exerted through the regulation of gut microbiota and thus the improvement of BAs metabolism.

Aldh1a1 and Scl25a30 in diaphragmatic dysfunction.

New methods to prevent ventilator-induced diaphragmatic dysfunction (VIDD) are urgently needed, and the cellular basis of VIDD is poorly understood. This study evaluated whether transvenous phrenic nerve stimulation (PNS) could prevent VIDD in rabbits undergoing mechanical ventilation (MV) and explored whether oxidative stress-related genes might be candidate molecular markers for VIDD. Twenty-four adult male New Zealand white rabbits were allocated to control, MV, and PNS groups (n = 8 in each group). Rabbits in the MV and PNS groups underwent MV for 24 h. Intermittent bilateral transvenous PNS was performed in rabbits in the PNS group. Transdiaphragmatic pressure was recorded using balloon catheters. The diameters and cross-sectional areas (CSAs) of types I and II diaphragmatic fibers were measured using immunohistochemistry (IHC) techniques. Genes associated with VIDD were identified by RNA sequencing (RNA-seq), differentially expressed gene (DEG) analysis, and weighted gene co-expression network analysis (WGCNA). Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and IHC analyses were carried out to verify the transcriptome profile. Pdi60Hz, Pdi80Hz, and Pdi100Hz were significantly higher in the PNS group than in the MV group at 12 and 24 h (P < 0.05 at both time points). The diameters and CSAs of types I (slow-twitch) and II (fast-twitch) fibers were significantly larger in the PNS group than in the MV group (P < 0.05). RNA-seq, RT-PCR, Western blotting, and IHC experiments identified two candidate genes associated with VIDD: Aldh1a1 and Scl25a30. The MV group had significantly higher mRNA and protein expressions of Aldh1a1/ALDH1A1 and significantly lower mRNA and protein expressions of Scl25a30/SCL25A30 than the control or PNS groups (P < 0.05). We have identified two candidate genes involved in the prevention of VIDD by transvenous PNS. These two key genes may provide a theoretical basis for targeted therapy against VIDD.

SMYD5 acts as a potential biomarker for hepatocellular carcinoma.

Determining the prognosis of patients remains a challenge due to the phenotypic and molecular diversities of hepatocellular carcinomas (HCC). We aimed to evaluate the role of SMYD5 in HCC. Wilcoxon signed-rank test and logistic regression analyzed the relationship between clinical pathologic features and SMYD5. We found that increased expression of SMYD5 in HCC was closely associated with high histologic grade, stage, T stage and nodal stage. Kaplan-Meier method, Cox regression, univariate analysis and multivariate analysis detected overall survival of TCGA-HCC patients. It turned out that high expression of SMYD5 predicted a worse prognosis in HCC. Gene Set Enrichment Analysis (GSEA) was applied via TCGA data set, which indicated that complement and coagulation cascades, fatty acid metabolism, primary bile acid biosynthesis, drug metabolism cytochrome P450, PPAR signaling pathway and retinol metabolism were differentially enriched in SMYD5 high expression phenotype. Interestingly, we proved that SMYD5 upregulation in HCC cells was induced by promoter hypo-methylation. Moreover, functional experiments demonstrated that SMYD5 silencing abrogated cell proliferation, migration and invasion and enhanced paclitaxel sensitivity in HCC. All findings implied that SMYD5 might be an underlying biomarker for prognosis and treatment of HCC.

Mir-204 Regulates LPS-Induced A549 Cell Damage by Targeting FOXK2.

Objective: To assess whether miR-204 and HA affect A549 cell injury induced by lipopolysaccharide. Material and Methods. A549 cells were treated with hirsutanol A, and cell damage was induced by LPS followed by analysis of cell proliferation by CCK-8, cell apoptosis by flow cytometry, apoptosis-related protein expression by western blot, downstream target of miR-20 by dual-luciferase reporter gene, and inflammatory factors by ELISA and PCR. Results: LPS can significantly inhibit the viability of A549 cells, induce cell apoptosis, and promote the release of IL-6, IL-1ß, and TNF-α, while HA pretreatment can target FOXK2 by upregulating miR-204 levels, thereby alleviating apoptosis and promoting cell viability and at the same time inhibiting the release of inflammatory factors by inhibiting the activation of NF-κB. Conclusions: miR-204 participates in the protection of HA acute lung injury by targeting FOXK2.

Protective Effects of Emodin on Lung Injuries in Rat Models of Liver Fibrosis.

OBJECTIVE: The aim of this study is to investigate the protective effects of emodin (EMD) on the lung injuries in the rat models of liver fibrosis. METHODS: Liver fibrosis was established in rats and the effect of intervention using EMD treatment was determined. Liver and lung weight coefficients were measured and lung content of TNF-α (tumor necrosis factor α), MDA (malondialdehyde), NO (nitric oxide), and ONOO- (peroxynitrite) were determined. Finally, histopathological changes were evaluated. RESULTS: Compared with the normal control group, the lung weight coefficient was significantly increased in the fibrosis model group. Moreover, pulmonary edema and inflammatory responses were observed. Levels of TNF-α, MDA, NO, and ONOO- in the lung homogenate were significantly increased in the fibrosis model group. After EMD treatment, the lung weight coefficients were significantly reduced. Moreover, pathological changes in the lung tissue were dramatically alleviated. Levels of TNF-α, MDA, NO, and ONOO- were significantly decreased. CONCLUSION: EMD exhibits protective effects against lung injuries in a rat model of liver fibrosis.

Correction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

[This corrects the article DOI: 10.1371/journal.pone.0175807.].

Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

BACKGROUND: Skeletal myoblast transplantation seems a promising approach for the repair of myocardial infarction (MI). However, the low engraftment efficacy and impaired angiogenic ability limit the clinical efficiency of the myoblasts. Gene engineering with angiogenic growth factors promotes angiogenesis and enhances engraftment of transplanted skeletal myoblasts, leading to improved infarction recovery in myocardial ischemia. The present study evaluated the therapeutic effects of hepatocyte growth factor (HGF) gene-engineered skeletal myoblasts on tissue regeneration and restoration of heart function in a rat MI model. METHODS AND RESULTS: The skeletal myoblasts were isolated, expanded, and transduced with adenovirus carrying the HGF gene (Ad-HGF). Male SD rats underwent ligation of the left anterior descending coronary artery. After 2 weeks, the surviving rats were randomized into four groups and treated with skeletal myoblasts by direct injection into the myocardium. The survival and engraftment of skeletal myoblasts were determined by real-time PCR and in situ hybridization. The cardiac function with hemodynamic index and left ventricular architecture were monitored; The adenovirus-mediated-HGF gene transfection increases the HGF expression and promotes the proliferation of skeletal myoblasts in vitro. Transplantation of HGF-engineered skeletal myoblasts results in reduced infarct size and collagen deposition, increased vessel density, and improved cardiac function in a rat MI model. HGF gene modification also increases the myocardial levels of HGF, VEGF, and Bcl-2 and enhances the survival and engraftment of skeletal myoblasts. CONCLUSIONS: HGF engineering improves the regenerative effect of skeletal myoblasts on MI by enhancing their survival and engraftment ability.

CXCR2 is involved in pulmonary intravascular macrophage accumulation and angiogenesis in a rat model of hepatopulmonary syndrome.

Hepatopulmonary syndrome (HPS) is a lung complication in various liver diseases, with high incidence, poor prognosis and no effective non-surgical treatments in patients with hepatocirrhosis. Therefore, assessing HPS pathogenesis to explore proper therapy strategies is clinically relevant. In the present study, male Sprague-Dawley rats underwent sham operation or common bile duct ligation (CBDL). Two weeks post-surgery, the following groups were set up for 2 weeks of treatment: sham + normal saline, CBDL + CXCR2 antagonist SB225002, CBDL + tumour necrosis factor α (TNF-α) antagonist PTX and CBDL + normal saline groups. Liver and lung tissues were collected after mean arterial pressure (MAP) and portal venous pressure (PVP) measurements. Haematoxylin and eosin (H&E) staining (lung) and Masson staining (liver) were performed for pathological analyses. Finally, pulmonary tissue RNA and total protein were assessed for target effectors. The mRNA and protein levels of CXCR2 were significantly increased in the pulmonary tissue of CBDL rats. What's more, CXCR2 inhibition by SB225002 reduced the expression of CD68 and von Willebrand factor (vWf) in CBDL rats. Importantly, CXCR2 inhibition suppressed the activation of Akt and extracellular signal-regulated kinase (ERK) in CBDL rats. Antagonization of TNF-α with PTX down-regulated the expression of CXCR2. During HPS pathogenesis in rats, CXCR2 might be involved in the accumulation of pulmonary intravascular macrophages and angiogenesis, possibly by activating Akt and ERK, with additional regulation by TNF-α that enhanced pulmonary angiogenesis by directly acting on the pulmonary tissue. Finally, the present study may provide novel targets for the treatment of HPS.

[Dynamic changes of TGF-α and TGF-ß1 in rats with liver cirrhosis induced by multiple pathogenic factors].

OBJECTIVE: To explore the dynamic changes of transforming growth factor-α (TGF-α) and transforming growth factor-ß1 (TGF-ß1) of liver cirrhosis induced by multiple pathogenic factors in rats. METHODS: Animals in the cirrhosis group were fed a mixture of maize flour, lard, cholesterol and alcohol plus subcutaneously injection with carbon tetrachloride (CCl4), the CCl4(0.5 ml/100 g · w) was injected at the first day of experiment and the 40% CCl4oil solution (0.3 ml /100 g · w) was injected at an interval of three days. The thirty-six male SD rats were randomly divided into liver cirrhosis group of the 4th, 6th and 8 th week, and normal control group of the 4th, 6th and 8th week. The contents of alanine transferase (ALT), endotoxin, tumor necrosis factor-α (TNF-α) and homocysteine (Hcy) in plasma were evaluated. Histopathological changes of the liver were observed under microscope with the staining of HE. The expressions of TGF-α and TGF-ß1 were analyzed by the method of immunohistochemistry. RESULTS: Compared with the corresponding normal control group, the levels of ALT, endotoxin, TNF-α and Hcy in plasma were gradually significantly increased in liver cirrhosis group of the 4th, 6th and 8th week (P < 0.05); the expression of TGF-α in the liver tissues was significantly increased at the 4th week (P < 0.05); the expression of TGF-ß1 in the liver tissues was gradually significantly increased in every model group (P < 0.05). CONCLUSION: In the formation process of cirrhosis, the expression of TGF-α was increased in liver of cirrhosis group at the 4th week, and later it was suppressed; the expression of TGF-ß1 was continuously increased. The characteristic dynamic changes of TGF-α and TGF-ß1 might be related to sustained endotoxemia, the high level of TNF-α and hyperhomocysteinemia.

Effect of artesunate supplementation on bacterial translocation and dysbiosis of gut microbiota in rats with liver cirrhosis.

AIM: To evaluate the effect of artesunate (AS) supplementation on bacterial translocation (BT) and gut microbiota in a rat model of liver cirrhosis. METHODS: Fifty-four male Sprague-Dawley rats were randomly divided into a normal control group (N), a liver cirrhosis group (M) and a liver cirrhosis group intervened with AS (MA). Each group was sampled at 4, 6 and 8 wk. Liver cirrhosis was induced by injection of carbon tetrachloride (CCl4), intragastric administration of 10% ethanol, and feeding a high fat diet. Rats in the MA group were intragastrically administered with AS (25 mg/kg body weight, once daily). Injuries of the liver and intestinal mucosa were assessed by hematoxylin-eosin or Masson's trichrome staining. Liver index was calculated as a ratio of the organ weight (g) to body weight (g). The gut microbiota was examined by automated ribosomal intergenic-spacer analysis of fecal DNA. BT was assessed by standard microbiological techniques in the blood, mesenteric lymph nodes (MLNs), liver, spleen, and kidney. RESULTS: Compared to group N, the body weight was reduced significantly in groups M and MA due to the development of liver cirrhosis over the period of 8 wk. The body weight was higher in group MA than in group M. The liver indices were significantly elevated at 4, 6 and 8 wk in groups M and MA compared to group N. AS supplementation partially decreased the liver indices in group MA. Marked histopathologic changes in the liver and small intestinal mucosa in group M were observed, which were alleviated in group MA. Levels of pro-inflammatory interleukin-6 and tumor necrosis factor-α were significantly elevated at 8 wk in ileal homogenates in group M compared to group N, which were decreased after AS supplementation in group MA. The dysbiosis of gut microbiota indicated by the mean diversity (Shannon index) and mean similarity (Sorenson index) was severe as the liver cirrhosis developed, and AS supplementation had an apparent intervention effect on the dysbiosis of gut microbiota at 4 wk. The occurrence of BT was increased in the liver of group M compared to that of group N. AS supplementation reduced BT in group MA at 8 wk. BT also occurred in the MLNs, spleen, and kidney, which was reduced by AS supplementation. BT was not detected in the blood in any group. CONCLUSION: Dysbiosis of gut microbiota, injury of intestinal mucosal barrier and BT occurred as liver cirrhosis progressed, which might enhance inflammation and aggravate liver injury. AS may have other non-antimalarial effects that modulate gut microbiota, inhibit BT and alleviate inflammation, resulting in a reduction in CCl4, alcohol and high fat-caused damages to the liver and intestine.

Increased expression of 78 kD glucose-regulated protein promotes cardiomyocyte apoptosis in a rat model of liver cirrhosis.

AIMS: This study was to investigate the role and underlying mechanism of 78 kD glucose-regulated protein (GRP78) in cardiomyocyte apoptosis in a rat model of liver cirrhosis. METHODS: A rat model of liver cirrhosis was established with multiple pathogenic factors. A total of 42 male SD rats were randomly divided into the liver cirrhosis group and control group. Cardiac structure analysis was performed to assess alterations in cardiac structure. Cardiomyocytes apoptosis was detected by TdT-mediated dUTP nick end labeling method. Expression of GRP78, CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) and B cell lymphoma-2 (Bcl-2) was detected by immunohistochemical staining. RESULTS: The ratios of left ventricular wall thickness to heart weight and heart weight to body weight were significantly increased with the progression of liver cirrhosis (P < 0.05). Apoptosis index of cardiomyocytes was significantly increased with the progression of liver cirrhosis (P < 0.05). The expression levels of GRP78, CHOP and caspase-12 were significantly increased in the progression of liver cirrhosis (P < 0.05). The expression levels of NF-κB p65 and Bcl-2 were highest in the 4-wk liver cirrhosis, and they were decreased in the 6-wk and 8-wk in the progression of liver cirrhosis. GRP78 expression levels were positively correlated with apoptosis index, CHOP and caspase-12 expression levels (P < 0.05). CHOP expression levels were negatively correlated with NF-κB p65 and Bcl-2 expression levels (P < 0.05). CONCLUSION: Increased expression of GRP78 promotes cardiomyocyte apoptosis in rats with cirrhotic cardiomyopathy.

Artesunate alleviates hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats.

The current study was performed in order to explore the effect of artesunate (Art) on experimental hepatic fibrosis and the potential mechanism involved. Art, a water-soluble hemisuccinate derivative of artemisinin extracted from the Chinese herb Artemisia Annua, is a safe and effective antimalarial drug. Hepatic fibrosis was induced in SD rats by multiple pathogenic factors. Rats were treated concurrently with Art (28.8 mg/kg) given daily by oral gavage for 6 or 8 weeks to evaluate its protective effects. Our data demonstrated that Art treatment obviously attenuated hepatic fibrosis, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM). Art remarkably decreased endotoxin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels as well. Art significantly downregulated protein and mRNA expression of α-smooth muscle actin (α-SMA), toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88) and transforming growth factor beta 1 (TGF-ß1). Art also significantly inhibited the nuclear transcription factor kappa B p65 (NF-κB p65) translocation into the nucleus. In addition, there were no remarkable differences between the N group and the NA group. In conclusion, we found that Art could alleviate hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats, suggesting that Art may be a potential candidate for the therapy of hepatic fibrosis.

[Protective effect of tanshinol on the hepatopulmonary syndrome in rat].

OBJECTIVE: To explore the mechanism of tanshinol on alleviate the inflammatory injury of lung tissue in rat hepatopulmonary syndrome (HPS). METHODS: SD rats were randomly divided into normal control group (n = 8), hepatopulmonary syndrome (HPS) group (n = 11) and tanshinol intervention group (n = 9). HE staining was used to observe the histopathology changes of pulmonary and hepatic tissues, and to count the number of macrophages in lung tissues. The activity of alanine transferase (ALT) and concentrations of endotoxin, tumor necrosis factor-a (TNF-alpha) and homocystein (Hcy) in plasma were detected. The concentrations of TNF-alpha, nitric oxide (NO) and malondialdehyde (MDA) and the activity of inducible nitric oxide synthase (iNOS) in the lung tissues were measured, respectively. RESULTS: Thickened alveolar septum and increased macrophages were observed in lungs in HPS rat. After administered with tanshinol, the pulmonary pathological changes were alleviated and the number of macrophages in lung tissue was decreased compared with HPS group. The activity of ALT and the concentrations of endotoxin, TNF-alpha and Hcy in plasma ,and TNF-alpha, iNOS, NO and MDA in lung tissue in HPS group were higher than those of normal control group; meanwhile, those tanshinol group were less those that of HPS group. CONCLUSION: Tanshinol may play an important role in delaying the development of HPS through protecting liver or directly antagonizing the effect of intestinal endotoxemia so as to alleviate the inflammatory reaction in lung tissue.

Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome.

OBJECTIVE: This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development. METHODS: The rat models with liver cirrhosis and HPS were induced by multiple pathogenic factors for 4 to 8 wk. The concentrations of alanine transferase (ALT) and endotoxin in plasma were detected in the models, followed by the detection of GRP78 expression. RT-PCR, quantitative real-time PCR and Western blotting were employed to assess the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), respectively. Immunohistochemistry staining was used to examine the expression of a specific vascular marker, factor VIII-related antigen (FVIII-RAg), and several cell proliferation- and apoptosis-related proteins, including CHOP/GADD153, caspase-12, Bcl-2 and nuclear factor (NF)-κB. RESULTS: The levels of endotoxin and ALT in plasma were gradually increased as the disease progressed, so did GRP78, which were in a positive correlation. The expression levels of VEGF (both mRNA and protein) and FVIII-RAg were significantly elevated in the HPS models, indicating active angiogenesis, which was also positively correlated with GRP78 expression. Furthermore, the expression levels of the pro-apoptotic proteins of CHOP/GADD153 and caspase-12 were dramatically decreased, while the anti-apoptotic proteins of Bcl-2 and NF-κB were significantly elevated, in the HPS models. There were also close correlation between these proteins and GRP78. CONCLUSIONS: Over-expression of GRP78 in lungs may be the critical pathogenic factor for HPS. Through promoting cell proliferation and survival and inhibiting apoptosis, GRP78 may promote the pulmonary microvascular remodeling in HPS pathogenesis. Our results provide a potential therapeutic target for clinical prevention and treatment for HPS and related complications.

Expression of the 78 kD glucose-regulated protein is induced by endoplasmic reticulum stress in the development of hepatopulmonary syndrome.

OBJECTIVE: This study is to explore the role of 78 kD glucose-regulated protein (GRP78) in the development of hepatopulmonary syndrome (HPS) in rats. METHODS: The rat model of liver cirrhosis and HPS were induced with multiple pathogenic factors. Hematoxylin and eosin (H & E) staining was performed to detect the pathological changes of the lung and liver tissues. The levels of alanine transferase (ALT), endotoxin, and tumor necrosis factor-α (TNF-α) in plasma and TNF-α and malondialdehyde (MDA) in lung tissues were detected. RT-PCR and Western blotting were conducted to detect the mRNA and protein expression levels of GRP78 in lungs. RESULTS: The plasma endotoxin level was gradually increased as HPS developed, and the mRNA and protein expression levels of GRP78 in lungs were also increased as the disease progressed. The levels of ALT and TNF-α in plasma and the contents of TNF-α and MDA in lung tissues were gradually increased along with the disease progression, with a strong positive correlation. Compared with controls, the plasma TNF-α level and the mRNA and protein expression levels of GRP78 in lung tissues were significantly higher in rats with HPS. The levels of endotoxin and ALT in plasma and the level of MDA in lungs were significantly higher in rats with HPS than controls. CONCLUSIONS: The increased GRP78 expression is indicative of endoplasmic reticulum stress response during HPS, which may play an important role in the disease pathogenesis.