Protective role of pretreatment with Anisodamine against sepsis-induced diaphragm atrophy via inhibiting JAK2/STAT3 pathway.

There is an increasing tendency for sepsis patients to suffer from diaphragm atrophy as well as mortality. Therefore, reducing diaphragm atrophy could benefit sepsis patients' prognoses. Studies have shown that Anisodamine (Anis) can exert antioxidant effects when blows occur. However, the role of Anisodamine in diaphragm atrophy in sepsis patients has not been reported. Therefore, this study investigated the antioxidant effect of Anisodamine in sepsis-induced diaphragm atrophy and its mechanism. We used cecal ligation aspiration (CLP) to establish a mouse septic mode and stimulated the C2C12 myotube model with lipopolysaccharide (LPS). After treatment with Anisodamine, we measured the mice's bodyweight, diaphragm weight, fiber cross-sectional area and the diameter of C2C12 myotubes. The malondialdehyde (MDA) levels in the diaphragm were detected using the oxidative stress kit. The expression of MuRF1, Atrogin1 and JAK2/STAT3 signaling pathway components in the diaphragm and C2C12 myotubes was measured by RT-qPCR and Western blot. The mean fluorescence intensity of ROS in C2C12 myotubes was measured by flow cytometry. Meanwhile, we also measured the levels of Drp1 and Cytochrome C (Cyt-C) in vivo and in vitro by Western blot. Our study revealed that Anisodamine alleviated the reduction in diaphragmatic mass and the loss of diaphragmatic fiber cross-sectional area and attenuated the atrophy of the C2C12 myotubes by inhibiting the expression of E3 ubiquitin ligases. In addition, we observed that Anisodamine inhibited the JAK2/STAT3 signaling pathway and protects mitochondrial function. In conclusion, Anisodamine alleviates sepsis-induced diaphragm atrophy, and the mechanism may be related to inhibiting the JAK2/STAT3 signaling pathway.

Glutathione S-Transferase α4 Alleviates Hyperlipidemia- Induced Vascular Neointimal Hyperplasia in Arteriovenous Grafts via Inhibiting Endoplasmic Reticulum Stress.

Neointimal hyperplasia causes the failure of coronary artery bypass grafting (CABG). Our previous studies have found that endothelial dysfunction is one candidate for triggering neointimal hyperplasia, but which factors are involved in this process is unclear. Glutathione S-transferase α4 (GSTA4) play an important role in metabolizing 4-hydroxynonenal (4-HNE), a highly reactive lipid peroxidation product, which causes endothelial dysfunction or death. Here, we investigated the role of GSTA4 in neointima formation after arteriovenous grafts (AVGs) with or without high-fat diet (HFD). Compared with normal diet (ND), HFD caused endothelial dysfunction and increased neointima formation, concomitantly accompanied by downregulated expression of GSTA4 at the mRNA and protein levels. In vitro, overexpression of GSTA4 attenuated 4-HNE-induced endothelial dysfunction and knockdown of GSTA4 aggravated endothelial dysfunction. Furthermore, silencing GSTA4 expression facilitated the activation of 4-HNE induced endoplasmic reticulum stress (ERS) and inhibition of ERS pathway alleviated 4-HNE-induced endothelial dysfunction. Additionally, compared with wild-type (WT) mice, mice with knockout of endothelial-specific GSTA4 (GSTA4 EC KO) exhibited exacerbated vascular endothelial dysfunction and increased neointima formation caused by HFD. Together, these results demonstrate the critical role of GSTA4 in protecting the function of endothelial cells and in alleviating hyperlipidemia-induced vascular neointimal hyperplasia in arteriovenous grafts.

Endoplasmic reticulum stress promotes sepsis-induced muscle atrophy via activation of STAT3 and Smad3.

Endoplasmic reticulum (ER) stress is involved in skeletal muscle atrophy in various conditions, but the role of ER stress in sepsis-induced muscle atrophy is not well understood. In this study, we conducted experiments in wild-type (WT) mice and C/EBP homologous protein knockout (CHOP KO) mice to explore the role and mechanism of ER stress in sepsis-induced muscle atrophy. Cecal ligation and puncture (CLP) was used to establish a mouse model of sepsis. In WT mice, the body weight, muscle mass, and cross-sectional area of muscle fibers in CLP group both decreased significantly compared with sham group, which revealed that sepsis-induced dramatic muscle atrophy. Additionally, sepsis activated the ubiquitin-proteasome system (UPS), accompanied by the activation of ER stress. In vitro, inhibition of ER stress suppressed the activity of E3 ubiquitin ligases and alleviated the myotube atrophy. In vivo, CHOP KO also reduced the expression of E3 ubiquitin ligases and UPS-mediated protein degradation, and significantly attenuated sepsis-induced muscle atrophy. Deletion of CHOP also decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and Smad3, and inhibition of STAT3 and Smad3 partly reduced proteolysis caused by ER stress in vitro. These findings confirm that ER stress activates UPS-mediated proteolysis and promotes sepsis-induced muscle atrophy, which is partly achieved by activating STAT3 and Smad3.

Endoplasmic Reticulum Stress-induced Endothelial Dysfunction Promotes Neointima Formation after Arteriovenous Grafts in Mice on High-fat Diet.

OBJECTIVE: Endothelial dysfunction is one candidate for triggering neointima formation after arteriovenous grafts (AVGs), but the factors mediating this process are unclear. The purpose of this study was to investigate the role of endoplasmic reticulum stress (ERS)-induced endothelial dysfunction in neointima formation following AVGs in high-fat diet (HFD) mice. METHODS: CCAAT-enhancer-binding protein-homologous protein (CHOP) knockout (KO) mice were created. Mice were fed with HFD to produce HFD model. AVGs model were applied in the groups of WT ND, WT HFD, and CHOP KO HFD. Human umbilical vein endothelial cells (HUVECs) were cultured with oxidized low density lipoprotein (ox-LDL) (40 mg/L) for the indicated time lengths (0, 6, 12, 24 h). ERS inhibitor tauroursodeoxycholic acid (TUDCA) was used to block ERS. Immunohistochemical staining was used to observe the changes of ICAM1. Changes of ERS were detected by real-time RT-PCR. Protein expression levels and ERS activation were detected by Western blotting. Endothellial cell function was determined by endothelial permeability assay and transendothelial migration assay. RESULTS: HFD increased neointima formation in AVGs associated with endothelial dysfunction. At the same time, ERS was increased in endothelial cells (ECs) after AVGs in mice consuming the HFD. In vitro, ox-LDL was found to stimulate ERS, increase the permeability of the EC monolayer, and cause endothelial dysfunction. Blocking ERS with TUDCA or CHOP siRNA reversed the EC dysfunction caused by ox-LDL. In vivo, knockout of CHOP (CHOP KO) protected the function of ECs and decreased neointima formation after AVGs in HFD mice. CONCLUSION: Inhibiting ERS in ECs could improve the function of AVGs.

Astragaloside IV alleviates sepsis-induced muscle atrophy by inhibiting the TGF-ß1/Smad signaling pathway.

BACKGROUND: Muscle atrophy occurs in patients with sepsis and increases mortality and disability. Remission of muscle atrophy may improve the quality of life in patients with sepsis. Astragaloside IV (ASIV) has been shown to have excellent anti-inflammatory and anti-fibrotic effects and to reduce organ damage caused by sepsis. However, the effect of ASIV on sepsis-induced muscle atrophy has not been reported. Therefore, this study explored the pharmacological effects and mechanisms of ASIV in sepsis-induced muscle atrophy. METHODS: Cecal ligation and puncture (CLP) was used to establish a mouse model of sepsis and lipopolysaccharide (LPS)-stimulated C2C12 myotubes. After administration of ASIV, the body weight, tibialis anterior (TA) and gastrocnemius muscle weight and fiber cross-sectional area of the mice were measured. The diameter of myotubes was observed by immunofluorescence staining. ELISA was used to assess inflammatory factors in plasma and cell culture supernatants. RT-PCR and Western blotting were used to detect the expression of MuRF1, Atrogin-1 and TGF-ß1/Smad signaling pathway components in TA and C2C12 myotubes. RESULTS: Our study found that ASIV reduced serum inflammatory factors and improved survival in septic mice. ASIV alleviated muscle mass reduction, myofiber cross-sectional area reduction, and C2C12 myotube atrophy by inhibiting the expression of the E3 ubiquitin ligases MuRF1 and atrogin-1. In addition, we observed that ASIV inhibited TGF-ß1/Smad signaling. Inhibition of the TGF-ß1/Smad signaling pathway partly blocked the anti-muscle atrophy effect of ASIV. CONCLUSION: ASIV can alleviate sepsis-induced muscle atrophy, which may be related to the inhibition of the TGF-ß1/Smad signaling pathway.

The effects of uncut Roux-en-Y anastomosis on laparoscopic radical gastrectomy patients' postoperative complications and quality of life.

OBJECTIVES: This study discusses and analyzes the effects of uncut Roux-en-Y anastomosis on the postoperative complications and quality of life of gastric cancer patients after they undergo laparoscopic total gastrectomies. METHODS: 86 patients admitted to our hospital for laparoscopic digestive tract reconstruction after distal gastrectomies were recruited as the study cohort and divided into group A (n=41) or group B (n=45) on the basis of the different digestive tract reconstruction surgery each patient underwent. The group-A patients underwent traditional Roux-en-Y anastomosis, and the group-B patients underwent uncut Roux-en-Y anastomosis. The operation outcomes, the early and late postoperative complications, the decline in the postoperative nutrition status, and the changes in the postoperative quality of life scores were compared between the two groups. RESULTS: The intraoperative anastomosis times and the blood losses, the postoperative ventilation, the liquid food intake times, and the hospitalization durations in group B were lower than they were in group A (P<0.05). The incidences of early and late postoperative complications in group A was significantly higher than they were in group B (P<0.05). The decrease of the TP, ALB, and Hb levels in group B was dramatically lower than it was in group A at 6 months after the surgeries (P<0.05), and no significant difference in the TP, ALB, or Hb levels was observed between the two groups at 12 months after the surgeries (P>0.05). The QOL scores in the two groups of patients were increased at 6 and 12 months after the surgeries compared with 1 month after the surgeries (P<0.05). The QOL scores in group B were notably higher than they were in group A at 6 and 12 months after the surgeries (P<0.05). CONCLUSION: Uncut Roux-en-Y anastomosis after laparoscopic total gastrectomy is simple to carry out. The method can promote patients' early postoperative recovery, reduce their early and late complications, and maintain their postoperative nutritional status, thus improving their postoperative quality of life, so it is worthy of clinical promotion.

Effectiveness of central venous pressure versus stroke volume variation in guiding fluid management in renal transplantation.

OBJECTIVE: The aim of this study was to compare the effectiveness of central venous pressure (CVP) versus stroke volume variation (SVV) to guide fluid management in renal transplantation. METHODS: The clinical data of 97 patients who underwent allogeneic renal transplantation in our hospital were collected retrospectively. Based on the method of intraoperative infusion monitoring, they were divided into group A, which received guided fluid management by monitoring CVP, and group B which received guided fluid management by monitoring SVV. The changes in intraoperative hemodynamic indicators, urine volume, blood loss, and total blood transfusion volume, total fluid volume, urine output at different time points after surgery, renal function indicators, blood purification rate, length of stay, and postoperative complications were compared between the two groups. RESULTS: CVP values at T1 (5 min before surgery), T2 (external iliac vein obstruction), T3 (establishment of vessel access), and T4 (end of surgery) in group B were higher than those in group A (P<0.05). The two groups showed no significant difference in intraoperative urine volume, blood loss and length of hospital stay (P>0.05). The total fluid volume and total infusion volume at 3 days after surgery in group B were less than those in group A (P<0.05). The urine volume did not differ at time points 0 h, 24 h, 48 h and 72 h postoperatively (P>0.05). Serum creatinine levels in group B at 0 h, 24 h, 48 h and 72 h postoperatively were lower than those in group A (P<0.05). After renal transplantation, the rate of blood purification was 4.08% in group B, which was lower than 25.00% in group A (P<0.05). The rate of respiratory failure in group B was 4.08%, which was not significantly different from 6.25% in group A (P>0.05). CONCLUSION: Compared with CVP, fluid management guided by monitoring SVV during renal transplantation can reduce intraoperative fluid volume, optimize the renal perfusion, reduce postoperative blood purification, and facilitate postoperative recovery.

Protective role of down-regulated microRNA-31 on intestinal barrier dysfunction through inhibition of NF-κB/HIF-1α pathway by binding to HMOX1 in rats with sepsis.

BACKGROUND: Intestinal barrier dysfunction is a significant clinical problem, commonly developing in a variety of acute or chronic pathological conditions. Herein, we evaluate the effect of microRNA-31 (miR-31) on intestinal barrier dysfunction through NF-κB/HIF-1α pathway by targeting HMOX1 in rats with sepsis. METHODS: Male Sprague-Dawley rats were collected and divided into the sham group, and the cecum ligation and perforation group which was subdivided after CACO-2 cell transfection of different mimic, inhibitor, or siRNA. Levels of serum D-lactic acid, diamine oxidase and fluorescence isothiocyanate dextran, FITC-DX concentration, and bacterial translocation were detected. Superoxidedismutase (SOD) activity and malondialdehyde (MDA) content were evaluated using the colorimetric method and an automatic microplate reader, respectively. Additionally, the levels of tumor necrosis factor, interleukin (IL)-6, and IL-10 were tested using enzyme-linked immunosorbent assay. The expression of miR-31, HMOX1, NF-κB, HIF-1α, IκB, ZO-1 and Occludin were assessed by reverse transcription quantitative polymerase chain reaction and Western blot analysis. RESULTS: Inhibition of miR-31 decreased intestinal mucosal permeability and intestinal barrier function. The increased levels of miR-31 could cause oxidative damage and affect the expression of inflammatory factors in intestinal tissue of rats. HMOX1 was confirmed as a target gene of miR-31. MiR-31 affected intestinal mucosal permeability and intestinal barrier function, as well as oxidative damage and inflammation level by regulating HMOX1. Down-regulation of miR-31 inhibited NF-κB/HIF-1α pathway related genes by regulating HMOX1 expression. Furthermore, inhibition of miR-31 increased survival rates of rats. CONCLUSION: Overall, the current study found that inhibition of miR-31 protects against intestinal barrier dysfunction through suppression of the NF-κB/HIF-1α pathway by targeting HMOX1 in rats with sepsis.

Candidate genes and pathogenesis investigation for sepsis-related acute respiratory distress syndrome based on gene expression profile.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years, the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment. This study aimed to predict some genes or pathways associated with sepsis-related ARDS based on a public microarray dataset and to further explore the molecular mechanism of ARDS. RESULTS: A total of 122 up-regulated DEGs and 91 down-regulated differentially expressed genes (DEGs) were obtained. The up- and down-regulated DEGs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. Protein-protein interaction network of ARDS analysis revealed 20 hub genes including cyclin B1 (CCNB1), cyclin B2 (CCNB2) and topoisomerase II alpha (TOP2A). A total of seven transcription factors including forkhead box protein M1 (FOXM1) and 30 target genes were revealed in the transcription factor-target gene regulation network. Furthermore, co-cited genes including CCNB2-CCNB1 were revealed in literature mining for the relations ARDS related genes. CONCLUSIONS: Pathways like mitotic cell cycle were closed related with the development of ARDS. Genes including CCNB1, CCNB2 and TOP2A, as well as transcription factors like FOXM1 might be used as the novel gene therapy targets for sepsis related ARDS.

Candidate genes and pathogenesis investigation for sepsis-related acute respiratory distress syndrome based on gene expression profile

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years, the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment. This study aimed to predict some genes or pathways associated with sepsis-related ARDS based on a public microarray dataset and to further explore the molecular mechanism of ARDS. RESULTS: A total of 122 up-regulated DEGs and 91 down-regulated differentially expressed genes (DEGs) were obtained. The up- and down-regulated DEGs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. Protein-protein interaction network of ARDS analysis revealed 20 hub genes including cyclin B1 (CCNB1), cyclin B2 (CCNB2) and topoisomerase II alpha (TOP2A). A total of seven transcription factors including forkhead box protein M1 (FOXM1) and 30 target genes were revealed in the transcription factor-target gene regulation network. Furthermore, co-cited genes including CCNB2-CCNB1 were revealed in literature mining for the relations ARDS related genes. CONCLUSIONS: Pathways like mitotic cell cycle were closed related with the development of ARDS. Genes including CCNB1, CCNB2 and TOP2A, as well as transcription factors like FOXM1 might be used as the novel gene therapy targets for sepsis related ARDS

Screening genes associated with myocardial infarction and transverse aortic constriction using a combined analysis of miRNA and mRNA microarray.

Myocardial infarction (MI) and transverse aortic constriction (TAC) are two models of cardiac hypertrophy. To study mechanisms of MI and TAC, GSE415 and GSE14267 were downloaded from Gene Expression Omnibus. GSE415 included left ventricle (LV) and intraventricular septum samples from mice that underwent MI, TAC or sham operation. GSE14267 included normal and MI samples from non-transgenic mice. Differentially expressed genes (DEGs) and microRNAs (miRNAs) were screened using limma package. Functional enrichment analysis was performed for DEGs using DAVID. Common DEGs of different groups were conducted for protein-protein interaction (PPI) analysis using STRING and visualized in PPI network by Cytoscape. Furthermore, targets were predicted for differentially expressed miRNAs using TarMir database. Totally, 277 DEGs, 31 common DEGs (e.g. SFRP2), 6 differentially expressed miRNAs (e.g. mmu-miR-448) and 1 miRNA-mRNA pair (mmu-miR-448→SIM2) were screened out. DEGs were significantly enriched in biological processes related to muscle development and ion transportation. In the PPI network for common DEGs, LOX (degree=7), POSTN (degree=5), SPARC (degree=4) and TIMP1 (degree=3) were with higher degrees. In addition, they might function by interacting with each other (e.g. LOX-TIMP1, LOX-POSTN, SPARC-TIMP1 and SPARC-POSTN). In conclusion, LOX, POSTN, SPARC, TIMP1 and SFRP2 might affect MI and TAC.3.

The effect of oxLDL on aortic valve calcification via the Wnt/ ß-catenin signaling pathway: an important molecular mechanism.

BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve disease (CAVD) is a commonly acquired valvular disease. Although previous studies have shown valve calcification to be mediated by a chronic inflammatory disease process, with many similarities to atherosclerosis that included inflammatory cell infiltrates, lipoproteins, lipids, extracellular bone-matrix proteins, and bone minerals, little is known of the mechanisms of the cellular and molecular components and processes. It has recently been hypothesized that the calcific aortic valve is a product of active inflammation, similar to the atherosclerosis pathological process. Thus, the cessation of statin therapy should, in theory, have an effect on the treatment of CAVD and on aortic valve myofibroblasts (AVMFs), which play an important role in aortic valvular calcification. The study aim was to determine if oxidized low-density lipoprotein (oxLDL) could stimulate the apoptosis of AVMFs and the calcific-related pathway, and whether atorvastatin could inhibit the effects of AVMFs induced by oxLDL. The Wnt/GSK-3ß/ß-catenin signaling pathway may play a key role in this process, thereby making a major contribution to aortic valve calcification. METHODS: AVMFs were successfully acquired using a combination of trypsin and collagenase enzyme digestion, and made phenotypic for the identification for alpha-smooth muscle actin (α-SMA). Cell apoptosis was monitored using flow cytometry, bone protein expression by Western blot, and related gene expression by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: A positive identification of α-SMA, a myofibroblast marker, confirmed the successful harvesting of myofibroblasts. OxLDL significantly induced cell apoptosis (p < 0.05), and this became even more obvious after 48 h (p < 0.01). OxLDL also significantly increased the protein expression of all differentiation markers (p < 0.05), as confirmed through Western blotting and RT-PCR, while atorvastatin significantly reduced the effects of oxLDL (p < 0.05). CONCLUSION: Among the mechanisms of the cellular and molecular components and processes, oxLDL increased the valve calcification-related signaling pathway by increasing extracellular bone-matrix protein that produces osteoblastic gene markers via the Wnt/GSK-3ß/ß-catenin pathway. And atorvastatin also prevented any oxLDL-induced effects through the same pathway, this may represent a new therapeutic target for CAVD, as an alternative to traditional valve replacement surgery.

Structure-based grafting, mutation, and optimization of peptide inhibitors to fit in the active pocket of human secreted phospholipase A2: find new use of old Peptide agents with anti-inflammatory activity.

Phospholipase A2 (PLA2) is a key enzyme in the production of diverse mediators of inflammatory conditions, which possesses an open active pocket that is physicochemically compatible with a variety of small-molecule substrates and peptide inhibitors. Although various peptides and peptide analogues have been identified to have inhibitory activity against PLA2 originated from animals and plants, only very few were designed for human secreted PLA2 (hsPLA2), an attractive target of inflammatory arthritis. Considering that the catalytic domains of PLA2 family members across different species are highly conserved in primary sequence, advanced structure, and biological function, in this study, we proposed a synthetic pipeline to implement structure-based grafting, mutation, and optimization of peptide ligands from the snake PLA2-peptide complex crystal structures into the active pocket of apo hsPLA2 structure to computationally generate a large number of potential peptide inhibitors for hsPLA2, and the hsPLA2 inhibitory potency of few highly promising candidates arising from the theoretical analysis was determined. As might be expected, three peptides FLSFK, FLVYK, and FISYR showed relatively high inhibitory capability against hsPLA2, and other three ALSYK, LVFYA, and KGAILGFM were also modestly potent as they can suppress the enzymatic activity with observable doses. Further, the designed peptide FLVYK with highest potency was carried out with structure-guided modification based on its atomic interactions with hsPLA2 using the computationally modeled structure data, consequently resulting in a dual-point mutant ELIYK with significantly increased activity.

Effects of tanshinone IIA on the transforming growth factor ß1/Smad signaling pathway in rat cardiac fibroblasts.

OBJECTIVES: This study explores the mechanism of tanshinone IIA (TSN)-mediated inhibition of myocardial fibrosis by investigating the effect of TSN on transforming growth factor ß1 (TGFß1) signal transduction in rat cardiac fibroblasts (CFs). MATERIALS AND METHODS: CFs were isolated from neonatal Sprague-Dawley rats by trypsin digestion and differential adhesion and stimulated with 5 ng/mL TGFß1 and TSN (10(-6), 10(-5), or 10(-4) mol/L). The expression of fibronectin (FN) mRNA in the CFs was determined using reverse transcriptase-polymerase chain reaction and the protein expression of FN and Smads in CFs was detected using Western blot. The intracellular expression and localization of Smads in the CFs were analyzed using immunocytochemistry. RESULTS: TGFß1 induced the expression of FN and Smads in a time-dependent manner. At the end of the culture treatment, the mRNA expression of FN and the expression of phosphorylated Smad2/3 (p-Smad2/3) increased significantly (P < 0.01). TSN pretreatment (10(-5) and 10(-4) mol/L) reduced the expression of FN and p-Smad2/3 (P < 0.01) following TGFß1 stimulation and led to a significant decrease in the nuclear staining intensity and a positive rate of p-Smad2/3 (P < 0.05 and P < 0.01, respectively). CONCLUSION: The inhibitory effect of TSN on myocardial fibrosis may be associated with its inhibition of TGFß1-induced Smad2/3 phosphorylation and p-Smad2/3 nuclear translocation, which blocks the TGFß1/Smad signaling pathway in CFs.

Upregulation of sestrin-2 expression via P53 protects against 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity.

Sestrin-2 (SESN2) is a conserved antioxidant protein that is activated upon oxidative stress and protects cells against reactive oxygen species (ROS). However, the role of SESN2 in neurodegenerative diseases, especially in Parkinson's disease (PD), has not yet been elucidated. In this study, we found that expression of SESN2 is elevated in the midbrain of patients with PD. Moreover, in vitro experiments display that the drug 1-methyl-4-phenylpyridinium (MPP+) induces the expression of SESN2 in SH-SY5Y cells in a time- and dose-dependent manner. Our results show that p53 is activated by MPP+. Importantly, inhibition of p53 using small RNA interferences abolishes the increased SESN2 levels induced by MPP+, suggesting that the inductive effect of MPP+ on SESN2 is mediated by p53. Furthermore, knockdown of SESN2 using small RNA interferences promotes MPP+-related neurotoxicity by attenuating oxidative stress, mitochondrial dysfunction, and apoptosis. All these data imply that the induction of SESN2 produces a protective effect in PD.

Inhibitory effect of tanshinone II A on TGF II-ß1-induced cardiac fibrosis.

This study examined the effect of tanshinone II A (TSN II A) on the cardiac fibrosis induced by transforming growth factor ß1 (TGF-ß1) and the possible mechanisms. Cardiac fibroblasts were isolated from cardiac tissues of neonatal Sprague-Dawley (SD) rats by the trypsin digestion and differential adhesion method. The cells were treated with 5 ng/mL TGF-ß1 alone or pretreated with TSN II A at different concentrations (10(-5) mol/L, 10(-4) mol/L). Immunocytochemistry was used for cell identification, RT-PCR for detection of the mRNA expression of connective tissue growth factor (CTGF) and collagen type I (COL I), Western blotting for detection of the protein expression of Smad7 and Smad3, and immunohistochemistry and immunofluorescence staining for detection of the protein expression of phosphorylated Smad3 (p-Smad3), CTGF and COLI. The results showed that TGF-ß1 induced the expression of CTGF, COL I, p-Smad3 and Smad7 in a time-dependent manner. The mRNA expression of CTGF and COL I was significantly increased 24 h after TGF-ß1 stimulation (P<0.01 for all). The protein expression of p-Smad3 and Smad7 reached a peak 1 h after TGF-ß1 stimulation, much higher than the baseline level (P<0.01 for all). Pretreatment with high concentration of TSN A resulted in a decrease in the expression of p-Smad3, CTGF and COL I (P<0.01). The protein expression of Smad7 was substantially upregulated after pretreatment with two concentrations of TSN II A as compared with that at 2 h post TGF-ß1 stimulation (P<0.05 for low concentration of TSN I IA; P<0.01 for high concentration of TSN II A). It was concluded that TSN II A may exert an inhibitory effect on cardiac fibrosis by upregulating the expression of Smad7, suppressing the TGF-ß1-induced phosphorylation of Smad3 and partially blocking the TGF-ß1-Smads signaling pathway.

Intravenous lipid emulsions combine extracorporeal blood purification: a novel therapeutic strategy for severe organophosphate poisoning.

Organophosphorus (OP) pesticide self-poisoning is a major clinical problem in rural Asia and it results in the death of 200,000 people every year. At present, it is lack of effective methods to treat severe organophosphate poisoning. The high mortality rate lies on the amount of toxic absorption. Intravenous lipid emulsions can be used as an antidote in fat-soluble drug poisoning. The detoxification mechanism of intravenous lipid emulsions is "lipid sink", which lipid emulsions can dissolve the fat-soluble drugs and separate poison away from the sites of toxicity. Most of organophosphorus pesticides are highly fat-soluble. So, intravenous lipid emulsions have the potentially clinical applications in treatment of OP poisoning. Extracorporeal blood purification especially charcoal hemoperfusion is an efficient way to eliminate the poison contents from the blood. We hypothesize that the combination of intravenous lipid emulsions and charcoal hemoperfusion can be used to cure severe organophosphate poisoning. This novel protocol of therapy comprises two steps: one is obtained intravenous access to infuse lipid emulsions as soon as possible; another is that charcoal hemoperfusion will be used to clear the OP substances before the distribution of OP compounds in tissue is not complete. The advantages of this strategy lie in three points. Firstly, it will alleviate the toxic effect of OP pesticide in the patients by isolation and removal the toxic contents. Secondly, the dosage of antidotes can be reduced and its side-effects will be eased. Thirdly, a large bolus of fatty acids provide energy substrate for the patients who are nil by mouth. We consider that it would become a feasible, safe and efficient detoxification intervention in the alleviation of severe organophosphate poisoning, which would also improve the outcome of the patients.

Changes of c-fos and c-jun mRNA expression in angiotensin II-induced cardiomyocyte hypertrophy and effects of sodium tanshinone IIA sulfonate.

The changes of proto-oncogene c-fos and c-jun mRNA expression in angiotensin II (Ang II)-induced hypertrophy and effects of sodium tanshinone IIA sulfonate (STS) in the primary culture of neonatal rat cardiomyocytes were investigated. Twelve neonatal clean grade Wistar rats were selected. The cardiomyocytes were isolated, cultured and divided according to different treatments in the medium. The cardiomyocyte size was determined by phase contrast microscope, and the rate of protein synthesis was measured by [3H]-Leucine incorporation. The c-fos and c-jun mRNA expression in cardiomyocytes was detected by reverse transcription polymerase chain reaction (RT-PCR). It was found after cardiomyocytes were treated with Ang II for 30 min, the c-fos and c-jun mRNA expression in cardiomyocytes was increased significantly (P<0.01). After treatment with Ang II for 24 h, the rate of protein synthesis in Ang II group was significantly increased as compared with control group (P<0.01). After treatment with Ang II for 7 days, the size of cardiomyocytes in Ang II group was increased obviously as compared with control group (P<0.05). After pretreatment with STS or Valsartan before Ang II treatment, both of them could inhibit the above effects of Ang II (P<0.05 or P<0.01). It was suggested that STS could ameliorate Ang II-induced cardiomyocyte hypertrophy by inhibiting c-fos and c-jun mRNA expression and reducing protein synthesis rate of cardiomyocytes.

Effects of tanshinone IIA on transforming growth factor beta1-Smads signal pathway in renal interstitial fibroblasts of rats.

The effects of tanshinone IIA (TSN) on transforming growth factor beta1 (TGFbeta1) signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFbeta1 and pretreated with 10(-6), 10(-5), 10(-4) mol/L TSN respectively. The mRNA levels of fibronectin (FN) were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFbeta1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively (P<0.01, P<0.01), and the protein expression of phosphorylated Smad2/3 (p-Smad2/3) increased by 7 times at the end of TGFbeta1 stimulation (P<0.01). TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10(-6) mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression (both P>0.05). After pretreatment with 10(-5) and 10(-4) mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively (P<0.05, P<0.01), the FN protein levels were decreased by 40% and 44% respectively (P<0.05, P<0.05), and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively (P<0.05, P<0.01). The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFbeta1-Smads signal pathway in renal interstitial fibroblasts.

[Changes of c-fos, c-jun mRNA expressions in cardiomyocyte hypertrophy induced by angiotensin II and effects of tanshinone II A].

OBJECTIVE: To investigate the changes of proto-oncogene c-fos, c-jun mRNA expression in angiotensin II (Ang II)-induced hypertrophy and effects of tanshinone II A (Tan) in the primary culture of neonatal rat cardiomyocytes. METHOD: Twelve neonatal Wistar rats aged one day old of clean grade and both sexes were selected to isolate and culture cardiomyocytes. The cardiomyocytes were divided into: normal control group, Ang II (10(-6) mol x L(-1)) group, Ang II (10(-6) mol x L(-1)) +Tan (10(-8) g x L(-1)) group, Ang II (10(-6) mol x L(-1)) + valsartan (10(-6) mol x L(-1)) group, Tan (10(-8) g x L(-1)) group, valsartan (10(-6) mol x L(-1)) group. The cardiomyocyte size was determined by phase contrast microscope, the rate of protein synthesis in cardiomyocytes was measured by 3H-leucine incorporation. The c-fos, c-jun mRNA expression of cardiomyocytes were assessed using reverse transcription polymerase chain reaction (RT-PCR). RESULT: Ang II was added to the culture medium and 30 min later, the c-fos, c-jun mRNA expression of cardiomyocytes increased significantly (P < 0. 01). After Ang II took effect for 24 h, the rate of protein synthesis in Ang II group increased more prominently than that in normal control group (P < 0.01). After Ang II took effect for 7 days, the size of cardiomyocyte in Ang II group increased obviously (P < 0. 05). If tanshinone II or valsartan was added to the culture medium before Ang II, both of them could inhibit the increase of c-fos, c-jun mRNA expression (P < 0.01), cardiomyocyte protein synthesis rate (P < 0.01), and cardiomyocyte size (P < 0.05) induced by Ang II. CONCLUSION: Tanshinone II could ameliorate Ang II-induced cardiomyocytes hypertrophy by inhabiting c-fos, c-jun mRNA expression.