Hippo pathway activation mediates chemotherapy-induced anti-cancer effect and cardiomyopathy through causing mitochondrial damage and dysfunction.

Rationale: Chemotherapy is a common clinical strategy for cancer treatment. However, the accompanied cardiomyopathy renders cancer patients under risk of another life-threatening condition. Whereas Hippo pathway is known to play key roles in both cancerogenesis and heart disease, it remains unclear whether Hippo pathway activation mediates chemotherapy-induced cardiomyopathy. Methods and Results: In human breast cancer cells, doxorubicin (DOX) significantly induced upregulation of Hippo kinase Mst1, inhibitory phosphorylation of YAP, mitochondrial damage, reduced cell viability and increased apoptosis. Hippo pathway inactivation by Mst1-siRNA transfection effectively improved cell survival and mitigated mitochondrial damage and cell apoptosis. Another anti-cancer drug YAP inhibitor verteporfin also induced lower cancer cell viability, apoptosis and mitochondrial injury. Chronic treatment with DOX in vivo (4 mg/kg/week for 6 weeks) caused mitochondrial damage and dysfunction, oxidative stress and cardiac fibrosis, while acute DOX treatment (16 mg/kg single bolus) also induced myocardial oxidative stress and mitochondrial abnormalities. Chronic treatment with verteporfin (2 months) resulted in cardiomyopathy phenotypes comparable to that by chronic DOX regimen. In transgenic mice with cardiac overexpression of kinase-dead mutant Mst1 gene, these adverse cardiac effects of DOX were significantly attenuated relative to wild-type littermates. Conclusions: Anti-cancer action of both DOX and verteporfin is associated with Hippo pathway activation. Such action on cardiac Hippo pathway mediates mitochondrial damage and cardiomyopathy.

Sappanone A: A natural PDE4 inhibitor with dual anti-inflammatory and antioxidant activities from the heartwood of Caesalpinia sappan L.

ETHNOPHARMACOLOGICAL RELEVANCE: Sumu (Lignum sappan), the dry heartwood of Caesalpinia sappan L., is a traditional Chinese medicine used as an analgesic and anti-inflammatory agent. AIM OF THE STUDY: The study aspired to discover natural phosphodiesterase 4 (PDE4) inhibitors with dual anti-inflammatory and antioxidant activities from Sumu for the treatment of chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: To accurately and efficiently identify natural PDE4 inhibitors from Sumu, molecular docking and molecular dynamics (MD) analysis methods were used for structure-based virtual screening of a self-built database of primary polyphenols in Sumu. According to the previous studies of Sumu and the free radical scavenging mechanism of polyphenols, the reported antioxidant components from Sumu and the potential antioxidants with the antioxidant pharmacophore of catechol and π-conjugated moieties were selected from the potential PDE4 inhibitors predicted by docking. Sappanone A, a potential PDE4 inhibitor with antioxidant activity from Sumu, was selected, calculated and synthesized to evaluate its dual anti-inflammatory and antioxidant functions in vitro and in vivo studies. Herein sappanone A was assayed for its inhibitory effects against PDE4 enzyme activity, tumor necrosis factor-alpha (TNF-α) production induced by lipopolysaccharide (LPS) in RAW264.7 macrophages and malondialdehyde (MDA) production induced by Fe2+ in mouse lung homogenate; sappanone A was also assayed for its abilities of radical (DPPH) scavenging, reducing Fe3+ and complexing Fe2+ in vitro. Additionally, LPS-induced acute lung injury (ALI) in mice was used to evaluate its anti-inflammatory activity as a PDE4 inhibitor in vivo, and the levels of TNF-α and total protein in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) activity in the lung were assayed. RESULTS: The present study predicted and validated that sappanone A was a promising PDE4 inhibitor from Sumu with dual anti-inflammation and antioxidant activities from Sumu. In vitro, sappanone A remarkably inhibited PDE4 enzyme activity and reduced TNF-α production induced by LPS in RAW264.7 macrophages and MDA production induced by Fe2+ in mouse lung homogenate. Meanwhile, it showed outstanding abilities of scavenging DPPH radicals, reducing Fe3+ and complexing Fe2+. In vivo, sappanone A (25 mg/kg and 50 mg/kg, i.p., twice daily for 7 days) distinctly prevented LPS-induced ALI in mice by reducing the levels of TNF-α and total protein in BALF and MPO activity in the lung. CONCLUSION: Sappanone A is a natural PDE4 inhibitor with dual anti-inflammatory and antioxidant activities from the traditional Chinese medicine Sumu, which may be a promising therapeutic agent to prevent the vicious cycle of COPD inflammation and oxidative stress.

Antibody prevalence and immunotherapy response in Chinese patients with epilepsy and encephalopathy scores for patients with different neuronal surface antibodies.

BACKGROUND: The scale assessment was helpful in predicting the presence of antibodies to autoimmune encephalitis. This study aimed to evaluate the application of antibody prevalence in Chinese patients with epilepsy and encephalopathy (APE2-CHN) and response to immunotherapy in Chinese patients with epilepsy and encephalopathy (RITE2-CHN) for patients with different neuronal surface antibodies. METHODS: A total of 1365 patients with epileptic seizures as the prominent feature in Xuanwu Hospital, Capital Medical University, from June 2016 to June 2020 were enrolled in our study. Of these, 915 patients with epilepsy of unknown etiology whose serum and/or cerebrospinal fluid samples were examined for autoimmune antibodies were selected. All patients were scored with antibody prevalence in patients with epilepsy and encephalopathy (APE2), response to immunotherapy with epilepsy and encephalopathy (RITE2), APE2-CHN, and RITE2-CHN scores. RESULTS: Of the 915 patients, 191 patients were positive for neural-surface specific antibodies (115 N-methyl-D-aspartate receptor (NMDAR) Ab, 47 leucine-rich glioma-inactivated protein 1 (LGI1) Ab, 8 contactin-associated protein 2 (CASPR2) Ab, 4 AMPA2R-Ab, and 11 GABAR-B-Ab; 3 CASPR2-Ab and LGI1-Ab, 2 NMDAR-Ab and CASPR2-Ab, and 1 NMDAR-Ab and myelin-oligodendrocyte glycoprotein [MOG] Ab). The sensitivity and specificity of APE2 ≥4 in predicting the presence of neural-surface specific antibodies in our study were 74.35% and 81.77%, respectively, and the sensitivity and specificity of APE2-CHN ≥4 were 75.92% and 84.53%, respectively. Eight cases had an APE2 score <4 and APE2-CHN score ≥5; all these patients had memory decline as the prominent manifestation. We divided the patients into six groups according to the different antibodies. APE2-CHN scores showed higher sensitivity for the prediction of NMDAR-Ab, but lower sensitivity for LGI1-Ab. A total of 187/191 (97.91%) patients received immunotherapy and 142/191 (74.35%) patients benefited from the treatments. The patients who were positive for LGI1-Ab with RITE2-CHN ≥8 responded well to immunotherapy. CONCLUSIONS: APE2-CHN had the highest value for predicting the positivity of NMDAR-Ab and RITE2-CHN evaluated the response of immunotherapy for anti-LGI1 encephalitis appropriately. However, RITE2 and RITE2-CHN do not appear to be good predictors of immunotherapy outcomes for patients with specific neuronal-surface antibodies and high APE2-CHN scores are often indicative of a poor response to immunotherapy.

Long-term efficacy and patient satisfaction of Le Fort colpocleisis for the treatment of severe pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: The objective was to investigate the long-term efficacy and patient satisfaction of Le Fort colpocleisis for the treatment of severe pelvic organ prolapse. METHODS: This was a retrospective study of patients who underwent Le Fort colpocleisis from January 2007 to August 2018 in our hospital. Follow-up was conducted via outpatient visits or the telephone. Records were reviewed for anatomical recurrence, complications, urinary and intestinal symptoms post-operation, reoperation rate, patient satisfaction, Patient Global Impression of Improvement (PGI-I) score, regret rate etc. RESULTS: A total of 208 patients underwent follow-up. The follow-up time was 60.7 ± 34.18 (12-140) months. There were no intraoperative complications. Postoperative urinary retention occurred in 3.8% of patients (8 out of 208). There was no anatomical recurrence. New or more severe urinary symptoms occurred in 8.7% of patients (18 out of 208); new or more severe intestinal symptoms occurred in 1.9% of patients (4 out of 208). The reoperation rate was 1.44% (3 out of 208). Three cases of reoperation occurred for the following reasons: a case of severe stress urinary incontinence, a case of abscess in the vaginal septum, and a case of uterine malignancy after 2 years of colpocleisis. Patient satisfaction was as follows: 98.6% (205 out of 208) of patients were very satisfied. The PGI-I score was very much improved or improved in 99.5% (207 out of 208) of patients. A total of 0.96% (2 out of 208) of patients regretted undergoing colpocleisis. CONCLUSIONS: The long-term follow-up results showed that Le Fort colpocleisis was a safe and effective surgical procedure associated with high satisfaction. There was a very low regret rate, but the procedure should be taken seriously.

Sexually Inactive Status in Women With Pelvic Organ Prolapse Before Colpocleisis and Postoperative Satisfaction and Regret Rate.

PURPOSE: The aim of the study was to investigate the sexually inactive status of patients with pelvic organ prolapse before colpocleisis and postoperative satisfaction and regret rate. METHODS: A retrospective study of patients with pelvic organ prolapse who underwent colpocleisis was conducted in our hospital from January 2007 to April 2019. Records were reviewed before surgery for general clinical characteristics, duration, and reasons for being sexually inactive. Follow-up was conducted by telephone about patient satisfaction, Patient Global Impression of Improvement score, and regret rate after surgery. RESULTS: The mean age of the 247 patients was 73.8 ± 5.58 years. A total of 76.9% (190/247) described the duration of being sexually inactive, and the mean time was 12.6 ± 8.69 years. The 247 patients gave the following reasons for being sexually inactive: 52.2% (129/247) were widowed and 37.2% (92/247) reported the physical health factors of their spouses or sexual partners. The first male factor was nervous system disease (37.0%, 34/92). A total of 5.3% (13/247) were patient-related factors and 5.3% (13/247) were factors of both the male and female. A total of 195 patients underwent follow-up, the rate was 78.9% (195/247), and the follow-up time was 39.7 ± 37.5 (2-140) months. A total of 98.5% (192/195) of patients were very satisfied. A total of 98.9% (193/195) of patients were very much improved or improved in Patient Global Impression of Improvement score. A total of 1.02% (2/195) of patients regretted having colpocleisis nearly 2 years later. CONCLUSIONS: The main reason for being sexually inactive was having been widowed. Colpocleisis was associated with high satisfaction rates and low regret rate.

Proton Transfer Can Govern Regioselectivity Assisted by Iron Catalysis.

Ortho-selective aromatic C-H functionalization is frequently used in organic synthesis and chemical/pharmaceutical industries. However, this reaction relies heavily on the use of directing groups suffering from limited substrate scope and extra steps to put on and remove the directing/protecting groups. Herein we present the previously neglected concept that enables good to nearly complete selective ortho position. Proton transfer was utilized to tune the electron density on the aryl ring and determine the positional selectivity of electrophilic substitution. Consistently with deuteration experiments and DFT studies, this work demonstrates that acid-promoted proton transfer directs accelerated ortho-selective halogenation of NH/OH contained aromatic amines/phenols with excellent selectivity (>40 examples; up to 98:2 ortho/para selectivity). The application potential of this Fe-catalyzed method is demonstrated by the convenient synthesis of three alkaloids and tizanidine. This report raises the possibility that proton transfer could serve as the basis of developing new selective C-H functionalization reactions.

Total flavones of Dracocephalum moldavica L. protect astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway.

BACKGROUND: The active components of Dracocephalum moldavica L. (TFDM) can inhibit myocardial ischemia by inhibiting oxidative stress. However, the effects of TFDM on astrocytes have not been investigated in vitro. The current study aimed to explore whether TFDM protects astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway. METHODS: The human glioma cell line U87 was used to investigate the ability of TFDM to protect astrocytes against H2O2-induced apoptosis. The cell counting kit-8 assay and flow cytometry were used to detect cell viability, apoptosis, MMP, Ca2+ influx and reactive oxygen species (ROS). Lactate dehydrogenase (LDH) and malonic dialdehyde (MDA) levels were measured by ELISA. In addition, protein and mRNA expression changes were detected by Western blotting and qRT-PCR. RESULTS: TFDM (0.78~200 µg/ml) had limited cytotoxic effects on the viability of U87 cells. Compared with the model group (treated with H2O2 only), cells treated with medium- and high-dose TFDM exhibited reduced MDA concentrations (P < 0.05) and ROS production (P < 0.05) and decreased MMP (P < 0.05) and reduced apoptosis (P < 0.05). The percentage of annexin V-FITC-stained cells was markedly suppressed by TFDM, confirming its anti-apoptotic properties. WB results showed that protein expression of Bcl-2-associated X protein (BAX), Caspase-3, Caspase-9, Caspase-12, and B-cell leukemia/lymphoma 2 (Bcl2) was reduced in the TFDM group compared with that in the model group (P < 0.05) and that expression of these proteins was normalized by TFDM treatment in a dose-dependent manner. According to RT-qPCR results, TFDM pretreatment resulted in reduced mRNA expression of BAX, Caspase-9, Caspase-12, p38MAPK, and CaMKII and increased mRNA expression of mTOR compared with the model group. CONCLUSIONS: The current study revealed the protective effects of TFDM on U87 cells under oxidative stress conditions through the inhibition of a mitochondria-dependent pathway that is associated with the CaMKII/P38MAPK/ERK1/2 and PI3K/AKT/mTOR pathways.

[Improving Bioremediation of Crude Oil-contaminated Soil by Mild Pre-oxidation].

To obtain a mild Fenton pre-oxidation method, which could promote the efficient degradation of total petroleum hydrocarbons (TPH) in subsequent bioremediation, the differences in the characteristics of the hydroxyl radical (·OH), nutrient consumption, activity of indigenous bacteria (CO2), and TPH removal were investigated during subsequent bioremediation after different Fenton pre-oxidation treatments. The results showed that high biodegradation of TPH was observed after mild Fenton pre-oxidation (·OH existence time:73 h; H2O2 concentration:225 mmol·L-1), because of the high activity of residual bacteria. Moreover, the same TPH removal with the addition of bacteria could be achieved without adding bacteria (the TPH removal rate 38%) because the activity of the residual bacteria was strong after mild Fenton pre-oxidation. Under the condition of no additional bacteria source, mild Fenton pre-oxidation TPH removal (approximately 38%) was higher than that after ordinary Fenton pre-oxidation (15.32%-33.15%). Further analysis of the removal efficiency of each chain of hydrocarbons revealed that the mild pre-oxidation group could reduce the inhibition of the chain hydrocarbon components (C17-C21) in the subsequent bioremediation stage. Comparing the activity of the indigenous bacteria in each group, revealed that mild pre-oxidation could appropriately stimulate the growth and increase the activity of indigenous microorganisms, all of which are beneficial to the removal of TPH.

[Ecological risk assessment of landslide disasters based on potential loss of ecosystem services]. / 基于生态系统服务潜在损失的滑坡灾害生态风险评价.

Landslides are common geological calamities in mountainous regions, which not only threaten social and economic development and residents' safety but also cause ecosystem damage, with consequences on human welfare. A more comprehensive and systematic reference for disaster prevention and mitigation with ecosystem services loss as an index for the potential damage of ecosystem could aid the progress of landslide ecological risk assessment. Five provinces in Southwest China (Sichuan, Yunnan, Guizhou, Guangxi and Chongqing) have diverse landforms, complex stratum and lithology, and active geological tectonic movements, which are the most landslide prone areas in China. In this study, the ecological risk assessment framework, model and indicator were constructed from three dimensions, including disaster risk, vulnerability and potential loss of the ecosystem. Disaster risk was based on the comprehensive analysis of factors such as geology, topo-graphy, landform, precipitation, and their mutual relationship. The vulnerability of the ecosystem was characterized by landscape patterns indices. The potential loss was measured by ecosystem service to evaluate the ecological risk associated with landslide hazards in the five provinces of Southwest China. The results showed that the areas with high potential loss of ecosystem services were mainly distributed in the south of Ailao Mountain in Yunnan Province, Qionglai Mountain of Sichuan Pro-vince, Hengduan Mountains, Dadu River Basin, Northwest Guangxi Autonomous Region, and eastern area of Dayao Mountain. The high ecological risk of landslide hazard in the study area mainly distributed in the areas of Min Mountain, Qionglai Mountain, Wuliang Mountain, Ailao Mountain, Miao Ridge, Leigong Mountain, and Dadu River basin. With respect to altitude, 500-1500 m was the main high-risk areas, accounting for 37.9% of the high-risk area. In terms of ecosystem types, forests are the high-risk areas, accounting for 66.4% of the high-risk areas. Landslide monitoring and early warning in the high ecological risk areas should be strengthened, through strengthening ecosystem protection in the region and improving the stability and resistance of ecosystems.

[Relationship of Cystic Fibrosis Transmembrane Conductance Regulator Expression with Clinical Features and Prognosis in Patients with Acute Leukemia].

OBJECTIVE: To investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in patients with acute leukemia and its relationship to clinical features and prognosis of acute leukemia. METHODS: A total of115 patients with acute leukemia were enrolled in the experimental group and 20 healthy individuals were used as control. Peripheral blood or bone marrow samples were collected, and mononuclear cells were isolated. The expression of CFTR protein was detected by Western blot. The relationships of CFTR protein expression to clinical features and prognosis was analyzed. RESULTS: The expression of CFTR protein was not detected in peripheral blood mononuclear cells of normal control, while it was positive in more than half of acute leukemias including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), but negative in the patients with acute promyelocytic leukemia (M3). In the patients with AML, there was no difference in peripheral white blood cells (WBC), peripheral blast cells, platelet and hemoglobin (HGB) between CFTR-positive and CFTR-negative patients. There was no relationship between the expression of CFTR protein and gene mutations such as NPM1, CEBPA, FLT3-ITD, and C-Kit. Complete remission (CR) rate after two course in CFTR-negative patients was slightly higher than that in positive patients. The survival time of CFTR-negative patients was little longer than that of positive patients, but the difference was not statistically significant. CONCLUSIONS: The expression of CFTR protein seems not associated with clinical features, treatment response and prognosis in the patients with acute leukemia.

Contribution of acid sphingomyelinase to angiotensin II-induced vascular adventitial remodeling via membrane rafts/Nox2 signal pathway.

AIMS: Vascular adventitial fibroblasts (AFs) in the vascular remodeling during atherosclerosis are increasing arousing attention. Acid sphingomyelinase (ASM) is a soluble glycoprotein which is involved in the development and progression of atherosclerosis. However, it remains unknown if ASM is expressed in vascular AFs and regulates vascular adventitial remodeling and underlying mechanisms. MAIN METHODS AND KEY FINDINGS: ASM downregulation with gene silencing was used in the rat AFs treated with angiotensin (Ang) II, which is universally demonstrated to induce vascular adventitia remodeling. It was showed that ASM was indeed expressed in vascular AFs and ASM downregulation resulted in a significant decrease in the protein level of PCNA and collagen I and cell migration under Ang II stimulation. Such improvement of adventitial remodeling was not further augmented by Ang-(1-7), which is deemed as an endogenous Ang II blocker. We further found that ASM downregulation blocked the Nox2-dependent superoxide (O2-) generation, which regulated vascular remodeling in AFs under Ang II. ASM siRNA decreased the aggregation of membrane rafts (MRs) and the consequent recruiting of ceramide and Nox2 in MRs. SIGNIFICANCE: In conclusion, these results suggested that ASM downregulation could improve vascular adventitial remodeling which was attributed to inhibiting MRs/Nox2 redox signaling pathway in AFs. Thus, these data supported the idea that ASM is a potential therapeutic target for diabetic vascular complication.

Effects of cadmium stress on the antioxidant system and chlorophyll fluorescence characteristics of two Taxodium clones.

KEY MESSAGE: The T.118 and T.406 seedlings showed strong adaptability under Cd concentrations ≤ 50 µM. The mechanisms of photoprotection in T.118 and T.406 differed in high-Cd concentrations. To explore the physiological response characteristics of Taxodium hybrids to cadmium (Cd) stress and provide basis for screening of Cd-tolerant species, the hydroponic cultivation of T.118 and T.406 seedlings was conducted to demonstrate the effects of Cd stress on seedling growth, antioxidant system, and chlorophyll fluorescence parameters. After 35 days of Cd stress at a concentration ≤ 50 µM, the dry weight biomass of the two clones did not significantly differ from that of the control. T.406 exhibited a significant increase in POD activity compared to T.118 and maintained high SOD activity after exposure to high concentrations of Cd, whereas MDA levels showed little changes. Under low-Cd stress, chlorophyll content and fluorescence parameters remained stable, especially for T.406. Under high-Cd concentration stress, the above parameters were lower than the control, with a more significant decrease in T.118 than in T.406. The non-photochemical quenching coefficient (NPQ) of both clones increased with increasing Cd concentration. T.118 showed a greater increase than T.406, particularly under high-Cd concentration stress. The T.118 and T.406 seedlings adapted to low-Cd concentration stress by enhancing their antioxidant enzyme activity to maintain the balance of reactive oxygen metabolism and reduce cellular damage. The photochemical activity of mesophyll cells remained high to maintain photosynthetic capacity and normal seedling growth. T.406 showed stronger resistance to Cd than T.118. T.406 prevented photodamage by promoting the photochemical utilization of the excitation energy and maintaining a strong antioxidant stress ability. Enhancement of heat dissipation capability may be the main photoprotection mechanism of T.118.

Regioselectivity inversion tuned by iron(iii) salts in palladium-catalyzed carbonylations.

Impactful regioselectivity control is crucial for cost-effective chemical synthesis. By using cheap and abundant iron(iii) salts, the hydroxycarbonylations of both aromatic and aliphatic alkenes were significantly enhanced in both reactivity and selectivity (iso/n or n/iso up to >99 : 1). Moreover, Pd-catalyzed carbonylation selectivity can be switched from branched to linear by using different Fe(iii) salts. In addition, similar results were obtained for the carbonylation of secondary alcohols.

Crocetin inhibits the proliferation, migration and TGF-ß2-induced epithelial-mesenchymal transition of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells, the major cell type in the fibrotic membrane of proliferative vitreoretinopathy, display enhanced proliferative and migratory capacities and epithelial-mesenchymal transition (EMT). In this study, we investigated the potential impact of crocetin on the proliferation, migration and EMT of cultured ARPE-19 cells. The cells were treated with crocetin alone or in combination with transforming growth factor-ß2 (TGF-ß2). Cell proliferation was examined using the CCK-8 assay. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining. The expression levels of proliferating cell nuclear antigen (PCNA), p21 and p53 were examined by Western blot analysis. Cell migration was assessed by in vitro scratch and Transwell assays. Real-time PCR, Western blotting and immunofluorescence were used to assess EMT features. Treatment of ARPE-19 cells with crocetin (50-200µM) significantly inhibited their proliferation and migration in a concentration- and time-dependent manner. Crocetin induced G1 arrest, reduced PCNA protein expression and increased the p21 and p53 accumulation in ARPE-19 cells. Crocetin inhibited TGF-ß2-induced EMT in ARPE-19 cells by maintaining the expression of E-cadherin and ZO-1 and by reducing the expression of vimentin and α-SMA through the suppression of phosphorylation of p38. These results indicate that crocetin is an effective inhibitor of the proliferation, migration and TGF-ß2-mediated EMT of ARPE-19 cells.

Reactive oxygen species mediated NF-κB/p38 feedback loop implicated in proliferation inhibition of HFLS-RA cells induced by 1,7-dihydroxy-3,4-dimethoxyxanthone.

1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is a bioactive compound isolated from Securidaca inappendiculata Hassk. and exerts the inhibitory effects on fibroblast-like synoviocytes by targeting NF-κB and p38. This study was designed to elucidate mechanisms underlying the divergent regulation on the two pathways in HFLS-RA cells by XAN. Expressions of hallmark proteins and transcription of GADD45α mRNA were determined by Western-blot and RT-qPCR methods, respectively. Fluorescence staining was employed to evaluate intracellular oxidative stress. Effects of XAN and N-acetyl-l-cysteine (NAC) on the proliferation of cells were investigated by MTT assay, and pro-apoptotic effects of XAN were assessed by Annexin V-FITC/PI method. It was found XAN blocked NF-κB signaling in HFLS-RA cells shortly after treatment. Moreover, it up-regulated both transcription and expression of GADD45α, and subsequently activated p38 pathway. As time went on, XAN significantly promoted the generation of reactive oxygen species (ROS), which accompanied with sustained up-regulation of p-p38 and increased apoptosis. 48H later, dual-effects of XAN on NF-κB and p38 were reversed. As activation of p38 and increased apoptosis induced by XAN were antagonized by NAC, they were deemed as ROS mediated effects. Furthermore, the accumulated ROS should also account for the activation of NF-κB in the late stage of treatments via interfering in p38/MSK1/NF-κB feedback. Altogether, these findings suggested XAN-induced ROS contributed great importance to the proliferation inhibition of HFLS-RA cells by mediating NF-κB/p38 feedback loop and apoptosis, which provided us a panoramic view of potential target in the therapy of RA by XAN.

Modulation of binding to vascular endothelial growth factor and receptor by heparin derived oligosaccharide.

We investigated the mechanism of heparin-derived oligosaccharide on the proliferation of vascular smooth muscle cell (VSMC) induced by vascular endothelial growth factor (VEGF). Expression levels of VEGFR 1 and VEGFR 2 were examined by RT-PCR, and the corresponding protein expression levels were detected by Western blotting and immunocytochemistry. Western blotting was taken to identify the expression levels of mechanism proteins. The binding of VEGF and VEGFR 2 was measured by co-IP. Besides, HS competition assay was to detect the ability of HDO to compete with Heparin for VEGF165. HDO showed an inhibitory effect on the expression of VEGFR1/2 proteins and PKC, MAPK, PI3K/Akt pathways. In addition, HDO affected the binding of VEGF-VEGFR, which may be one of the most important mechanisms of HDO suppress the cell proliferation induced by growth factors. Thus HDO showed the ability as a VEGF antagonist.

Heparin-derived oligosaccharide inhibits vascular intimal hyperplasia in balloon-injured carotid artery.

The aims of the present study were to determine the effects of heparin-derived oligosaccharides (HDOs) on vascular intimal hyperplasia (IH) in balloon-injured carotid artery and to elucidate the underlying mechanisms of action. An animal model was established by rubbing the endothelia within the common carotid artery (CCA) in male rabbits. The rabbits were fed a high-cholesterol diet. Arterial IH was determined by histopathological changes to the CCA. Serum lipids were detected using an automated biochemical analysis. Expressions of mRNAs for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), scavenger receptor class B type I (SR-BI), and ATP-binding cassette transporter A1 (ABCA-1) were analyzed using reverse transcription polymerase chain reaction assays. Expressions of VEGF, VCAM-1, MCP-1, SR-BI and ABCA-1 proteins were analyzed by Western blotting. Enzyme-linked immunosorbent assays were used to quantify expression levels of VEGF and bFGF. Our results showed that administration of HDO significantly inhibited CCA histopathology and restenosis induced by balloon injury. The treatment with HDOs significantly decreased the mRNA and protein expression levels of VEGF, bFGF, VCAM-1, MCP-1, and SR-BI in the arterial wall; however, ABCA-1 expression level was elevated. HDO treatment led to a reduction in serum lipids (total cholesterol, triglycerides, high-density and low-density lipoproteins). Our results from the rabbit model indicated that HDOs could ameliorate IH and underlying mechanism might involve VEGF, bFGF, VCAM-1, MCP-1, SR-BI, and ABCA-1.

Multiple H+ sensors mediate the extracellular acidification-induced [Ca2+]i elevation in cultured rat ventricular cardiomyocytes.

Acidosis has been known to cause "Ca2+ transients", however, the mechanism is still uncertain. Here, we demonstrated that multiple H+ sensors, such as ASICs, TRPV1 and proton-sensing G protein coupled receptors (GPCRs) are involved in extracellular acidification-induced intracellular calcium ([Ca2+]i) elevation. By using calcium imaging measures, we observed that both ASIC and TRPV1 channels inhibitors suppressed the [Ca2+]i elevation induced by extracellular acidosis in cultured rat cardiac myocytes. Then, both channels mRNA and proteins were identified by RT-PCR, western blotting and immunofluorescence. ASIC-like and TRPV1-like currents were induced by extracellular acidification, suggesting that functional ASIC and TRPV1 channels jointly mediated extracellular calcium entry. Furthermore, either pre-exhaustion of sarcoplasmic reticulum (SR) Ca2+ with thapsigargin or IP3 receptor blocker 2-APB or PLC inhibitor U73122 significantly attenuated the elevation of [Ca2+]i, indicating that the intracellular Ca2+ stores and the PLC-IP3 signaling also contributed to the acidosis-induced elevation of [Ca2+]i. By using genetic and pharmacological approaches, we identified that ovarian cancer G protein-coupled receptor 1 (OGR1) might be another main component in acidosis-induced release of [Ca2+]i. These results suggest that multiple H+-sensitive receptors are involved in "Ca2+ transients" induced by acidosis in the heart.

A novel thymidine phosphorylase mutation in a Chinese MNGIE patient.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with mitochondrial alterations. MNGIE is characterized by severe gastrointestinal dysmotility, cachexia, ophthalmoplegia, ptosis, peripheral neuropathy, and leukoencephalopathy. The condition is caused by mutation of the TYMP gene. We studied the clinical and biochemical characteristics of a family with MNGIE. The proband was a 48-year-old male presenting with diarrhea and progressive weight loss. He also had ptosis and exhibited eyeball fixation. His blood and cerebrospinal fluid lactate levels were elevated. Magnetic resonance imaging of the brain revealed diffuse leukoencephalopathy. Ragged red fibers and cytochrome c oxidase-deficient fibers were apparent on muscle biopsy. His vision and ptosis deteriorated significantly during follow-up. Our clinical diagnosis of MNGIE was confirmed by TYMP gene analysis. We discovered a homozygous TYMP c.1193-1216 dup-GGGCGCTGCCGCTGGCGCTGGTGC mutation (a duplication). Some of the family members were heterozygous for the mutation but had no clinical features. We predicted the function of this mutation using PredictProtein and found that the secondary structure had changed in the region of the helix and strand, the transmembrane region, and the protein-protein binding sites. The family described herein exhibited biochemically, genetically, and functionally confirmed MNGIE syndrome.