Sirtuin 3 improves fatty acid metabolism in response to high nonesterified fatty acids in calf hepatocytes by modulating gene expression.

Sirtuin 3 (SIRT3), a mitochondrial deacetylase, is a key regulator of energy metabolism in the liver. In nonruminants, the hepatic abundance of SIRT3 is decreased in individuals with nonalcoholic fatty liver diseases, and recovery of SIRT3 alleviates hepatic triacylglycerol (TG) deposition. However, the level of SIRT3 expression and its effects on lipid metabolism in dairy cows have not been characterized. Here we studied the hepatic expression of SIRT3 in cows with fatty liver and the role of SIRT3 in fatty acid metabolism in bovine hepatocytes. This in vivo study involved 10 healthy cows and 10 cows with fatty liver, from which we collected samples of liver tissue and blood. Primary hepatocytes were isolated from Holstein calves and treated with 0, 0.5, or 1.0 mM nonesterified fatty acids (NEFA) for 24 h or transinfected with SIRT3 overexpression adenovirus (Ad-SIRT3)/SIRT3-short interfering (si)RNA for 48 h. Cows with fatty liver displayed lower serum glucose concentrations but higher serum NEFA and ß-hydroxybutyrate concentrations relative to healthy cows. Cows with fatty liver also had significant lower mRNA and protein abundance of hepatic SIRT3. Incubation of primary hepatocytes with NEFA reduced SIRT3 abundance in primary hepatocytes in a dose-dependent manner. Fatty acid (1 mM) treatment also markedly increased the abundance of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) but significantly decreased the abundance of carnitine palmitoyltransferase I (CPT1A), carnitine palmitoyltransferase II (CPT2), and acyl-CoA oxidase (ACO). Knockdown of SIRT3 by SIRT3-siRNA downregulated the mRNA abundance of CPT1A, CPT2, and ACO. In contrast, overexpression of SIRT3 by Ad-SIRT3 upregulated the mRNA abundance of CPT1A, CPT2, and ACO; downregulated the mRNA abundance of ACC1 and FAS; and consequently, decreased intracellular TG concentrations. Overexpression of SIRT3 ameliorated exogenous NEFA-induced TG accumulation by downregulating the abundance of ACC1 and FAS and upregulating the abundance of CPT1A, CPT2, and ACO in calf hepatocytes. Our data demonstrated that cows with fatty liver had lower hepatic SIRT3 contents, and an increase in SIRT3 abundance by overexpression mitigated TG deposition by modulating the expression of lipid metabolism genes in bovine hepatocytes. These data suggest a possible role of SIRT3 as a therapeutic target for fatty liver disease prevention in periparturient dairy cattle.

Association between serum vitamin D levels and benign paroxysmal positional vertigo: a systematic review and meta-analysis of observational studies.

OBJECTIVE: Benign paroxysmal positional vertigo (BPPV) was the most common neuro-otological disorder manifests as recurrent positional vertigo, but its risk factors are elusive. Recent studies suggest that decreased Vitamin D level may be a risk factor, but the literature is inconsistent. METHODS: The databases PubMed, Web of Science, Chinese National Knowledge Infrastructure, Wanfang, SinoMed, and Embase were systematically searched for studies on the association between BPPV and  serum Vitamin D levels published up to June 2019. Data from eligible studies were meta-analyzed using Stata 12.0. RESULTS: A total of 18 studies were included in the analysis. Serum Vitamin D levels were significantly lower in individuals with BPPV than in controls (WMD - 2.46, 95% CI - 3.79 to - 1.12, p < 0.001). Subgroup analysis by geographical area showed that vitamin D level was significantly lower in BPPV than in controls in China (WMD - 3.27, 95% CI - 4.12 to - 2.43, p < 0.001), but not outside China (WMD - 0.90, 95% CI - 4.36 to 2.56, p = 0.611). Vitamin D levels were significantly lower in recurrent than non-recurrent BPPV across all countries in the sample (WMD 2.59, 95% CI 0.35-4.82, p = 0.023). Vitamin D deficiency emerged as an independent risk factor of BPPV (OR 1.998, 95% CI 1.400-2.851, p < 0.001). CONCLUSION: The available evidence suggests that BPPV is associated with decreased levels of serum Vitamin D, and vitamin D deficiency was an independent risk factor for BPPV.

Hepatic nuclear factor kappa B signaling pathway and NLR family pyrin domain containing 3 inflammasome is over-activated in ketotic dairy cows.

Ketosis is an important metabolic disease that can negatively affect the production efficiency of dairy cows. Earlier studies have revealed metabolic and inflammatory alterations in the blood associated with ketosis; however, a link between ketosis and hepatic inflammation has not been well documented. The objective of this study was to investigate whether the nuclear factor kappa B (NF-κB) signaling pathway and NLR family pyrin domain containing 3 (NLRP3) inflammasome were activated in the liver of ketotic cows. Liver and blood samples were collected from healthy (n = 15, control group) and ketotic (n = 15, ketosis group) cows that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Results showed that serum levels of fatty acids, ß-hydroxybutyrate (BHB), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were higher and glucose was lower in ketotic cows. Concentrations of serum proinflammatory cytokines IL18, tumor necrosis factor (TNF)-α, and IL1B were greater and the anti-inflammatory cytokine IL10 was lower in the ketosis group. Cows with ketosis had triacylglycerol accumulation in the liver. Upregulation of phosphorylated (p)-NF-κB and p-inhibitor of κB (IκB)α protein abundance in cows with ketosis indicated that the hepatic NF-κB signaling pathway was overactivated. The mRNA abundance of TNFA, inducible nitric oxide synthase (NOS2), IL18, and IL1B were greater and IL10 was lower in ketotic cows. More importantly, the mRNA and protein abundance of NLRP3 and caspase-1 (CASP1) along with CASP1 activity were greater in the liver of cows with ketosis. Overall, the data indicate that the onset of ketosis is accompanied by activation of the NF-κB signaling pathway and NLRP3 inflammasome, resulting in a state of inflammation.

[Spectrum-effect relationships on enriching blood activities of aerial parts of Angelica sinenis].

Ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to establish the chromatography fingerprint for aerial parts of Angelica sinenis(AAS) from 10 different places. Acetyl-phenyl-hydrazine(APH) was used to duplicate the mouse model of blood deficiency. Then partial least squares regression was used to investigate the spectrum-effect relationship between the relative contents and the data of enriching blood pharmacodynamics efficacy. The results showed that the three groups of high, medium and low doses of AAS had certain enriching blood activities(P<0.05), and the high dose group had the best effect(P<0.01). The contribution degree of the AAS to enriching blood activities of each common peaks were determined by PLS regression coefficient. Among them, 7 common peaks, including P17(unknown), P18(unknown), P19(unknown), P28(alisol B 23-acetate or its isomer), N5(luteolin), N11(1-caffeoylquinicacid,1-O-caffeoylquinic acid) and N14(unknown), contributed significantly to the effect of enriching blood activities. This paper dealed with the investigation on the spectrum-effect relationship between enriching blood activities and LC-MS chromatography fingerprint of AAS, and determination of the effective compositions of AAS with enriching blood activities. It provided theoretical foundation for the comprehensive development and utilization of AAS.

[Spectrum-effect relationships on enriching blood activities of fresh and dry roots of Angelica sinensis].

Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(LC-MS) was used to establish the chromatography fingerprint for fresh(FRAS) and dry(RAS) roots of Angelica sinensis from 10 different places. The rat model of blood deficiency was established by acetyl-phenyl-hydrazine(APH) and cyclophosphamide(CTX). Then grey relational analysis(GRA) and partial least squares regression(PLS) were used to investigate the spectrum-effect relationship between the relative contents and the data of enriching blood pharmacodynamics efficacy. The results showed that the FRAS and RAS had certain enriching blood activities(P<0.05). The contribution degree of the FRAS and RAS to enriching blood activities of each common peaks were determined by regression coefficient. Among them, 4 common peaks contributed significantly to the effect of enriching blood activities, P1(unknown), P2(unknown), P7(ferulic acid), and P11(senkyunolide A) respectively. This paper investigated the spectrum-effect relationship between enriching blood activities and LC-MS chromatography fingerprint of RAS and FRAS, and determined the effective compositions of RAS and FRAS with enriching blood activities. It lays a theoretical foundation for the comprehensive development and utilization of A. sinensis.

Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution.

Somatic L1 retrotransposition events have been shown to occur in epithelial cancers. Here, we attempted to determine how early somatic L1 insertions occurred during the development of gastrointestinal (GI) cancers. Using L1-targeted resequencing (L1-seq), we studied different stages of four colorectal cancers arising from colonic polyps, seven pancreatic carcinomas, as well as seven gastric cancers. Surprisingly, we found somatic L1 insertions not only in all cancer types and metastases but also in colonic adenomas, well-known cancer precursors. Some insertions were also present in low quantities in normal GI tissues, occasionally caught in the act of being clonally fixed in the adjacent tumors. Insertions in adenomas and cancers numbered in the hundreds, and many were present in multiple tumor sections, implying clonal distribution. Our results demonstrate that extensive somatic insertional mutagenesis occurs very early during the development of GI tumors, probably before dysplastic growth.

Adaptations of hepatic lipid metabolism and mitochondria in dairy cows with mild fatty liver.

The inevitable deficiency in nutrients and energy at the onset of lactation requires an optimal adaptation of the hepatic metabolism to overcome metabolic stress. Fatty liver is one of the main health disorders after parturition. Therefore, to investigate changes in hepatic lipid metabolic status and mitochondria in dairy cows with mild fatty liver, liver and blood samples were collected from healthy cows (n = 15) and cows with mild fatty liver (n = 15). To determine the effects of palmitic acids (PA), one of the major component of fatty acids, on lipid metabolism and mitochondria in vitro, calf hepatocytes were isolated from healthy calves and treated with various concentrations of PA (0, 50, 100, and 200 µM). Dairy cows with mild fatty liver displayed hepatic lipid accumulation. The protein levels of sterol regulatory element-binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor-α (PPARα) and mRNA levels of acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), acyl-CoA oxidase (ACO), and carnitine palmitoyltransferase 1A (CPT1A) were significantly higher in dairy cows with mild fatty liver than in control cows. The hepatic mitochondrial DNA content, mRNA levels of oxidative phosphorylation complexes I to V (CO 1-V), protein levels of cytochrome c oxidase subunit IV (COX IV), voltage dependent anion channel 1 (VDAC1), peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) and nuclear respiratory factor 1 (NRF1), and adenosine triphosphate (ATP) content were all markedly increased in the liver of dairy cows with mild fatty liver compared with healthy cows. The PA treatment significantly increased lipid accumulation; protein levels of SREBP-1c and PPARα; and mRNA levels of ACC1, FAS, ACO, and CPT1A in calf hepatocytes. Moreover, the mitochondrial DNA content, mRNA levels of CO 1-V, protein levels of COX IV, VDAC1, PGC-1α, NRF1, mitochondrial transcription factor A, and ATP content were significantly increased in PA-treated hepatocytes compared with control hepatocytes. The protein level of mitofusin-2 was significantly decreased in PA-treated groups. In conclusion, lipid synthesis and oxidation, number of mitochondria, and ATP production were increased in the liver of dairy cows with mild fatty liver and PA-treated calf hepatocytes. These changes in hepatic mitochondria and lipid metabolism may be the adaptive mechanism of dairy cows with mild fatty liver.

Impaired hepatic autophagic activity in dairy cows with severe fatty liver is associated with inflammation and reduced liver function.

The ability of liver to respond to changes in nutrient availability is essential for the maintenance of metabolic homeostasis. Autophagy encompasses mechanisms of cell survival, including capturing, degrading, and recycling of intracellular proteins and organelles in lysosomes. During negative nutrient status, autophagy provides substrates to sustain cellular metabolism and hence, tissue function. Severe negative energy balance in dairy cows is associated with fatty liver. The aim of this study was to investigate the hepatic autophagy status in dairy cows with severe fatty liver and to determine associations with biomarkers of liver function and inflammation. Liver and blood samples were collected from multiparous cows diagnosed as clinically healthy (n = 15) or with severe fatty liver (n = 15) at 3 to 9 d in milk. Liver tissue was biopsied by needle puncture, and serum samples were collected on 3 consecutive days via jugular venipuncture. Concentrations of free fatty acids and ß-hydroxybutyrate were greater in cows with severe fatty liver. Milk production, dry matter intake, and concentration of glucose were all lower in cows with severe fatty liver. Activities of serum aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and γ-glutamyl transferase were all greater in cows with severe fatty liver. Serum concentrations of haptoglobin and serum amyloid A were also markedly greater in cows with severe fatty liver. The mRNA expression of autophagosome formation-related gene ULK1 was lower in the liver of dairy cows with severe fatty liver. However, the expression of other autophagosome formation-related genes, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 3, ATG5, and ATG12, did not differ. More important, ubiquitinated proteins, protein expression of sequestosome-1 (SQSTM1, also called p62), and microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3)-II was greater in cows with severe fatty liver. Transmission electron microscopy revealed an increased number of autophagosomes in the liver of dairy cows with severe fatty liver. Taken together, these results indicate that excessive lipid infiltration of the liver impairs autophagic activity that may lead to cellular damage and inflammation.

Adherence reporting in clinical trials of type 2 Diabetes Metellius in the field of Traditional Chinese Medicine.

OBJECTIVE: To assess the medication adherence reporting in clinical trials the field of Traditional Chinese Medicine (TCM) and the impact factors of medication adherence. METHODS: Reviewed and evaluated were all randomized clinical trials in the field of TCM in treatment of type 2 diabetes mellitus published in Chinese journals in 2012, in terms of their medication adherence, adherence measurement, and impacted factors of adherence. RESULTS: Finally 124 studies were included. None studies reported the medication adherence. The factors impacting medication adherence couldn'tbe analyzed due to none reporting adherence. CONCLUSION: Medication adherence reporting was poor in clinical trials in TCM research. Establishing standards for adherence assessment and reporting may be one of the important steps to improve the quality of clinical studies.

Endothelial expression of hypoxia-inducible factor 1 protects the murine heart and aorta from pressure overload by suppression of TGF-ß signaling.

Chronic systemic hypertension causes cardiac pressure overload leading to increased myocardial O(2) consumption. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of O(2) homeostasis. Mouse embryos lacking expression of the O(2)-regulated HIF-1α subunit die at midgestation with severe cardiac malformations and vascular regression. Here we report that Hif1a(f/f);Tie2-Cre conditional knockout mice, which lack HIF-1α expression only in Tie2(+) lineage cells, develop normally, but when subjected to pressure overload induced by transaortic constriction (TAC), they manifest rapid cardiac decompensation, which is accompanied by excess cardiac fibrosis and myocardial hypertrophy, decreased myocardial capillary density, increased myocardial hypoxia and apoptosis, and increased TGF-ß signaling through both canonical and noncanonical pathways that activate SMAD2/3 and ERK1/2, respectively, within endothelial cells of cardiac blood vessels. TAC also induces dilatation of the proximal aorta through enhanced TGF-ß signaling in Hif1a(f/f);Tie2-Cre mice. Inhibition of TGF-ß signaling by treatment with neutralizing antibody or pharmacologic inhibition of MEK-ERK signaling prevented TAC-induced contractile dysfunction and pathological remodeling. Thus, HIF-1 plays a critical protective role in the adaptation of the heart and aorta to pressure overload by negatively regulating TGF-ß signaling in endothelial cells. Treatment of wild-type mice with digoxin, which inhibits HIF-1α synthesis, resulted in rapid cardiac failure after TAC. Although digoxin has been used for decades as an inotropic agent to treat heart failure, it does not improve survival, suggesting that the countertherapeutic effects of digoxin observed in the TAC mouse model may have clinical relevance.

Brazilin-Ce nanoparticles attenuate inflammation by de/anti-phosphorylation of IKKß.

Inflammation is associated with a series of diseases like cancer, cardiovascular disease and infection, and phosphorylation/dephosphorylation modification of proteins are important in inflammation regulation. Here we designed and synthesized a novel Brazilin-Ce nanoparticle (BX-Ce NPs) using Brazilin, which has been used for anti-inflammation in cardiovascular diseases but with narrow therapeutic window, and Cerium (IV), a lanthanide which has the general activity in catalyzing the hydrolysis of phosphoester bonds, to conferring de/anti-phosphorylation of IKKß. We found that BX-Ce NPs specifically bound to Asn225 and Lys428 of IKKß and inhibited its phosphorylation at Ser181, contributing to appreciably anti-inflammatory effect in cellulo (IC50 = 2.5 µM). In vivo mouse models of myocardial infarction and sepsis also showed that the BX-Ce NPs significantly ameliorated myocardial injury and improved survival in mice with experimental sepsis through downregulating phosphorylation of IKKß. These findings provided insights for developing metal nanoparticles for guided ion interfere therapy, particularly synergistically target de/anti-phosphorylation as promising therapeutic agents for inflammation and related diseases.

Peritoneal Amyloid as a Presenting Manifestation of AL Amyloid.

Amyloid is a systemic disease characterized by extracellular deposition of misfolded protein. Gastrointestinal and peritoneal deposition of light chain (AL) amyloid is an under-recognized manifestation of this systemic disease, usually as a late sequela. Here we present a case of recently diagnosed AL peritoneal amyloid that presented in the context of recurrent, acute onset abdominal discomfort and was found to have bowel obstruction complicated by perforation in the setting of AL-mediated gastrointestinal tract infiltration and dysmotility.

Hypoxia and hypoxia-inducible factor in the burn wound.

The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF's downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products vascular endothelial growth factor and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and vascular endothelial growth factor all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.

Effectiveness and Safety of Traditional Chinese Medicine in Treating COVID-19: Clinical Evidence from China.

When the outbreak of COVID-19 occurred in 2020, the Chinese government promptly undertook a series of preventive control and medical treatment measures that have effectively reduced the infection and mortality rates of COVID-19. In the process of preventing COVID-19 transmission and treating COVID-19, the Chinese government actively promotes the use of traditional Chinese medicine (TCM), and a series of studies have been carried out to determine the efficacy of TCM for COVID-19. Chinese physicians have accumulated rich experiences and created three Chinese patent medicines (i.e., Lianhua Qingwen Capsule, Jinhua Qinggan Granules, and Xuebijing Injection) and three herbal prescriptions (i.e., Xuanfeibaidu Recipe, Huashi Paidu Recipe, and Qingfei Paidu Decoction), as well as other strategies based on TCM theory to effectively treat COVID-19. Studies have reported that TCM treatment plays a significant role in improving the clinical symptoms, shortening the duration of hospitalization, reducing the overall mortality rate, obtaining favorable treatment outcomes in patients with severe COVID-19, preventing disease progression, improving quality of life and immunity, and reducing the positive rate of viral nucleic acid testing. TCM treatment has a fairly high degree of safety, but the level of evidence needs to be further improved.

Sirtuin 3 Restores Synthesis and Secretion of Very Low-Density Lipoproteins in Cow Hepatocytes Challenged with Nonesterified Fatty Acids In Vitro.

Fatty liver is closely associated with elevated concentrations of nonesterified fatty acids (NEFA) and a low level of very low-density lipoproteins (VLDL) in blood of dairy cows. High NEFA inhibit the VLDL synthesis and assembly, and cause hepatic triacylglycerol (TAG) deposition. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, antagonizes NEFA-induced TAG accumulation through modulating expressions of fatty acid synthesis and oxidation genes in cow hepatocytes. However, the role of SIRT3 in the VLDL synthesis and assembly was largely unknown. Here we aimed to test whether SIRT3 would recover the synthesis and assembly of VLDL in cow hepatocytes induced by high NEFA. Primary cow hepatocytes were isolated from 3 Holstein cows. Hepatocytes were infected with SIRT3 overexpression adenovirus (Ad-SIRT3), SIRT3-short interfering (si) RNA, or first infected with Ad-SIRT3 and then incubated with 1.0 mM NEFA (Ad-SIRT3 + NEFA). Expressions of key genes in VLDL synthesis and the VLDL contents in cell culture supernatants were measured. SIRT3 overexpression significantly increased the mRNA abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100 (ApoB100) and ApoE (p < 0.01), and raised VLDL contents in the supernatants (p < 0.01). However, SIRT3 silencing displayed a reverse effect in comparison to SIRT3 overexpression. Compared with NEFA treatment alone, the Ad-SIRT3 + NEFA significantly upregulated the mRNA abundance of MTP, ApoB100 and ApoE (p < 0.01), and increased VLDL contents in the supernatants (p < 0.01). Our data demonstrated that SIRT3 restored the synthesis and assembly of VLDL in cow hepatocytes challenged with NEFA, providing an in vitro basis for further investigations testing its feasibility against hepatic TAG accumulation in dairy cows during the perinatal period.

Hypoxia preconditioning protection of corneal stromal cells requires HIF1alpha but not VEGF.

PURPOSE: Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor). METHODS: Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1alpha and VEGF in transcriptional and translational levels. RESULTS: During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha. CONCLUSIONS: These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.

Hypoxia reduces TGFbeta1-induced corneal keratocyte myofibroblast transformation.

PURPOSE: The purpose of this study was to determine whether transient hypoxia had an effect on transforming growth factor beta1 (TGFbeta1)-induced rabbit corneal keratocyte myofibroblast transformation. METHODS: Primary isolated rabbit corneal keratocytes were cultured in a serum-free medium. The effect of transient hypoxia treatment (1% oxygen, 4 h/day) on TGFbeta1 (5 ng/ml)-induced alpha-smooth muscle actin (alpha-SM actin) expression was examined by immunofluorescence, flow cytometry, and immunocytochemistry 72 h after treatment. We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1. To explore the possible mechanism for this effect, we screened for the effects of hypoxia on several early TGFbeta-dependent signaling events including activated pSmad3, CREB (cAMP response element binding) binding protein (CBP), MAPKs (Mitogen-activated protein kinase), and RhoA by co-immunoprecipitation and western blotting. RESULTS: Hypoxia alone increased alpha-SM actin expression and the association of pSmad3 to CBP, but it did not induce the myofibroblast phenotype. The levels of pERK (the extracellular signal-regulated protein kinase) and pSmad3 or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by hypoxia whereas the activation of RhoA induced by TGFbeta1 was significantly reduced. CONCLUSIONS: We conclude that hypoxia can inhibit TGFbeta1-induced corneal myofibroblast transformation and alpha-SM actin expression. Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.

Increased risk of carotid atherosclerosis for long-term exposure to indoor coal-burning pollution in rural area, Hebei Province, China.

Smoky coal burning is a predominant manner for heating and cooking in most rural areas, China. Air pollution is associated with the risk of atherosclerosis, however, the link between indoor air pollution induced by smoky coal burning and atherosclerosis is not very clear. Therefore, we designed a cross-sectional study to evaluate the association of long-term exposure to smoky coal burning pollutants with the risk of atherosclerosis. 426 and 326 participants were recruited from Nangong, China and assigned as the coal exposure and control group according to their heating and cooking way, respectively. The indoor air quality (PM2.5, CO, SO2) was monitored. The association between coal burning exposure and the prevalence of atherosclerosis was evaluated by unconditional logistic regression analysis, adjusted for confounding factors. The inflammatory cytokines mRNAs (IL-8, SAA1, TNF-α, CRP) expression in whole blood were examined by qPCR. People in the coal exposure group had a higher risk of carotid atherosclerosis compared with the control (risk ratio [RR], 1.434; 95% confidence interval [95%CI], 1.063 to 1.934; P = 0.018). The association was stronger in smokers, drinkers and younger (<45 years old) individuals. The elevation of IL-8 (0.24, 95%CI, 0.06-0.58; P < 0.05), CRP (0.37, 95%CI, 0.05-0.70; P < 0.05), TNF-α (0.41, 95%CI, 0.14-0.67; P < 0.01) mRNAs expression in whole blood were positively related to coal exposure. Our results suggested long-term exposure to smoky coal burning emissions could increase the risk of carotid atherosclerosis. The potential mechanism might relate that coal burning emissions exposure induced inflammatory cytokines elevation which had adverse effects on atherosclerotic plaque, and then promoted the development of atherosclerosis.

Nobiletin alleviates palmitic acid­induced NLRP3 inflammasome activation in a sirtuin 1­dependent manner in AML­12 cells.

The NOD­like receptor family pyrin domain containing 3 (NLRP3) inflammasome has been reported to contribute to palmitic acid (PA)­induced lipotoxicity. Nobiletin (Nob) is a polymethoxylated flavonoid derived from citrus fruits that has been reported to exert antioxidant and antitumor effects. However, its protective and regulatory mechanisms in PA­induced lipotoxicity remain unclear. Therefore, the aim of the present study was to investigate the protective effects of Nob in AML­12 cells against lipotoxicity and examine the underlying mechanism. Western blotting, reverse transcription­quantitative polymerase chain reaction and ELISA assays were performed to investigate the activation of the NLRP3 inflammasome. Sirtuin 1 (SIRT1) small interfering RNA was used to knockdown SIRT1 expression in AML­12 cells. The results demonstrated that PA effectively activated NLRP3 inflammasome and increased the expression and secretion of interleukin (IL)­1ß and IL­18. Notably, the PA­induced inflammasome activation was reversed by Nob, as indicated by the decreased expression levels of NLRP3, Caspase­1, IL­1ß and IL­18. Furthermore, Nob treatment with or without PA enhanced the expression of SIRT1 in AML­12 cells, while knockdown of SIRT1 with SIRT1­small interfering RNA reversed the anti­inflammatory effects of Nob. Overall, the results of the present study indicated that Nob alleviated PA­induced lipotoxicity in AML­12 cells via the suppression of NLRP3 inflammasome activation in a SIRT1­dependent manner. These results provide a possible basis of the underlying mechanism and, in turn, the potential application of Nob in the treatment of non­alcoholic fatty liver disease.

Lack of association between the P413L variant of chromogranin B and ALS risk or age at onset: a meta-analysis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS), the most common motor neuron disease, is thought to result from interaction of genetic and environmental risk factors. Whether the potentially functional exonic P413L variant in the chromogranin B gene influences ALS risk and age at onset is controversial. METHOD: We meta-analysed or other studies assessing the association between the P413L variant and ALS risk or age at ALS onset indexed in Web of Science, PubMed, Embase, Chinese National Knowledge Infrastructure, Wanfang, and SinoMed databases. RESULTS: Five case-control studies were analysed, involving 2639 patients with sporadic ALS, 201 with familial ALS and 3381 controls. No association was detected between risk of either ALS type and the CT + TT genotype or T-allele of the P413L variant. Age at ALS onset was similar between carriers and non-carriers of the T-allele. CONCLUSION: The available evidence suggests that the P413L variant of chromogranin B is not associated with ALS risk or age at ALS onset. These results should be validated in large, well-designed studies.