Guanxinning tablets improve myocardial hypertrophy by inhibiting the activation of MEK-ERK1/2 signaling pathway.

Myocardial hypertrophy may lead to heart failure and sudden death. As traditional Chinese medicine, Guanxinning tablets (GXN) have significant pharmacological effects in the prevention and treatment of cardiovascular diseases. However, the anti-cardiac hypertrophy efficacy of GXN and its mechanism of action are still unclear. Therefore, we established a heart failure rat model and isolated primary cardiomyocytes of neonatal rat to observe the protective effect of GXN on heart failure rat model and the intervention effect on myocardial cell hypertrophy, and to explore the possible mechanism of GXN preventing and treating myocardial hypertrophy. The results of in vivo experiments showed that GXN could significantly reduce the degree of cardiac hypertrophy, reduce the size of cardiomyocytes, inhibit the degree of myocardial remodeling and fibrosis, and improve cardiac function in rats with early heart failure. The results of in vitro experiments showed that GXN was safe for primary cardiomyocytes and could improve cardiomyocyte hypertrophy and reduce the apoptosis of cardiomyocytes in pathological state, which may be related to the inhibition of the over-activation of MEK-ERK1/2 signaling pathway. In conclusion, GXN may inhibit cardiac hypertrophy and improve early heart failure by inhibiting the over-activation of MEK-ERK1/2 signaling pathway.

Bioinformatics analysis of vascular RNA-seq data revealed hub genes and pathways in a novel Tibetan minipig atherosclerosis model induced by a high fat/cholesterol diet.

BACKGROUND: Atherosclerosis is a major contributor to cardiovascular events, however, its molecular mechanism remains poorly known. Animal models of atherosclerosis can be a valuable tool to provide insights into the etiology, pathophysiology, and complications of atherosclerosis. In particular, Tibetan minipigs are a feasible model for studying diet-related metabolic and atherosclerotic diseases. METHODS: We used vascular transcriptomics to identify differentially expressed genes (DEGs) in high fat/cholesterol (HFC) diet-fed Tibetan minipig atherosclerosis models, analyzed the DEGs gene ontology (GO) terms, pathways and protein-protein interactions (PPI) networks, and identified hub genes and key modules using molecular complex detection (MCODE), Centiscape and CytoHubba plugin. The identified genes were validated using the human carotid atherosclerosis database (GSEA 43292) and RT-PCR methods. RESULTS: Our results showed that minipigs displayed obvious dyslipidemia, oxidative stress, inflammatory response, atherosclerotic plaques, as well as increased low-density lipoprotein (LDL) and leukocyte recruitment after 24 weeks of HFC diet feeding compared to those under a regular diet. Our RNA-seq results revealed 1716 DEGs in the atherosclerotic/NC group, of which 1468 genes were up-regulated and 248 genes were down-regulated. Functional enrichment analysis of DEGs showed that the HFC diet-induced changes are related to vascular immune-inflammatory responses, lipid metabolism and muscle contraction, indicating that hypercholesterolemia caused by HFC diet can activate innate and adaptive immune responses to drive atherosclerosis development. Furthermore, we identified four modules from the major PPI network, which are implicated in cell chemotaxis, myeloid leukocyte activation, cytokine production, and lymphocyte activation. Fifteen hub genes were discovered, including TNF, PTPRC, ITGB2, ITGAM, VCAM1, CXCR4, TYROBP, TLR4, LCP2, C5AR1, CD86, MMP9, PTPN6, C3, and CXCL10, as well as two transcription factors (TF), i.e. NF-ĸB1 and SPI1. These results are consistent with the expression patterns in human carotid plaque and were validated by RT-PCR. CONCLUSIONS: The identified DEGs and their enriched pathways provide references for the development and progression mechanism of Tibetan minipig atherosclerosis model induced by the HFC diet.

Cathelicidin-BF, a Novel Antimicrobial Peptide from Bungarus fasciatus, Attenuates Disease in a Dextran Sulfate Sodium Model of Colitis.

Antimicrobial peptides are molecules of innate immunity. Cathelicidin-BF is the first cathelicidin peptide found in reptiles. However, the immunoregulatory and epithelial barrier protective properties of C-BF have not been reported. Inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, can lead to colon cancer, the third most common malignant tumor. The objective is to develop the new found cathelicidin-BF as a therapeutic to patients of ulcerative colitis. The morphology of the colon epithelium was observed by H&E staining; apoptosis index and infiltration of inflammatory cells in colonic epithelium were measured by TUNEL and immunohistochemistry; the expression level of endogenous mCRAMP was analyzed by immunofluorescence; and phosphorylation of the transcription factors c-jun and NF-κB in colon were analyzed by Western blot. Our results showed that the morphology of the colon epithelium in the C-BF+DSS group was improved compared with the DSS group. Apoptosis and infiltration of inflammatory cells in colonic epithelium were also significantly attenuated in the C-BF+DSS group compared with the DSS group, and the expression level of endogenous mCRAMP in the DSS group was significantly higher than other groups. DSS-induced phosphorylation level of c-jun and NF-κB while C-BF effectively inhibited phosphorylation of NF-κB (p65). The barrier protective effect of C-BF was still excellent. In conclusion, C-BF effectively attenuated inflammation and improved disrupted barrier function. Notably, this is the first report to demonstrate that C-BF attenuates DSS-induced UC both through the regulation of intestinal immune and retention of barrier function, and the exact pathway was through NF-κB.

Vasodilatory Effect of Guanxinning Tablet on Rabbit Thoracic Aorta is Modulated by Both Endothelium-Dependent and -Independent Mechanism.

Vasodilatory therapy plays an important role in the treatment of cardiovascular diseases, especially hypertension and coronary heart disease. Previous research found that Guanxinning tablet (GXNT), a traditional Chinese compound preparation composed of Salvia miltiorrhiza (Danshen) and Ligusticum chuanxiong (Chuanxiong), increase blood flow in the arteries, but whether vasodilation plays a role in this effect remains unclear. Here, we found that GXNT significantly alleviated the vasoconstriction of isolated rabbit thoracic aorta induced by phenylephrine (PE), norepinephrine (NE), and KCl in a dose-dependent manner with or without endothelial cells (ECs). Changes in calcium ion levels in vascular smooth muscle cells (VSMCs) showed that both intracellular calcium release and extracellular calcium influx through receptor-dependent calcium channel (ROC) declined with GXNT treatment. Experiments to examine potassium channels suggested that endothelium-denuded vessels were also regulated by calcium-activated potassium channels (Kca) and ATP-related potassium channels (KATP) but not voltage-gated potassium channels (kv) and inward rectifying potassium channels (KIR). For endothelium-intact vessels, the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contents in vascular tissue obviously increased after GXNT treatment, and pretreatment with the NO synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) or guanylyl cyclase inhibitor methylthionine chloride (MB) significantly inhibited vasodilation. An assessment of NO-related pathway protein expression revealed that GXNT enhanced the expression of phosphorylated endothelial NO synthase (eNOS) in a dose-dependent manner but had no effect on total eNOS, p-Akt, Akt, or PI3K levels in human umbilical vein ECs (HUVECs). In addition to PI3K/AKT signaling, Ca2+/calmodulin (CaM)-Ca2+/CaM-dependent protein kinase II (CaMKII) signaling is a major signal transduction pathway involved in eNOS activation in ECs. Further results showed that free calcium ion levels were decreased in HUVECs with GXNT treatment, accompanied by an increase in p-CaMKII expression, implying an increase in the Ca2+/CaM-Ca2+/CaMKII cascade. Taken together, these findings suggest that the GXNT may have exerted their vasodilative effect by activating the endothelial CaMKII/eNOS signaling pathway in endothelium-intact rings and calcium-related ion channels in endothelium-denuded vessels.

Differential metabolic and hepatic transcriptome responses of two miniature pig breeds to high dietary cholesterol.

AIMS: Pigs are increasingly used as human metabolic disease models; however, there is insufficient research on breed-related genetic background differences. This study aimed to investigate the differential metabolic responses to high-fat and high-cholesterol (HFC) diet-induced non-alcoholic fatty liver disease (NAFLD) of two miniature pig breeds and explore the molecular mechanisms involved. MAIN METHODS: Male Wuzhishan (WZSP) and Tibetan pigs (TP) were randomly fed either a standard or an HFC diet for 24 weeks. Weight, serum lipids, bile acid, insulin resistance, liver function, liver histology, and hepatic lipid deposition were determined. RNA-Seq was used to detect the hepatic gene expression profiles. Western blot, immunohistochemistry, and qRT-PCR were used to detect the lipid and glucose metabolism-related gene expressions. KEY FINDINGS: The HFC diet caused obesity, hypertension, severe hypercholesterolemia, liver injury, increased hepatocellular steatosis and inflammation, and significantly increased serum insulin levels in both pig breeds. This diet led to higher serum and hepatic cholesterol level concentrations in WZSP and elevated fasting glucose levels in TP. Transcriptome analysis revealed that the genes controlling hepatic cholesterol metabolism and the inflammatory response were consistently regulated; lipid metabolism and insulin signaling related genes were uniquely regulated by the HFC diet in the WZSP and TP, respectively. SIGNIFICANCE: Our study demonstrated that the genetic background affects profoundly pigs' metabolic and hepatic responses to an HFC diet. These results deepened our understanding of the molecular mechanisms of HFC diet-induced NAFLD and provided a foundation for selecting the appropriate pig breeds for metabolic studies in the future.

[Expression of Sirtuin 1 in visceral adipose tissue in Tibetan mini-pigs with obesity and insulin resistance induced by high fat/cholesterol diet].

OBJECTIVE: To investigate the role of Sirt1 in visceral adipose tissue in Tibetan mini-pigs with obesity and insulin resistance induced by high fat/cholesterol diet. METHODS: Twelve male Tibetan mini-pigs were divided into 2 groups randomly: normal control (NC) group, high-fat/cholesterol (HFC) diet group, 6 in each group. After 16 weeks of modeling, fasting body weight and body mass index (BMI) were measured. Total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured in anterior venous blood, and atherosclerosis index (AI) was calculated. Meanwhile, intravenous glucose tolerance test was conducted to observe the changes of blood glucose and insulin, and the area under the curve (AUC) was calculated. After euthanasia, visceral fat rate was detected, and visceral fat tissue was taken for histopathological observation and fat cell diameter analysis. RT-PCR was used to observe the mRNA expression levels of Sirtuin1 (Sirt1), insulin-like growth factor-1 (IGF-1), glucose transporter 4 (GLUT4), peroxisome proliferator-activated receptor γ (PPARγ), peroxisome proliferator-activated receptor γ-assisted activator 1α (PGC-1α), forkhead box protein O1 (FoxO1), lipolysis-related gene hormone-sensitive lipase (HSL), and fat synthesis-related gene fatty acid synthase (FASN)changes in adipose tissue. RESULTS: Compared with the NC group, the body weight, BMI, TC, LDL-C, HDL-C, AI and visceral fat rate were significantly increased after 16 weeks of high-fat/cholesterol induction in Tibetan mini-pigs(P＜0.05,P＜0.01). Meanwhile, the glucose tolerance curve was significantly delayed and the area under the curve of blood glucose and insulin was significantly increased (P＜0.05). HE pathological observation and quantitative analysis showed that fat cells were hypertrophy and the average cell diameter was increased significantly (P＜0.01). In addition, the mRNA expression levels of Sirt1,PGC-1α, GLUT4, and HSL were all decreased in varying degrees in adipose tissue, among which the mRNA expressions of Sirt1 and HSL were significantly decreased (P＜0.05), while the mRNA expressions of FOXO1, IGF-1, PPARγ, and FASN were significantly increased (P＜0.05, P＜0.01). CONCLUSION: Tibetan mini-pigs were induced by high fat/cholesterol diet to form obesity model with phenotypic characteristics such as lipid disorder and insulin resistance, whereas Sirt1 plays a key role in visceral fat deposition and insulin sensitivity reduction in obese Tibetan mini-pigs.

Royal jelly causes hypotension and vasodilation induced by increasing nitric oxide production.

Among royal jelly's (RJ) various biological activities, its possible antihypertension and vasorelaxation effects deserve particular attention, but the underlying mechanisms of action remain unclear. Therefore, this study used the spontaneously hypertensive rats (SHR) hypertension model and the isolated rabbit thoracic aorta rings model to explore the mechanisms underlying the hypotension and vasorelaxation effects of RJ. Rats were divided into the following groups (n = 6): WKY-control group, SHR-control group, and SHR-RJ group. SHR-RJ group was received 1 g/kg of RJ via oral administration daily for 4 weeks. Systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and nitric oxide (NO) level were detected. In addition, the mechanism of vasodilation of RJ was investigated using an isolated rabbit aortic ring technique. RJ significantly reduced SBP and DBP as well as increased NO levels of SHR in vivo. RJ caused vasorelaxation of the isolated aorta rings, and this effect was inhibited by atropine (M3 receptor blocker), L-NAME (nitric oxide synthase inhibitor), methylene blue (guanylate cyclase inhibitor), and indomethacin (cyclooxygenase inhibitor). Moreover, RJ could markedly suppress the NE-induced intracellular Ca2+ releases and high K+-induced extracellular Ca2+ influx in denuded aortic rings. In addition, RJ can also increase cGMP levels and the production of NO in isolated aortic rings. The present study showed that RJ has antihypertensive effects and was associated with increased NO production. In addition, RJ contains muscarinic receptor agonist, possibly an acetylcholine-like substance, and induces vasodilation through NO/cGMP pathway and calcium channels.

Lower cardiovagal tone and baroreflex sensitivity associated with hepatic insulin resistance and promote cardiovascular disorders in Tibetan minipigs induced by a high fat and high cholesterol diet.

AIMS: A long-term high-fat/cholesterol (HFC) diet leads to hepatic insulin resistance (IR), which is associated with autonomic dysfunction and cardiovascular diseases risk increasing. However, whether this occurs in Tibetan minipigs remains unknown. We tested that a long-term HFC diet caused hepatic IR and promote cardiovascular disorders in Tibetan minipigs, and are associated with the reduction of cardiovagal tone and baroreflex sensitivity (BRS). METHODS: Male Tibetan minipigs were fed either a standard diet or a HFC diet, and were euthanized at 12 weeks. Thereafter, the minipigs were tested for biochemical blood indices, glucose tolerance, blood pressure, heart rate variability (HRV), BRS, and insulin receptor substrate (IRS)-associated gene and protein expression levels, as well as cardiac function. RESULTS: HFC-fed minipigs developed IR by increasing body weight, total cholesterol, fasting blood glucose and insulin levels, and nonesterified fatty acid (NEFA) and high sensitive C-reactive protein (hs-CRP) levels, glucose intolerance. Increased adipose cell size, hepatic fat deposition, malondialdehyde (MDA) content and NEFA level, down-regulation of IRS1, IRS2, PI3K, Akt, p-Akt, Glut2 and PGC1ɑ expression concomitant with up-regulation of mTOR, GSK3ß, TNF-ɑ, FOXO1, p-mTOR and p-p70S6K expression in the liver tissue, as well as hypertension and left ventricular diastolic dysfunction were observed in HFC-fed minipigs. HRV parameters and BRS values were further significantly reduced. Furthermore, multiple linear regression analysis showed that the development of hepatic IR toward cardiovascular disease was associated with low HFnu, RMSSD, BRS and LV -dp/dtmax, high NEFA, high hepatic TG content. CONCLUSION: These data suggest that HFC-fed Tibetan minipigs develop hepatic IR and promote cardiovascular disorders, and are associated with lower cardiovagal tone and BRS.

Effects of casein glycomacropeptide supplementation on growth performance, intestinal morphology, intestinal barrier permeability and inflammatory responses in Escherichia coli K88 challenged piglets.

Casein glycomacropeptide (CGMP) is a bioactive peptide derived from milk with multiple functions. This study was aimed at evaluating the effects of CGMP as a potential feed additive on growth performance, intestinal morphology, intestinal barrier permeability and inflammatory responses of Escherichia coli K88 (E. coli K88) challenged piglets. Eighteen weaning piglets were randomly assigned to three groups. Control group and K88 challenged group received a basal diet, and CGMP treated group received the basal diet supplemented with 1% of CGMP powder. The trail lasted for 12 days, K88 was orally administered to the piglets of K88 challenged group and CGMP treated group on days 8-10. The results showed that the diet containing 1% CGMP significantly alleviated the decrease in average daily gain (P < 0.05), increase in pathogenic bacteria amounts in intestinal contents (P < 0.05), intestinal morphology (P > 0.05) and barrier permeability damage (P < 0.05), and acute inflammatory response (P < 0.05) induced by E. coli K88 infection. In conclusion, CGMP supplementation in the diet protected the weaning piglets against E. coli K88 infection.

Expression pattern of porcine antimicrobial peptide PR-39 and its induction by enterotoxigenic Escherichia coli (ETEC) F4ac.

PR-39 is a gene-encoded, proline-arginine-rich porcine antimicrobial peptide with multiple biological functions. In the current study, the tissue-specific mRNA expression of PR-39 was investigated in Chinese Jinhua pigs, and the effect of enterotoxigenic Escherichia coli (ETEC) expressing F4ac (K88ac) fimbriae challenge on the mRNA expression of PR-39 in various tissues was compared between Jinhua and Landrace pigs. The three most stable expressed housekeeping genes were validated before evaluating PR-39 expression. PR-39 mRNA was predominantly expressed in the bone marrow compared with the spleen, thymus, MLN, liver and ileum. The ETEC F4ac challenge could up-regulate PR-39 mRNA expression in both Jinhua and Landrace pigs, but the changes were different between the two breeds. Jinhua pigs responded more strongly to ETEC F4ac challenge than did Landrace pigs, because the interaction between the breed and challenge significantly impact PR-39 mRNA in the thymus, liver and ileum. The PR-39 mRNA expression levels of challenged Jinhua pigs were significantly higher in the spleen, thymus, liver, ileum and MLN compared with challenged Landrace pigs. These differences in the mRNA expression of PR-39 could be a result of genetic differences in the resistance to ETEC F4ac infection between the two breeds, but this speculation requires further investigation.