Cardiac sympathetic overdrive, M2 macrophage activation and fibroblast heterogeneity are associated with cardiac remodeling in a chronic pressure overload rat model of HFpEF.

Heart failure with preserved ejection fraction (HFpEF) is a multifaceted pathogenesis disease and the exact mechanisms driving HFpEF have not been completely elucidated. Pressure overload hypertrophy (POH) related fibroblasts and M2 macrophages in HFpEF myocardium have been recently identified and are now of great interest. Sympathetic overdrive has also been implicated in HFpEF. This study is designed to dynamically observe the potential roles of aforementioned mechanisms in pathological remodeling and cardiac dysfunction in chronic PO rats. Surgical constriction of the abdominal aorta was used for induction of HFpEF. Echocardiography, electrocardiogram, hemodynamic measurement, hematoxylin and eosin staining, Masson staining, immunohistochemistry and immunofluorescence were performed to assess the changes in heart dysfunction, cardiac remodeling and driving mechanisms at different time points (2, 18, 24 weeks). The PO induced HFpEF model was well established, which was confirmed by the persistent increase in carotid artery systolic and diastolic blood pressure, and left ventricle hypertrophy at the corresponding postoperative stage. Meanwhile, PO hypertrophy gradually developed into HFpEF, associated with QT and QTc intervals prolongation, normal systolic (EF was maintained at >50%) but impaired diastolic function (increasing LVEDP and LV -dP/dtmin, abnormal E/A ratio), increased myocytes size, and observed relatively slight inflammatory infiltration but robust reactive fibrosis. IHC staining further confirmed that macrophages (CD68) but not neutrophils (MPO) or T cells (CD3) accounted for a predominant proportion of infiltrating cells. Mechanistically, we found that the infiltrating macrophages in the heart expressed high levels of CD206 which was simultaneously adjacent to POH fibroblasts appeared to overexpression of α-SMA in PO rats at late stages. Interestingly, we distinguished two different POHF sub-populations during PO induced HFpEF development, according to non overlapping signals of α-SMA and PDGFRα/ß proteins. Additionally, PO led to a pronounced exaggeration in sympathetic fibers at all time points. These findings suggest that the establishing model here begins with cardiac sympathetic overdrive, subsequently along with immune cells especially M2 macrophage accumulation and fibroblast heterogeneity at later stages is associated with the development of cardiac maladaptive remodeling and diastolic dysfunction thus further progression to HFpEF.

Niaodukang mixture inhibits micro-inflammation in CKD rats by enhancing MiR-146a levels in enterogenous exosomes.

ETHNOPHARMACOLOGICAL RELEVANCE: The Niaodukang mixture (NDK) is a preparation known for its ability to lower serum creatine levels in individuals with chronic kidney disease (CKD) and is commonly administered at medical facilities like the Zhongshan Hospital of Traditional Chinese Medicine. The initial use of NDK was mainly to treat CKD. Our hospital frequently utilizes NDK, which consists of Rheum officinaleBaill., Salvia miltiorrhiza Bunge., Astragalus aaronii (Eig) Zohary., Carthamus tinctorius L., and Sanguisorba officinalis L., for treating patients with CKD-MBD. It has the effects of eliminating dampness and turbidity and dredging kidney collaterals. However, The impact and process of NDK in chronic kidney disease remain unknown. AIM OF THE STUDY: To determine whether microRNA-146a (miR-146a) is associated with CKD micro-inflammationand whether NDK protects against CKD micro-inflammation by modulating the miR-146a/nuclear factor kappa-B (NF-κB) signaling pathway. MATERIALS AND METHODS: (1) An adenine-induced rat model of chronic kidney disease was created through the use of materials and methods. The levels of miR-146a in exosomes from plasma and ileum were determined by RT-PCR. (2) Human cloned colon adenocarcinoma (Caco-2)cellswere stimulated with lipopolysaccharide (LPS)and transfected with miR-146a mimic and inhibitor. Following that, the Western blot and RT-PCR techniques were used to measure the protein and mRNA quantities of Toll-like receptor 4 (TLR4), NF-κB, and TNF receptor-associated factor 6 (TRAF6). (3) Enzyme-linked immunosorbent assay (ELISA) was used to identify serum levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). (4) Plasma exosomes were extracted, and the exosomes in intestinal tissues were extracted via ultrahigh-speed centrifugation.Negative staining electron microscopy was used to analyze the morphology of exosomes and the ultrastructure of intestinal tissue and exosomes. The particle size of the exosomes was measured using nanoparticle tracking analysis. RESULTS: The pathological characteristics of CKD rats included those associated with systemic micro-inflammation, which may be associated with the release of exosomes in intestinal tissue. NDK suppressed the inflammatory response in Caco-2 cells and decreased the levels of IL-1ß, IL-6, and TNF-α in rats with CKD. The expression of miR-146a, which regulates inflammation, differed between plasma-derived and enterogenous exosomes in CKD rats, which may be due to stimulation of ileal exosome release into the blood. NDK effectively reduced the levels of TRAF6, NF-κB, and TLR4 in the ileum tissue of CKD rats. CONCLUSION: NDK can effectively improve micro-inflammation in CKD ratsby enhancing the release of enterogenous exosomes, thereby enhancing the release of exosome-associated miR-146a and inhibiting micro-inflammation.

Molecular characterization and structure basis of a malonyltransferase with both substrate promiscuity and catalytic regiospecificity from Cistanche tubulosa.

Enzymatic malonylation of natural glycosides provides a promising alternative method for drug-like malonylated glycosides supply. However, the catalytic potential and structural basis of plant malonyltransferase are far from being fully elucidated. This work identified a new malonyltransferase CtMaT1 from Cistanche tubulosa. It displayed unprecedented mono- and/or di-malonylation activity toward diverse glucosides with different aglycons. A "one-pot" system by CtMaT1 and a malonyl-CoA synthetase was established to biosynthesize nine new malonylated glucosides. Structural investigations revealed that CtMaT1 possesses an adequately spacious acyl-acceptor pocket capable of accommodating diverse glucosides. Additionally, it recognizes malonyl-CoA through strong electrotactic and hydrogen interactions. QM/MM calculation revealed the H167-mediated SN2 reaction mechanism of CtMaT1, while dynamic simulations detected the formation of stable hydrogen bonds between the glucose-6-OH group and H167, resulting in its high malonylation regiospecificity. Calculated energy profiles of two isomeric glycosides highlighted lower reaction energy barriers towards glucoside substrates, emphasizing CtMaT1's preference for glucosides. Furthermore, a mutant CtMaT1H36A with notably increased di-malonylation activity was obtained. The underlying molecular mechanism was illuminated through MM/GBSA binding free energy calculation. This study significantly advances the understanding of plant acyltransferases from both functional and protein structural perspectives, while also providing a versatile tool for enzymatic malonylation applications in pharmacology.

Discovery of plant chemical defence mediated by a two-component system involving ß-glucosidase in Panax species.

Plants usually produce defence metabolites in non-active forms to minimize the risk of harm to themselves and spatiotemporally activate these defence metabolites upon pathogen attack. This so-called two-component system plays a decisive role in the chemical defence of various plants. Here, we discovered that Panax notoginseng, a valuable medicinal plant, has evolved a two-component chemical defence system composed of a chloroplast-localized ß-glucosidase, denominated PnGH1, and its substrates 20(S)-protopanaxadiol ginsenosides. The ß-glucosidase and its substrates are spatially separated in cells under physiological conditions, and ginsenoside hydrolysis is therefore activated only upon chloroplast disruption, which is caused by the induced exoenzymes of pathogenic fungi upon exposure to plant leaves. This activation of PnGH1-mediated hydrolysis results in the production of a series of less-polar ginsenosides by selective hydrolysis of an outer glucose at the C-3 site, with a broader spectrum and more potent antifungal activity in vitro and in vivo than the precursor molecules. Furthermore, such ß-glucosidase-mediated hydrolysis upon fungal infection was also found in the congeneric species P. quinquefolium and P. ginseng. Our findings reveal a two-component chemical defence system in Panax species and offer insights for developing botanical pesticides for disease management in Panax species.

Enzymatic biosynthesis of novel 2-(2-phenylethyl)chromone glycosides catalyzed by UDP-glycosyltransferase UGT71BD1.

2-(2-Phenylethyl)chromones (PECs) are the main bioactive components of agarwood which showed diverse pharmaceutical activities. Glycosylation is a useful structural modification method to improve compounds' druggability. However, PEC glycosides were rarely reported in nature which largely limited their further medicinal investigations and applications. In this study, the enzymatic glycosylation of four naturally separated PECs 1-4 was achieved using a promiscuous glycosyltransferase UGT71BD1 identified from Cistanche tubulosa. It could accept UDP-Glucose, UDP-N-acetylglucosamine and UDP-xylose as sugar donors and conduct the corresponding O-glycosylation of 1-4 with high conversion efficiencies. Three O-glucosylated products 1a (5-hydroxy-2-(2-phenylethyl)chromone 8-O-ß-D-glucopyranoside), 2a (8-chloro-2-(2-phenylethyl)chromone 6-O-ß-D-glucopyranoside) and 3a (2-(2-phenylethyl)chromone 6-O-ß-D-glucopyranoside) were prepared and structurally elucidated as novel PEC glucosides based on NMR spectroscopic analyses. Subsequent pharmaceutical evaluation found that 1a showed remarkably improved cytotoxicity against HL-60 cells, whose cell inhibition rate was 19 times higher than that of its aglycon 1. The IC50 value of 1a was further determined to be 13.96 ± 1.10 µM, implying its potential as a promising antitumor-leading candidate. To improve the production of 1, docking, simulation and site-directed mutagenesis were performed. The important role of P15 in the glucosylation of PECs was discovered. Besides, a mutant K288A with a two-fold increased yield for 1a production was also afforded. This research reported the enzymatic glycosylation of PECs for the first time, and also provide an eco-friendly pathway for the alternative production of PEC glycosides for leading compounds discovery.

Chronic Administration of Methamphetamine Aggravates Atherosclerotic Vulnerable Plaques in Apolipoprotein E Knockout Mice Fed with a High-cholesterol Diet.

BACKGROUND: It has been observed previously that chronic methamphetamine (METH) administration could upregulate neuropeptide Y (NPY) expression and promote atherosclerotic formation in apolipoprotein E knockout (ApoE-/-) mice fed with a normal cholesterol or high diet and NPY might be involved in the pathogenesis of METH-induced atherogenic effects through NPY Y1 receptor pathway. Vulnerable coronary atherosclerotic plaque (VP) is a critical pathological finding responsible for the acute coronary syndrome (ACS). In this study, we explored whether METH abuse could aggravate the formation of VP in ApoE-/- mice fed with high cholesterol diet. OBJECTIVE: The purpose of this study was to observe if chronic METH administration could aggravate vulnerable plaque (VP) formation in ApoE-/- mice fed with a high-cholesterol diet. METHODS: Male ApoE-/- mice fed with a high-cholesterol diet were intraperitoneally injected with normal saline (NS) or 8 mg/kg/day METH (M8) for 24 weeks. Body weight was monitored from baseline to 24 weeks at 2 weeks intervals. After 24 weeks of treatment, plasma lipid variables were measured. Movat's staining and immunohistochemical staining were performed on frozen sections of the aortic roots to calculate VP percentage and intraplaque hemorrhage (IPH) percentage and detect expression of NPY, vascular endothelial growth factor (VEGF), and CD31. In vitro, the expressions of Y2R, VEGF, and CD31 were detected by immunofluorescence staining in aortic endothelial cells incubated with PBS, 100µM METH, 10nmol NPY, or 100µM METH plus 10nmol NPY for 12 hours. RESULTS: The CD31 positive area, percentage of IPH, VP, and the expressions of NPY and VEGF were significantly increased in the M8 group than in the NS group. In vitro, the expressions of Y2R, VEGF, and CD31 were significantly increased in the METH+NPY group than in the PBS, METH, and NPY groups and these effects could be blunted by treatment with a Y2R antagonist or DPPIV inhibitor. CONCLUSION: Chronic METH administration could aggravate VP in ApoE-/- mice fed with a high-cholesterol diet, possibly through upregulating vascular NPY and VEGF expression and promoting angiogenesis and vessel rupture in atherosclerotic plaques. Our findings indicated that increased VP formation might contribute to the development of acute coronary syndrome post-chronic METH abuse by activating DPPIV/NPY/Y2R pathway.

Rheum officinale and Salvia miltiorrhiza inhibit renal fibrosis via miR-21/PTEN/Akt signaling pathway in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of the main components of many famous Chinese herbal formulas, Rheum palmatum L. and Salvia miltiorhiza Bunge (RS) are extensively used to treat chronic kidney disease (CKD). RS has been proved to improve renal function and relieve renal fibrosis (RF), but the potential mechanism remains a mystery. AIM OF THE STUDY: The purpose of this study is to determine whether microRNA-21 (miR-21) is associated with RF progression, as well as whether RS protects against RF through miR-21/PTEN/AKT signaling. MATERIALS AND METHODS: (1) The rat model of RF was established using unilateral ureteral obstruction (UUO). After UUO surgery, miR-21 levels in plasma were detected by RT-PCR and RF scores were assessed by Masson's trichrome stain at days 3, 7, 14 and 21. The correlation analysis of the above two indexes was carried out by Spearman correlation analysis. (2) Human proximal tubular epithelial cells (HK-2) was transfected with miR-21 mimic and inhibitor, and then the levels of phosphatase and tensin homolog (PTEN) protein and mRNA were measured with Western blotting and RT-PCR, respectively. (3) TGF-ß (10 ng/mL) was added into HK-2 cells to induce fibrosis, followed by the intervention of RS-containing rat serum. PTEN and protein kinase-B (Akt) phosphorylation, as well as the expression of PTEN protein in HK-2 cells, were assessed by RT-PCR, Western blotting and immunofluorescence. (4) The rat models of RF were prepared by UUO and treated with RS. Serum creatinine and urea nitrogen levels were measured. RF score was determined by Masson's trichrome stain. RT-PCR was used to determine the expression of miR-21, PTEN, and Akt mRNA. Western blotting was used to determine the expression of PTEN and Akt proteins. RESULTS: A positive correlation was found between plasma miR-21 levels and RF scores of rats after UUO surgery at Days 3, 7, 14 and 21. It was confirmed that miR-21 targeted PTEN. RS drug-containing serum could rise the expression of PTEN and reduce Akt phosphorylation of HK-2 cells induced by TGF-ß. Moreover, RS drug-containing serum could increase PTEN expression and reduce Akt phosphorylation induced by miR-21 mimic in HK-2 cells. The rats treated with RS had significantly decreased serum creatinine and urea nitrogen levels and a lower RF score. RS also decreased miR-21 and Akt expressions, increased PTEN expression of UUO rats. CONCLUSION: There was a positive correlation between plasma miR-21 levels and RF scores. The inhibitory effect of RS on RF might be mediated by miR-21/PTEN/AKT signaling.

Self-Assembly 2D Ti3C2/g-C3N4 MXene Heterojunction for Highly Efficient Photocatalytic Degradation of Tetracycline in Visible Wavelength Range.

An ultrathin 2D Ti3C2/g-C3N4 MXene (2D-TC/CN) heterojunction was synthesized, using a facile self-assembly method; the perfect microscopic-morphology and the lattice structure presented in the sample with a 2 wt% content of Ti3C2 were observed by the field-emission scanning electron microscopy (SEM) and transmission electron microscope (TEM). The optimized sample (2-TC/CN) exhibited excellent performance in degrading the tetracycline (TC), and the degradation rate reached 93.93% in the conditions of 20 mg/L, 50 mL of tetracycline within 60 min. Except for the increased specific-surface area, investigated by UV-vis diffuse reflectance spectra (UV-vis DRS) and X-ray photoelectron microscopy (XPS) valence spectra, the significantly enhanced photocatalytic activity of the 2-TC/CN could also be ascribed to the formation of Ti-N bonds between Ti3C2 and g-C3N4 nanosheets, which reduced the width of the band gap through adjusting the position of the valence band, thus resulting in the broadened light-absorption. Furthermore, the facilitated electron transmission was also proved by time-resolved photoluminescence (TRPL) and electrochemical impedance spectroscopy (EIS), which is effective in improving the quantum efficiency of photo-generated electrons. In addition, the resulting radical-capture experiment suggested that superoxide radicals have the greatest influence on photodegradation performance, with the photodegradation rate of TC reducing from 93.16% to 32.08% after the capture of superoxide radicals, which can be attributed to the production of superoxide radicals only, by the 2-TC/CN composites with a high conduction-band value (-0.62 eV). These facilely designed 2D Ti3C2/g-C3N4 composites possess great application potential for the photodegradation of tetracycline and other antibiotics.

Biosynthesis Investigations of Terpenoid, Alkaloid, and Flavonoid Antimicrobial Agents Derived from Medicinal Plants.

The overuse of antibiotics in the past decades has led to the emergence of a large number of drug-resistant microorganisms. In recent years, the infection rate caused by multidrug-resistant microorganisms has been increasing, which has become one of the most challenging problems in modern medicine. Plant-derived secondary metabolites and their derivatives have been identified to display significant antimicrobial abilities with good tolerance and less adverse side effects, potentially having different action mechanisms with antibiotics of microbial origin. Thus, these phyto-antimicrobials have a good prospect in the treatment of multidrug-resistant microorganisms. Terpenoids, alkaloids, and flavonoids made up the predominant part of the currently reported phytochemicals with antimicrobial activities. Synthetic biology research around these compounds is one of the hotspot fields in recent years, which not only has illuminated the biosynthesis pathways of these phyto-antimicrobials but has also offered new methods for their production. In this review, we discuss the biosynthesis investigations of terpenoid, alkaloid, and flavonoid antimicrobial agents-using artemisinin and oleanolic acid (terpenoids), berberine and colchicine (alkaloids), and baicalin (flavonoids) as examples-around their antimicrobial action mechanisms, biosynthesis pathway elucidation, key enzyme identification, and heterologous production, in order to provide useful hints for plant-derived antimicrobial agent discovery and development.

Effects of triangle grass decoction on bone metabolism in rats with chronic kidney disease complicated with mineral and bone abnormalities.

ETHNOPHARMACOLOGICAL RELEVANCE: Triangle grass is a liliaceous Chlorophytum perennial herb of ChlorophytumlaxumR.Br. It is distributed mainly in Guangdong and Guangxi Provinces of China. The initial use of triangle grass was mainly to treat bone pain and swelling caused by a fall injury. Triangle grass tablets (NO. Z20070544) are also used as a preparation in our hospital because of their analgesic, anti-inflammatory, anti-snake venom and microcirculation improvement properties and other pharmacological effects (Mei et al., 2006). Triangle grass tablets have been widely used in our hospital to treat patients with bone pain from chronic kidney disease-mineral and bone disorder (CKD-MBD). However, the effects and mechanism of triangle grass on bone metabolism in chronic kidney disease complicated with mineral and bone abnormalities are unclear. AIM OF THE STUDY: The aim of the present study was to investigate the effects of a triangle grass decoction on bone metabolism in CKD-MBD rats. MATERIALS AND METHODS: CKD-MBD model rats were subjected to 5/6 nephrectomy combined with 0.5 g NaH2PO4/rat. Serum blood urea nitrogen (BUN), creatinine (Cr), phosphorus (P), calcium (Ca), and intact parathyroid hormone (iPTH) levels were measured with an automatic biochemical analyser. Bone mineral density was determined with a Viva CT 40 system. Bone morphogenetic protein 7(BMP-7),runt-related transcription factor 2 (Runx2) and Osterix protein levels were measured by Western blot analysis. Kidney, vertebra and thoracic aorta tissue samples were assessed by histopathology and immunohistochemistry (IHC). RESULTS: The degrees of membrane thickening, necrosis, swelling and cast deposition were significantly reduced in high-dose rats and Low-dose rats. Serum BUN levels were significantly reduced in the Pre-H group (P < 0.05). Hypocalcaemia and hyperphos phataemia were detected in triangle grass (P < 0.05, P < 0.05). In addition, iPTH levels were significantly increased in the Pre-H group (P < 0.05). Alkaline phosphatase (ALP)levels were significantly decreased in the Pre-H group (P < 0.05). The bone mineral density was improved in the Pre-H and Pre-L groups. BMP-7 protein levels were significantly increased in the Pre-H group (P < 0.05). The pathological changes in muscle fibres in the thoracic aorta middle membranes were significantly alleviated in rats in the Pre-H and Pre-L groups. Changes in SM22α and SMα-act in protein levels were significantly attenuated in the Pre-H group (P < 0.05, P < 0.05). Changes in Runx2 and Osterix protein levels were also significantly attenuated in the Pre-H and Pre-L groups (P < 0.05, P < 0.05). CONCLUSIONS: Triangle grass can simultaneously ameliorate vertebral bone loss and abnormal calcification in the thoracic aorta. Triangle grass has a definite effect on bone metabolism disorder in CKD-MBD rats.

Protective effect of miR378* on doxorubicin-induced cardiomyocyte injury via calumenin.

Doxorubicin (Dox) is a highly effective antitumor antibiotic, however myocardial toxicity severely limits its use clinically. The pathogenesis of doxorubicin-induced cardiomyopathy is unclear. In Dox cardiomyopathy mice, there is a decline in cardiac function, a change in myocardial pathology and a reduction in miR378* expression. Expression changes in calumenin, an endoplasmic reticulum stress (ERS) chaperone protein and pathway factor, as well as apoptosis, were observed in cardiomyocytes after doxorubicin-induced injury. However, miR378* increased calumenin expression, eased ERS, and reduced cardiomyocyte apoptosis, while, silencing miR378* reduced calumenin expression, aggravated ERS, and increased cardiomyocyte apoptosis. The above results indicate that miR378* alleviates ERS and inhibits the activation of the ERS-mediated apoptosis signaling pathway in cardiomyocytes via regulating calumenin expression, thereby reducing cardiomyocyte apoptosis after doxorubicin-induced injury. Increasing miR378* expression may be a new way to improve cardiac function and quality of life in patients with Dox cardiomyopathy.

MiR-378 modulates energy imbalance and apoptosis of mitochondria induced by doxorubicin.

Doxorubicin (DOX) is an effective anticancer drug, however its clinical application is limited due to its cardiotoxicity. Therefore, understanding the mechanisms of cardiotoxicity induced by DOX is essential. We found that the level of miR-378 was decreased in the hearts of DOX-treated rats. Increasing the expression of miR-378 resulted in a decrease of lactate dehydrogenase (LDH) upon DOX treatment in vitro by targeting lactate dehydrogenase A (LDHA). Furthermore, bioinformatics analysis indicated that cyclophilin A (PPIA), a regulator of apoptosis, is also a direct target gene of miR-378. We confirmed this by Western blot. Our results also showed that the overexpression of miR-378 inhibited the hyperactivation of ER stress signaling induced by DOX. In addition, MiR-378 overexpression was found to protect cardiomyocytes from DOX-induced energy imbalance and apoptosis of mitochondria. These results may allow for a therapeutic approach that overcomes the cardiotoxicity of DOX-based treatments for cancer.

Ibutilide treatment protects against ER stress induced apoptosis by regulating calumenin expression in tunicamycin treated cardiomyocytes.

BACKGROUND: Ibutilide, a class III antiarrhythmic agent has been shown to be cardioprotective in treating atrial fibrillation, promoting cardioconversion and recently this agent has been shown to protect against ER stress induced apoptosis in cardiomyocytes. In this study we begin to identify the mechanism by which ibutilide exerts its cardioprotection in tunicamycin treated cardiomyocytes. We examined ER stress markers including calumenin; a calcium binding ER chaperone protein that has recently been linked to ER stress in cardiomyocytes, in our treated cells. METHODS: To assess the effect of ibutilide we used the well characterized in vitro model of ER stress induced apoptosis in rat neonatal cardiomyocytes (RNC). RNC were treated with tunicamycin and the degree of ER stress was assessed by quantifying mRNA and protein levels of GRP78, GRP94 and calumenin, and examined the extent of apoptosis by assessing the protein levels of caspase-3/9/12, CHOP, ATF6, p-PERK, spliced XBP-1, the ratio of Bax/Bcl-2 and the percentage of deoxynucleotidyl-transferase- mediated dUTP nick end labeling (TUNEL) positive cells. RESULTS: We demonstrate ibutilide attenuated the up-regulation of ER stress markers GRP78 and GRP94 and rescued the decline in calumenin mRNA and protein levels in tunicamycin treated cardiomyocytes. The up-regulation of apoptotic markers caspase-3, CHOP, ATF6, p-PERK, spliced XBP-1, the ratio of Bax/Bcl-2 and the percentage of TUNEL positive cells were also attenuated after ibutilide treatment while the protein levels of Caspase-9 and Caspase-12 were unaffected. CONCLUSIONS: This study suggests another cardioprotective effect of the antiarrhythmic agent ibutilide whereby pretreatment leads to the attenuation of ER stress induced apoptosis by regulating calumenin expression. This study provides further evidence for the role of calumenin in the cardiomyocyte ER stress response.

[The correlation research on miRNA378* and calumenin,endoplasmic reticulum stress,apoptosis in suckling mouse myocardial cells infected with coxsackie virus B3].

OBJECTIVE: To investigate the effects of silencing miRNA378* on apoptosis, endoplasmic reticulum stress and calumenin of cardiomyocyte with coxsackie virus B3 (CVB3) infection. METHODS: Primary cultured suckling mouse myocardium were divided into control group (normal cell), coxsackie virus infection group (normal cell and coxsackie virus B3), miRNA378* control group (normal cell +coxsackie virus B3+miRNA378* empty plasmid), miRNA378* silencing plasmid group(normal cells + coxsackie virus B3 + miRNA378* silencing plasmid). Four groups of cells were transfected, infected and treated in CO2 incubator at 37℃. The α-SMA protein, cell apoptosis rate, calumenin, glucose regulated protein 78 (GRP78), activation transcription factor 6(ATF6) and transcription factors c/ebp homologue protein (CHOP) in endoplasmic reticulum were analyzed. RESULTS: By detecting α-SMA protein, the isolated suckling mouse ventricular myocardium were confirmed. TUNEL detection of different groups of ventricular cell apoptosis found that coxsackie virus group of ventricular myocytes apoptosis was significant. Compared with the coxsackie virus infection group of myocardial cells, miRNA378* silencing plasmid expression of cardiomyocyte apoptosis cells significantly reduced(P<0.01). The expressions of GRP78, ATF6 and CHOP were increased compared with those infected by Coxsackie virus infection (P<0.01), while the expressions of calumenin were decreased (P<0.01). CONCLUSIONS: CVB3 infected myocardial cells effected miRNA378* expression. It can trigger endoplasmic reticulum stress and activates signaling pathway factor and increase myocardial cell apoptosis.>.

[Effects of caspase inhibitor z-VAD-fmk on the expressions of calumenin, caspase-3, GRP78 and GRP94 in adriamycin-injured cardiomyocytes].

OBJECTIVE: To study the effects of Caspase broad spectrum inhibitor Z-VAD-FMK on the expressions of calumenin,caspase-3, GRP78 and GRP94 in adriamycin-injured cardiomyocytes and to discuss whether there is a regulation relationship between calumenin and endo-plasmic reticulum stress and myocardial apoptosis. METHODS: The primary cultured suckling mouse myocardium were randomly divided into control group (cardiomyocyte), adriamycin group (3 mg/L adriamycin + cardiomyocyte) and z-VAD-fm group (3 mg/L adriamycin + 0.1 µmol/L Z-VAD-fmk + cardiomyocyte), each group of cardiomyocytes were cultured in CO2 incubator at 37℃ for 24 h (n=3). The expres-sion ofα -SMA protein in ventricular myocytes was detected by immunohistochemical method. The expressions of calumenin, GRP78, GRP94 and Caspase-3 in the myocardial cells were detected by Western blot. RESULTS: Compared with the control group, the expression of calumenin in adriamycin induced myocardial cells was significantly decreased (P < 0.01), while the expressions of GRP78, GRP94 and Caspase-3 ex-pression were increased (P < 0.01). Compared with adriamycin group, the expression of calumenin in z-VAD-fm group was increased (P < 0.01), while the expressions of GRP78, GRP94 and caspase-3 were decreased (P < 0.01). CONCLUSIONS: The caspase inhibitor z-VAD-fmk inhibited the expression of caspase and increased the expression of calumenin in adriamycin induced myocardial cells, and thus alleviated the endoplasmic reticulum stress.

[The relationship between miRNA378, calumenin and endoplasmic reticulum stress in suckling mouse myocardium with myocarditis caused by adriamycin].

OBJECTIVE: To investigate the relationship between miRNA378, calumenin and endoplasmic reticulum stress in suckling mouse myocardium with myocarditis caused by adriamycin. METHODS: The suckling mouse myocardium of primary culture were randomly divided into control group, adriamycin group, lentivirus infection miRNA378 over expression control group, lentivirus infection miRNA378 over expression group, lentivirus infection miRNA378 silence control group and lentivirus infection miRNA378 silence group. Firstly to identify the suckling mouse myocardium, α-SMA was monitored by immunohistochemical method, and secondly the ventricular myocytes were transfered by lentivirus plasmids. Then the expression change of miRNA378, calumenin and glucose regulated protein 78(GRP78) mRNA were detected by Quantitative Real-time PCR. RESULTS: Compared with the adriamycin infection group, the expression of calumenin mRNA in lentivirus infection miRNA378 over expression group was increased(P<0.01), while the expression of GRP78 mRNA was reduced(P<0.01); Compared with the adriamycin infection group, the expressions of calumenin and GRP78 mRNA in lentivirus infection miRNA378 silence group did not change insignificantly. CONCLUSIONS: Adriamycin injection may cause expression of calumenin in suckling mouse myocardium with myocarditis reduced, which may lead to the endoplasmic reticulum stress. This effect is related with miRNA378.

Low responder T cell susceptibility to the suppressive function of regulatory T cells in patients with dilated cardiomyopathy.

OBJECTIVE: The pathogenesis of dilated cardiomyopathy (DCM) is closely connected with dysfunction of the autoimmune system, and CD4(+)CD25(high)CD127(low/-) regulatory T (Treg) cells have a vital role in maintaining self-tolerance. In this study, we compared the frequency and regulatory function of Treg cells between DCM patients and normal controls. METHODS AND RESULTS: The frequencies of CD4(+)CD25(+) T cells in DCM patients were statistically decreased compared with normal controls (p<0.05) by flow cytometry, and the levels of FOXP3 mRNA and protein expression in PBMCs (peripheral blood mononuclear cells) of DCM patients were lower than those of normal controls (p<0.01), using real-time RT-PCR assay and western blot. Notably, the suppressive capacity of CD4(+)CD25(high)CD127(low/-) regulatory T cells of DCM patients acting on autologous CD4(+)CD25(-) responder T (Tresp) cells seemed to be partially impaired (43.83+/-3.19% suppression versus 63.17+/-3.66% in normal controls, p=0.01). Surprisingly, Treg cells from DCM patients efficiently suppressed the proliferation of Tresp cells from normal subjects to the similar level as Treg cells from normal subjects on autologous Tresp cells (p=0.286), whereas Treg cells of normal subjects poorly inhibited the proliferation of Tresp cells from DCM patients. CONCLUSION: The defective capacity of Treg cells suppressing autologous Tresp cells is attributed to the increasing resistance of Tresp cells to inhibition of Treg cells in DCM patients. Therefore, strategies to improve the susceptibility of Tresp cells to Treg cell-mediated suppression might benefit DCM patients.