Cyanidin attenuates pressure overload-induced cardiac remodeling in mice via mitochondrial fusion enhancement

AIM To investigate the effect of cyanidin （Cyn） on pressure overload-induced cardiac remodeling and the underlying mechanism. METHODS Six-week-old male C57BL/6 mice （n=120） were divided into 4 groups： sham group （n=20）, sham+Cyn group （n=20）, transverse aortic constriction （TAC） group （n=40） and TAC+Cyn group （n=40）. The model of cardiac chronic pressure overload was induced by TAC, and the first day of TAC was defined as day 0. The animals in sham+Cyn group and TAC+Cyn group were treated with Cyn dissolved in DMSO and normal saline （5 mg·kg^-1·d^-1） for 5 d before TAC, while the animals in sham group and TAC group were treated with the same amount of DMSO and normal saline. Four weeks after TAC, the survival rate of the animals in each group was analyzed, the heart function of the mice was measured by ultrasound echocardiography, and the heart weight/body weight and lung weight/body weight were calculated. The cross-sectional area of the cardiomyocytes was measured by wheat germ agglutinin staining and hematoxylin-eosin staining. The degree of cardiac oxidative stress was evaluated by dihydroethidium staining and measurement of superoxide dismutase （SOD） and malondialdehyde （MDA） levels. The cardiomyocyte apoptosis was detected by TUNEL method. The mRNA expression levels of atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP） and β-myosin heavy chain （β-MHC） were detected by RT-qPCR, and the protein expression levels of Bax, Bcl-2, optic atrophy protein 1 （OPA1） and dynamin-related protein 1 （Drp1） were determined by Western blot. The mitochondrial morphological changes were observed by transmission electron microscopy. RESULTS Compared with TAC group, the survival rate of the mice in TAC+Cyn group was significantly increased （P<0.05）, the myocardial apoptosis, the cross-sectional area of myocardial cells, the heart weight/body weight, the lung weight/body weight, the level of reactive oxygen species and the MDA content were decreased （P<0.05）, and the SOD was activated （P<0.05）. M-mode ultrasound tests showed that Cyn treatment significantly increased left ventricular ejection fraction and left ventricular fractional shortening in the mice after TAC （P<0.05）, while left ventricular end-diastolic diameter and left ventricular posterior wall thickness in diastole were reduced （P<0.05）. Transmission electron microscopic observation showed that the number of myocardial mitochondria was increased and the mitochondrial area was decreased after TAC （P<0.05）, while treatment with Cyn increased the area of myocardial mitochondria and decreased the mitochondrial number （P<0.05）. Compared with sham group, the protein level of OPA1 in TAC group was significantly reduced （P<0.05）, while treatment with Cyn significantly increased the protein level of OPA1. CONCLUSION Cyanidin significantly increases the survival rate, improves the cardiac function and attenuates the cardiac remodeling of the mice after TAC. The mechanism may be related to the inhibition of myocardial mitochondrial OPA1 cleavage and the promotion of mitochondrial fusion.     

Metformin attenuates nerve injury in stroke rats by regulating SIRT1/PGC-1α signaling pathway via miR-29c

AIM To explore the inhibitory effect of metformin （MET） on nerve injury in rats with stroke and its mechanism. METHODS SD rats were randomly divided into sham group （n=15）, model group （n=30）, MET group （n=30）, MET+agomir-NC group （n=30） and MET+agomir group （n=30）. The modified Puisinelli four-vessel occlusion method was used to prepare the model of global ischemic stroke, while the blood vessels in sham rats were isolated without clamping the common artery. One week before modeling, the rats in MET group, MET+agomir-NC group and MET+agomir group were given intraperitoneal injection of 100 mg·kg^-1·d^-1 MET, 100 mg·kg^-1·d^-1 MET+40 nmol/d agomir-NC, 100 mg·kg^-1·d^-1 MET+40 nmol/d miR-29c agomir, respectively, and the rats in sham group and model group were given intraperitoneal injection of the same amount of normal saline. Each treatment in the above groups was given once a day, 0.2 mL each time, for 7 consecutive days. The neurological deficit scores were measured 24, 48 and 72 h after operation. HE staining was used to observe the morphological changes of the hippocampus, and the living neurons were counted. RT-qPCR was used to detect the expression level of miR-29c, and the mRNA levels of silent information regulator 1 （SIRT1） and peroxisome proliferator-activated receptor γ coactivator 1α （PGC-1α） in hippocampus. The protein expression levels of SIRT1 and PGC-1α were determined by Western blot. RESULTS At the same time point, compared with model group, the neurological deficit score in MET group was significantly decreased, and the survival rate of the neurons was significantly increased （P<0.05）. Compared with MET+agomir-NC group, the neurological deficit score in MET+agomir group was increased, and the survival rate of the neurons was significantly decreased （P<0.05）. With the prolongation of time, except for sham group, the neurological deficit score was increased and the survival rate of the neurons was decreased. At 72 h after operation, compared with sham group, the expression of miR-29c in hippocampus of model group was significantly increased, and the mRNA and protein expression levels of SIRT1 and PGC-1α were significantly decreased （P<0.05）. Compared with model group, the expression of miR-29c in hippocampus of MET group was significantly decreased, and the expression of SIRT1 and PGC-1α at mRNA and protein levels was significantly increased （P< 0.05）. Compared with MET+agomir-NC group, the expression of miR-29c in hippocampus of MET+agomir group was significantly increased, and the mRNA and protein expression of SIRT1 and PGC-1α was significantly decreased （P<0.05）. CONCLUSIONS MET alleviates nerve injury in stroke rats, which may be related to down-regulation of miR-29c and promotion of SIRT1/PGC-1α signaling pathway activation.     

Effects of Huanglian-jiedu decoction on TLR4/NF-κB signaling pathway and goblet cells of mucosa in rats with allergic rhinitis

AIM To investigate the effect of Huanglian-jiedu decoction （HLJD） on goblet cells and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB signaling pathway in allergic rhinitis rats. METHODS The rat model of allergic rhinitis was made by ovalbumin. The model rats were divided into model group, low-, medium- and high-dose HLJD groups, and positive control drug group. The rats in low-, medium- and high-dose HLJD groups were given different doses（5, 10 and 20 g/kg, respectively） of crude drug by intragastric administration, the rats in positive control group was given fluticasone propionate nasal spray （50 μg per side）, and the rats in control group and model group were given normal saline, once per day for 10 days. The behaviors were observed and scored after modeling and treatment, the weight of nasal secretion was measured after the treatment. The goblet cells in nasal mucosa were observed by periodic acid-Schiff （PAS） staining. The morphological changes of nasal mucosa were observed by hematoxylin-eosin （HE） staining. The interleukin （IL）-4 and IL-5 levels in nasal mucosa were measured by ELISA. The mRNA levels of mouse calcium-activated chloride channel 3 （mCLCA3） and mucin 5AC （MUC5AC） were detected by RT-qPCR. The protein expression of TLR4 and NF-κB p65 was determined by Western blot. RESULTS After modeling, compared with control group, the behavioral scores in model group, low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）. The eosinophils, neutrophils and lymphocytes in nasal mucosa were seriously infiltrated, inflammatory infiltration was obvious, and obvious small vessel dilatation and interstitial edema in model group were observed. With the increase in the dosage of HLJD, the lymphatic infiltration was obviously relieved, but the eosinophil and neutrophil infiltration still existed, and the inflammatory infiltration was relieved. Compared with control group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 and NF-κB p65 in the mucosa of model group were increased （P<0.05）. Compared with model group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 in the mucosa of low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）, and the protein expression of NF-κB p65 in the mucosa of medium- and high-dose HLJD groups and positive control group was decreased （P<0.05）. CONCLUSION Huanglian-jiedu decoction reduces the relative proportion of goblet cells in the nasal mucosa of rats with allergic rhinitis, which may be achieved by inhibiting TLR4/NF-κB signaling pathway.     

Curcumin attenuates rat lung ischemia/reperfusion injury by interfering with autophagy

AIM To investigate the role of curcumin （CUR） in lung ischemia/reperfusion （I/R） injury （LIRI） and its relationship with autophagy. METHODS 40 SD rats were randomly divided into sham operation （sham） group, I/R group, solvent （DMSO） group, CUR group and CUR+rapamycin （CUR-Rap） group. The rats were intraperitoneally injected with normal saline, DMSO, CUR or CUR+Rap before operation. After the rat LIRI model was established, the lung tissues were taken to measure W/D, TLW, IAR, and the contents of SOD and MDA were also measured to indicate the oxidative stress level. Light and electron microscopes were used to observed the morphology and ultrastrucure of lung tissues. The expression levels of autophagy-related proteins were determined by Western blot to evaluate autophagy levels. RESULTS Compared with sham group, wet weight/dry weight （W/D）, total lung water （TLW）, injured alveoli rate（IAR） and malondialdehyde （MDA） content in all other groups were increased, superoxide dismutase （SOD） activity was decreased, the levels of autophagy were increased （P<0.05）, and lung tissue injury and cell ultrastructural damage were aggravated in CUR group. Compared with DMSO group, W/D, TLW and IAR and MDA content were decreased, SOD activity was decreased, autophagy levels were also decreased （P<0.05）, and lung tissue and cell ultrastructural damage were attenuated. Compared with CUR group, W/D, TLW, IAR and MDA content were increased, SOD activity declined, the autophagy levels were increased （P<0.05）, and damage of lung tissues and cells were more serious in CUR-Rap group. CONCLUSION Curcumin attenuates the lung I/R injury in rats, and its mechanism may be related to the reduction of oxidative stress and the inhibition of autophagy.     

Effect of Tripterygium wilfordii multiglucoside on intestinal flora and immune function in IgA nephropathy rats based on C1GALT1/Cosmc pathway

AIM To investigate the effects of Triptergium wilfordii multiglucoside （TWM） on intestinal flora and immune function in IgA nephropathy （IgAN） rats based on core 1 β1,3-galactosyltransferase （C1GALT1） and its chaperone protein Cosmc （C1GALT1/Cosmc pathway）. METHODS The rat model of IgAN was established, and the animals were randomly divided into model group （IgAN group）, dexamethasone （Dex） group and TWM group. Normal rats served as normal control （NC） group. The levels of serum creatinine （SCr） and blood urea nitrogen （BUN）, 24-hour urinary total protein （24 h UTP） and the number of urinary red blood cells were measured by automatic biochemical analyzer. The levels of serum IgA1, and plasma tumor necrosis factor-α （TNF-α）, B-cell activating factor （Baff） and interleukin-17 （IL-17） were detected by ELISA. The level of galactose-deficient IgA1 （Gd-IgA1） was detected by Vicia villosa lectin affinity ELISA. The intestinal colony was cultured in selective bacterial medium. The ratio of CD4⁺ CD25⁺ regulatory T cells （Treg） to CD4⁺ T cells （Treg proportion） in peripheral blood mononuclear cells （PBMC） was detected by flow cytometry.Western blot was used to determine the protein expression of C1GALT1 and Cosmc in intestinal mucosa. RESULTS Compared with NC group, 24 h UTP, the number of urinary red blood cells, SCr, BUN, serum IgA1 and Gd-IgA1, the numbers of Enterobacteriaceae, Enterococcus and Bacteroides, and the levels of TNF-α, Baff and IL-17 in plasma in IgAN group were significantly increased （P<0.05）, while the numbers of Bifidobacteria and Lactobacilli, the Treg proportion in PBMC, and the protein expression levels of C1GALT1 and Cosmc in intestinal mucosa were significantly decreased （P<0.05）. Compared with IgAN group, 24 h UTP, the number of urinary red blood cells, SCr, BUN, serum IgA1 and Gd-IgA1, the numbers of Enterobacteriaceae, Enterococcus and Bacteroides, and the levels of TNF-α, Baff and IL-17 in plasma in Dex group and TWM group were significantly reduced （P<0.05）, and those in TWM group were lower than those in Dex group （P<0.05）. Moreover, the numbers of Bifidobacteria and Lactobacilli, the Treg proportion in PBMC, and the protein expression levels of C1GALT1 and Cosmc in intestinal mucosa were significantly elevated （P<0.05）, and those in TWM group were higher than those in Dex group （P<0.05）. CONCLUSION TWM reduces the abnormal glycosylation level of IgA in IgAN rats by promoting the activation of C1GALT1/Cosmc pathway, and attenuates the intestinal flora disorder and immune dysfunction in IgAN rats, thus exerting the therapeutic effect.     

Effect of Xiaozhongzhitong mixture on vascular regeneration of random flap in rats

AIM To observe the effect of Xiaozhongzhitong （detumescence and relieving pain） mixture on vascular regeneration and vascular endothelial growth factor （VEGF）-Dll4/Notch signaling pathway of random flap in rats. METHODS A total of 240 SD rats were randomly divided into 6 groups： blank group, sham group, model group, Xiaozhongzhitong mixture group （detumescence group）, Xiaozhongzhitong mixture+Notch blocker MK-0752 group （detumescence+MK group） and Xiaozhongzhitong mixture+VEGF receptor inhibitor axitinib group （detumescence+AXI group）. The capillary filling time, the number of new capillaries, the microvascular density, the microvascular diameter and the vascular survival area were observed by HE staining. The serum level of VEGF was measured by ELISA, and the mRNA expression of VEGFA, Notch and Dll4 in rat flap tissues was detected by RT-qPCR. RESULTS On the 10th day, compared with model group, the capillary filling time in detumescence group was decreased （P<0.05）. The capillary filling time in detumescence+AXI group was increased compared with detumescence group （P<0.05）. The flap tissue cells inmodel group were disordered and the nucleus was sparse, which showed obvious edema. Compared with model group, the number, diameter and area of microvessels, the serum content of VEGF, and the mRNA expression of VEGFA, Notch and Dll4 were significantly increased in detumescence group （P<0.05）. Compared with detumescence group, the number, density and area of microvessels, the serum content of VEGF, and the mRNA expression of VEGFA, notch and Dll4 was significantly decreased in detumescence+AXI group （P<0.05）. After the intervention with Notch blocker, the mRNA expression of VEGFA was increased, and the mRNA expression of Notch and Dll4 was inhibited （P<0.05）. CONCLUSION Xiaozhongzhitong mixture promotes the regeneration of blood vessels and enhances the survival rate of random flap, which may be related to the regulation of VEGF-Dll4/Notch signaling pathways.     

Quercetin activates mitochondrial autophagy via PINK1/parkin pathway to alleviate cerebral ischemia-reperfusion injury in rats

AIM To analyze the regulatory effect of quercetin （QUE） on PTEN-induced putative kinase 1 （PINK1）/parkin mitochondrial autophagy pathway, and to explore the mechanism of quercetin in relieving cerebral ischemia/reperfusion （I/R） injury. METHODS Sixty SD male rats were randomly divided into sham operation group, model group （I/R group）, QUE group,3-methyladenine （3-MA） group and QUE+3-MA group. Administration started in each group 3 days before modeling, once a day, at 30 min after the last administration,except sham group, the other groups used 4-vessel blockage method to establish the whole brain I/R model. On the day after modeling, the neural function was evaluated by neuropathy disability score （NDS）. The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride （TTC） staining. The morphological changes of mitochondria in hippocampus were observed by transmission electron microscopy. The contents of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in hippocampus were measured by ELISA. The activity of superoxide dismutase （SOD） and contents of malondialdehyde （MDA） in hippocampus were detected by xanthine oxidase method, thiobarbituric acid condensation method. Western blot was used to detect the proteinex pression of PINK1, parkin and LC3-II in brain tissue. RESULTS Compared with sham group, the hippocampus of the rats in I/R group and QUE+3-MA group showed swelling of mitochondria, destruction or disappearance of internal crista and other pathological damage,also the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA, the protein expression levels of PINK1, parkin and LC3-II were increased （P<0.05）, while NDS score and activity of SOD were decreased （P<0.05）. Compared with I/R group and QUE+3-MA group, the pathological damage degree of hippocampus in QUE group was reduced, the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA were decreased （P<0.05）, the proteinexpression levels of PINK1, parkin and LC3-II, and NDS score and activity of SOD were increased （P<0.05）.The above indexes in 3-MA group were opposite to QUE group. No significant difference in the above indexes between I/R group and QUE+3-MA group was observed （P>0.05）. CONCLUSION Quercetin activates mitochondrial autophagy and reduces cerebral I/R by regulating the expression of PINK1/parkin pathway proteins.     

High expression of BIRC5 enhances cell viability,inhibits apoptosis and is associated with poor prognosis in gastric cancer

AIM To investigate the expression of baculoviral inhibitor of apoptosis protein repeat-containing protein 5 （BIRC5） in gastric cancer tissue and its relationship with prognosis of gastric cancer patients, and to explore the effect of BIRC5 knock-down on the viability and apoptosis of gastric cancer cells. METHODS The expression of BIRC5 was detected by immunohistochemistry in 67 cases of gastric cancer tissues and paracancerous tissues for analyzing the relationships with clinicopathological characteristics. The mRNA and protein expression levels of BIRC5 in gastric carcinoma cell lines （AGS, MKN-1 and MGC-803） and normal gastric epithelial cell line GES-1 were detected by RT-qPCR and Western blot. The AGS cells were divided into blank group （no treatment）, Ctr-sh group （blank plasmid transfection） and BIRC5-sh group （BIRC5-shRNA plasmid transfection）. The interference efficiency of BIRC5-shRNA was evaluated by Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry, and the levels of apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. RESULTS BIRC5 was mainly expressed in cytoplasm, and the positive expression rate of BIRC5 in the gastric cancer tissues was higher than that in the adjacent tissues （P<0.01）. The positive rates of BIRC5 in the gastric cancer patients at TNM Ⅲ~Ⅳ stages and with lymph node metastasis were higher than those in the patients at TNM Ⅰ~Ⅱ stages and without lymph node metastasis, respectively （P<0.05）. The survival time of the patients with positive BIRC5 expression was shorter than that of the patients with negative BIRC5 expression （P=0.011 2）. The cell viability in BIRC5-sh group was lower than that in blank group and Ctr-sh group at time points of 48, 72 and 96 h. The apoptotic rate in BIRC5-sh group was increased compared with blank group and Ctr-sh group. The protein levels of cleaved caspase-3 and Bax in BIRC5-sh group were higher than those in blank group and Ctr-sh group, while the protein expression of Bcl-2 in BIRC5-sh group was lower than that in blank group and Ctr-sh group （P<0.05）. CONCLUSION High expression of BIRC5 in gastric cancer indicates poor prognosis. BIRC5 promotes the growth of gastric cancer cells and inhibits apoptosis.     

Effects of naringenin on PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways in rats with myocardial ischemia/reperfusion injury

AIM To observe the effect of naringenin on cardiac injury in ischemia/reperfusion （I/R） rats, and to explore whether the role of naringenin is involved in PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways. METHODS SD rats （n=48） were randomly divided into sham operation （sham） group, model （I/R） group, naringenin treatment （NAR） group and naringenin+LY294002 （NL） group. Myocardial I/R injury model was prepared by ligation of left anterior descending coronary artery of rats for 30 min followed by reperfusion for 120 min. After reperfusion, the serum levels of cardiac troponin I （cTnI） was measured by ELISA. HE staining, TTC staining and TUNEL staining were used to detect the myocardial histopathological changes, myocardial infarction area and myocardial cell apoptotic rate. The mRNA levels of endoplasmic reticulum stress-related indicators glucose-regulated protein 78 （GRP78）, C/EBP homologous protein （CHOP） and caspase-12 were detected by RT-qPCR. The protein levels of cleaved caspase-3, GRP78, CHOP, caspase-12, p-PI3K and p-AKT were determined by Western blot. RESULTS Compared with I/R group, the serum content of cTnI, myocardial pathological damage, myocardial infarction area and myocardial cell apoptotic rate were significantly reduced （P<0.05）, the protein levels of cleaved caspase-3, GRP78, CHOP and caspase-12 were decreased （P<0.05）, and the protein levels of p-PI3K and p-AKT were increased in NAR group （P<0.05）. LY294002 attenuated the protective effect of naringenin to some extent. CONCLUSION Naringenin reduces myocardial I/R injury in rats possibly by activating PI3K/AKT signaling pathway and subsequently regulating endoplasmic reticulum stress and its related apoptotic pathways.     

Role and potential mechanism of fecal microbiota transplantation in treatment of chronic hepatitis B

AIM To investigate the effect of fecal microbiota transplantation （FMT） on the treatment of chronic hepatitis B （CHB） and the potential mechanism. METHODS Fifty C57BL/6J mice （6~8 weeks old） were divided into 5 groups： control group, CHB group, entecavir （ETV） group, comprehensive treatment （ETV+FMT, EFMT） group, and blocker （TAK-242+ETV+FMT, EFMT-TAK） group. The mice in each group were given corresponding treatment. The general condition of the mice was observed daily, and fecal specimens were kept every 10 d. The mice were sacrificed after 12 weeks, and the liver tissues and blood samples were collected. HE staining was used for histological scoring. Serum hepatitis B surface antigen （HBsAg） and interleukin-18 （IL-18） levels were measured by ELISA. Toll-like receptor 4 （TLR4） expression was detected by flow cytometry. Intestinal flora diversity was analyzed by high-throughput sequencing. RESULTS （1） Compared with control group, the body weight of the mice in CHB group was significantly reduced （P<0.05）. The body weight loss of the mice in ETV group, EFMT group and EFMT-TAK group was reversed to some extent as compared with CHB group （P<0.05）. （2） The histological score of the mice in CHB group was significantly higher than that in control group （P<0.05）. The score in ETV group was lower than that in CHB group （P<0.05）. The scores in EFMT group and EFMT-TAK group were lower than that in ETV group （P<0.05）, and that in EFMT-TAK group had a further downward trend compared with EFMT group （P<0.05）. （3） Compared with control group, the serum level of HBsAg in the CHB mice was significantly increased （P<0.05） and decreased after ETV treatment （P<0.05）. The HBsAg level in both EFMT group and EFMT-TAK group was significantly lower than that in ETV group （P<0.05）. （4） The IL-18 level in CHB group was significantly higher than that in control group （P<0.05）. After ETV treatment, the IL-18 level was decreased （P<0.05）, and that in both EFMT group and EFMT-TAK group was decreased more than that in ETV group （P<0.05）. （5） TLR4 expression in CHB group was higher than that in control group （P<0.05）, that in ETV group was lower than CHB group （P<0.05）, and that in EFMT group was further decreased （P<0.05）. （6） The heat map analysis at the class level showed that the abundances of Gammaproteobacteria, Deltaproteobacteria and Negativicutes in CHB group were significantly higher than those in control group, and those of Deltaproteobacteria and Negativicutes in EFMT group were close to those in control group. The heat map analysis at the family level indicated that the abundances of Burkholderiaceae, Desulfovibrionaceae and Veillonellaceae in CHB group were significantly higher than those in control group, while those in ETV group and EFMT group gradually approached normal levels. The α diversity index in CHB group was significantly decreased, while the diversity in ETV group was increased, that in EFMT group was further increased, and that in EFMT-TAK group was the highest. CONCLUSION FMT plays an active role in the treatment of CHB. The mechanism may be related to reducing the level of IL-18 and improving the structure and diversity of intestinal flora. The TLR4 signaling pathway is involved.     

Effects of astragaloside on the levels of sex hormone and oxidative stress injury in rats with polycystic ovary syndrome

AIM To investigate the effects of astragaloside on the levels of sex hormone and oxidative stress in rats with polycystic ovary syndrome （PCOS）. METHODS Female SD rats （n=60） were randomly divided into normal control group, model group, Diane-35 （0.339 2 mg/kg） group, low dose astragaloside （12.5 mg/kg） group and high dose astragaloside （50 mg/kg） group, with 12 rats in each group. The PCOS model was induced by letrozole （1 mg/kg）, which was administered by gavage once a day for 3 weeks. After administration, the estrus cycle of the rats was observed by vaginal smear, and the ovarian index was calculated. HE staining was used to observe the histopathological changes of the ovaries. Serum levels of the sex hormones testosterone （T）, estradiol （E₂）, luteinizing hormone （LH） and follicle-stimulating hormone （FSH） were measured by ELISA. The levels of superoxide dismutase （SOD）, malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） in serum and ovarian tissue were detected by colorimetry, and the protein levels of steroidogenetic acute regulatory protein （StAR） and apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 in ovarian tissue were detected by Western blot. RESULT Compared with control group, the oestrous cycle of the rats in model group was disorder, and the ovarian index was increased, ovary was polycystic. The serum levels of T, LH and MDAwere significantly increased （P<0.05）, while the contents of E2, FSH and the activities of GSH-Px and SOD were significantly decreased （P<0.05）. The levels of MDA, StAR, cleaved caspase-3 and Bax proteins in ovarian tissue were significantly up-regulated （P<0.05）. GSH-Px and SOD activities and Bcl-2 protein levels were significantly down-regulated （P<0.05）. CONCLUSION Astragalosideeffectively balances the levels of sex hormone in PCOS rats and relieves the oxidative stress injury, the mechanism may be related to the inhibition of StAR expression.     

Destruction of autophagy marker LC3 rhythm is involved in β1-AA-induced H9c2 rat cardiomyocyte death

AIM To investigate the effect of β₁-adrenergic receptor autoantibodies （β₁-AA） on the rhythm of autophagy marker microtubule-associated protein 1 light chain 3 （LC3）, and the underlying mechanism of cardiomyocyte death. METHODS The test materials were Sprague-Dawley （SD） rats and H9c2 rat cardiomyocytes. The SD rats were randomly divided into immunization group and control group with 6 rats in each group. The H9c2 cells were randomly divided into control group, β₁-AA group, lentivirus （LV）-NC group, and LV-shPer2 group （n=6）. Affinity chromatography was used for purification of β₁-AA from rat serum. CCK-8 assay was used to observe the viability of cardiomyocytes treated with β₁-AA for 24 h. The cells were synchronized by dexamethasone and then treated with β₁-AA. The mRNA and protein levels of LC3 at different time points were determined by real-time PCR and Western blot, respectively. The Per2 protein level at different time points was also determined by by Western blot. JTK_CYCLE algorithm was used to estimate the circadian rhythm parameters. After destruction of LC3 circadian rhythm via LV-shPer2, CCK-8 assay was used to measure the viability of H9c2 cells. RESULTS High level of β₁-AA in rat serum was found after active immunization compared with control group （P<0.05）. The viability of H9c2 cells in β₁-AA group was significantly lower than that in control group （P<0.05）. The LC3 and Per2 rhythms were both disrupted in H9c2 cells induced by β₁-AA （JTK_CYCLE P<0.05）. After LV-shPer2 infection, the LC3 rhythm was disrupted （JTK_CYCLE P<0.05） and the cell viability was reduced （P<0.05）. CONCLUSION β₁-AA may induce the destruction of autophagy marker LC3 rhythm in rat cardiomyocytes and then promote cell death.     

菜薹BrNAP克隆及其在采后叶片衰老中的功能分析

在菜薹(Brassica rapa var. parachinensis)中克隆Br NAP1并分析其功能。Br NAP1编码区全长813 bp,编码270个氨基酸,具有NAP转录因子特有的保守结构域,属于NAP亚家族成员。Br NAP1表达量与叶片衰老程度呈正相关且受ABA诱导表达上调。亚细胞定位试验表明Br NAP1定位于细胞核。互补试验显示Br NAP1能使拟南芥atnap滞绿表型回复至野生型,过表达则能引起采后叶片早衰。双荧光素酶试验表明Br NAP1能够激活Br SAG113表达。这些说明Br NAP1是菜薹采后叶片衰老的正调控基因。     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Geniposide attenuates cognitive dysfunction in sleep-deprived rats by inhibiting TLR4/NF-κB signaling pathway

AIM To investigate the effects of geniposide （Gen） on Toll like receptor 4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and cognitive dysfunction in sleep deprived rats. METHODS Wistar rats （n=120） were randomly divided into normal control （NC） group, model （M） group, low-dose （5 g·kg^-1·d^-1） Gen （Gen-L） group, medium-dose （10 g·kg^-1·d^-1） Gen （Gen-M） group, high-dose （20 g·kg^-1·d^-1） Gen （Gen-H） group and Gen-H+LPS （0.4 mg·kg^-1·d^-1, tail vein injection） group. After 7 days of intervention, the sleep deprivation model of rats in M group, Gen-L, Gen-M, Gen-H and Gen-H+LPS group was established by improved small platform water environment. The escape latency of Morris water maze experiment and the behavior correct rate of Y maze experiment were measured. The serum levels of S100B and neuron-specific enolase （NSE）, and the levels of interleukin-1β （IL-1β）, IL-6 and tumor necrosis factor-α （TNF-α） in hippocampus were detected by ELISA. The mRNA levels of TLR4 and NF-κB p65 were detected by RT-qPCR, and the protein levels of TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with NC group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the mRNA and protein expression of TLR4 and NF-κB p65 were increased significantly in M group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. Compared with M group, the escape latency, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were decreased significantly in Gen-L, Gen-M and Gen-H groups （P<0.01）, and the behavior correct rate was increased in turn （P<0.01）. Compared with Gen-H group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were increased significantly in Gen-H+LPS group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. CONCLUSION Geniposide may inhibit the TLR4/NF-κB p65 signaling pathway to effectively improve cognitive function in sleep-deprived rats and reduce hippocampus inflammation.     

Isolation of exosomes and effect of stellate ganglion block on the number of exosomes in post-hemorrhagic shock mesenteric lymph of rats

AIM To isolate the exosomes in mesenteric lymph, verify the source of exosomes, and observe the effect of stellate ganglion block （SGB） on the number of exosomes in post-hemorrhagic shock mesenteric lymph （PHSML） of rats. METHODS Twenty-four male rats were randomly divided into sham, sham+SGB, shock, and shock+SGB groups. SGB was performed before the establishment of hemorrhagic shock model using the routine methods in our lab. The PHSML was drained for exosomes isolation. The exosomes were identified through particle size analysis and CD63 protein expression. The expression of epithelial cell adhesion molecule （EpCAM） was detected to identify whether the exosomes were derived from epithelial cell. The number of exosomes in various mesenteric lymphs was measured using the flow cytometry. RESULTS The diameter of granular material extracted from mesenteric lymph was about 100 nm. The positive expression of exosomes pecific protein CD63 indicated the successful isolation of exosomes, and the EpCAM expression verified the exosomes were derived from intestinal epithelial cells. The number of exosomes in mesenteric lymph isolated from the rats of Shock group was obviously increased compared to that from the Sham group （P<0.05）, while the exosomes from the Shock+SGB group was markedly decreased when compared to Shock group （P<0.05）. CONCLUSION The current study establishes the isolation technique of exosomes in mesenteric lymph, and proved the exosomes were derived from the intestinal epithelial cells. SGB treatment reduces the number of exosomes in PHSML.     

Effects of EGCG on GLUT2/G6PD/GS expression and blood glucose level in type 2 diabetic rats

AIM To investigate whether epigallocatechin gallate （EGCG） improves blood glucose in type 2 diabetic rats through glucose transporter 2 （GLUT2）-glucose-6-phosphate dehydrogenase （G6PD）-glycogen synthase （GS） pathway. METHODS Type 2 diabetes mellitus （T2DM） model was established in male Sprague-Dawley （SD） rats by feeding with high-fat diet and injection of streptozotocin （STZ）. The rats were divided into 5 groups （n=10）： control （Con） group, T2DM model （M） group, metformin （Met; 200 mg/kg, ig） group, T2DM+low-dose （50 mg/kg, ig） EGCG （EL） group, and T2DM+high-dose （100 mg/kg, ig） EGCG （EH） group. Diabetic rats were given drugs for 8 weeks. After 8 weeks of administration, the rats were killed, and the blood and liver tissues were collected. The levels of fasting blood glucose （FBG）, fasting serum insulin （FINS） and serum glycosylated hemoglobin were measured by biochemical tests. Liver glycogen were test by periodic acid-Schiff （PAS） staining. The mRNA expression of G6PD in the liver was detected by real-time PCR. The protein levels of GS and GLUT2 were determined by Western blot and immunohistochemistry. RESULTS T2DM rat model was established successfully. Compared with Con group, the levels of FBG, FINS and serum glycosylated hemoglobin in M group were increased significantly （P<0.05）, while the insulin sensitivity index （ISI）, the liver glycogen, the G6PD mRNA expression, and the protein levels of GS and GLUT2 were decreased significantly （P<0.05）. Compared with M group, the levels of FBG and serum glycosylated hemoglobin in Met group and EH group were decreased significantly （P<0.05）, while the ISI, the liver glycogen, the G6PD mRNA expression, and the protein levels of GS and GLUT2 were increased significantly （P<0.05）. CONCLUSION EGCG reduces the blood glucose level in T2DM rats, which may be related to the regulation of GLUT2-G6PD-GS signaling pathway.     

miR-92b-5p attenuates renal injury and inflammatory response in diabetic nephropathy rats by targeting HMGB1

AIM To investigate the effect of microRNA-92b-5p （miR-92b-5p） on renal injury and inflammatory response in diabetic nephropathy （DN） rats and its mechanism. METHODS The rats were divided into control group， DN group， lentiviral negative control （LV-NC） group， LV-miR-92b group， LV-high mobility group protein B1 （LV-HMGB1） group and miR-92b+HMGB1 group， with 15 rats in each group. After fasting for 12 h， the model rats were intraperitoneally injected with streptozotocin at dose of 60 mg/kg， and the control rats were intraperitoneally injected with an equal volume of citrate buffer. Three days later， the rats in each treatment group were intravenously injected with 100 μL LV-NC， LV-miR-92b and LV-HMGB1 （1×10¹¹ U/L） twice a week for 8 consecutive weeks. Urinary protein， blood glucose， blood urea nitrogen and serum creatinine were detected by an automatic biochemical analyzer. The expression of miR-92b-5p and HMGB1 mRNA was detected by RT-qPCR. The targeting relationship between miR-92b-5p and HMGB1 was verified by dual-luciferase reporter assay. HMGB1 expression in kidney tissue was detected by Western blot. The kidney damage was observed by HE staining. The apoptosis was detected by flow cytometry. The levels of interleukin-6 （IL-6）， IL-1β and tumor necrosis factor-α （TNF-α） in renal tissues were detected by ELISA. RESULTS In DN model rats， miR-92b-5p was down-regulated， while HMGB1 was highly expressed. There was a binding site between miR-92b-5p and HMGB1 3'-untranslated region. High expression of miR-92b-5p inhibited the luciferase activity of the wild-type HMGB1 plasmid （P<0.01）， but had no effect on the luciferase activity of the mutant HMGB1 plasmid. Compared with DN group， urinary protein， blood glucose， serum creatinine and blood urea nitrogen in LV-miR-92b group were significantly reduced （P<0.01）. The degree of hyperplasia， swelling and inflammatory cell infiltration of glomerular mesangium and basement membrane tubules， the apoptosis rate of renal tissues， and the content of IL-6， IL-1β and TNF-α in renal tissues were significantly decreased （P<0.01）. Co-transfection of LV-HMGB1 significantly reversed the effect of miR-92b-5p on DN rats. CONCLUSION miR-92b-5p reduces renal injury and inflammatory response in DN rats by targeting HMGB1 and down-regulating its expression.