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 intermittent hypoxia on bladder detrusor cell apoptosis and regulatory mechanism of Alpiniae oxyphyllae Fructus

AIM To investigate the effect of intermittent hypoxia （IH） on bladder detrusor cells apoptosis and calcium channel, and to discuss the regulatory mechanism of Alpiniae oxyphyllae Fructus （AOF）. METHODS IH model of bladder detrusor cells was established by treating the cells with 6 cycles of 5% O₂ for 60 min and 20% O₂ for 30 min. Human bladder detrusor cells were cultured in vitro, randomly divided into 6 groups, each group had 8 holes. P₂X₃ receptor antagonist + IH （A） group, M₃ receptor antagonist + IH （B） group, β₃ receptor antagonist + IH （C） group, AOF + IH （D） group, saline + IH control （NC） group and air simulation control （AC） group were set up. The cells density and morphology were identified by the methods of counting chamber and immunofluorescence light microscopy （LM） after interventions. The apoptosis was analyzed by flow cytometry. Calcium channel expression was detected by patch clamp. RESULTS （1） Compared with the cells in AC group, the cells density and activity were significantly increased in NC group （P<0.05）; some cells appeared protrusions, turned round and blur in cell borders. （2） The results of immunofluorescence for detecting α-SMA protein expression showed that, compared with the cells in group AC, the mean absorbance （MA） in group NC was significantly increased （F=3.25, P<0.05）; compared with the cells in group NC, that in group A and group D was both decreased significantly （P<0.05）. （3） Compared with the cells in group AC, the apoptotic rate was significantly decreased in group NC （P<0.05）; Compared with the cells in group NC, the apoptotic rates in group A and group D were both significantly increased （P>0.05）. （4） Compared with the cells in group AC, calcium ion channel expression was significantly decreased （P<0.05）. Compared with the cells in group NC, calcium ion channel expression in AOF （100 mg/L） and AOF （50 mg/L） group was significantly increased （P<0.05）. CONCLUSION IH regulates bladder detrusor cells proliferation and apoptosis through P₂X₃ bladder nerve receptors, high or moderate dose of AOF may change calcium channel and play a protective role in IH induced cell damage.     

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.     

Relationships between lncRNA HOTAIR/H19 SNPs and genetic susceptibility to gastric carcinoma/EBV-related gastric carcinoma

AIM To investigate the potential associations between the single nucleotide polymorphisms （SNPs） of long noncoding RNA （lncRNA） H19/HOTAIR and the susceptibility to gastric carcinoma, especially to Epstein-Barr virus （EBV）-associated gastric carcinoma （EBVaGC）. METHODS Peripheral blood samples from 65 cases of EBV-negative gastric carcinoma （EBVnGC）, 50 cases of EBVaGC and 115 cases of healthy people were collected. A total of 4 TagSNPs, H19 rs3024270 and rs3741219, as well as HOTAIR rs4759314 and rs874945, were selected. The Taq-Man MGB allele typing kit was used to detect the genotype of each SNP locus, and the experimental results were statistically analyzed. RESULTS （1） There were significant differences of both genotypic and allelic frequencies at H19 rs3024270 locus between gastric carcinoma group and control group （P<0.05）. Individuals carrying the G allele at H19 rs3024270 locus had significantly low risk of gastric carcinoma （P<0.01）, indicating that the G allele was protective. （2） People with the GG genotype at HOTAIR rs4759314 locus had significantly high risk of gastric carcinoma （P<0.05）. Carrying the G allele increased the risk of gastric carcinoma, which indicated that the risk gene for gastric carcinoma might be the G allele. （3） No significant difference of the genotypic and allelic frequencies at H19 rs3741219 and HOTAIR rs874945 loci between gastric carcinoma group and control group was observed （P>0.05）.（4） The G allele frequency at HOTAIR rs4759314 locus in EBVaGC group was significantly higher than that in EBVnGC group. However, no difference of the other 3 SNPs was found between EBVaGC group and EBVnGC group （P>0.05）. CONCLUSION The SNPs at H19 rs3024270 and HOTAIR rs4759314 loci are related to the risk of gastric carcinoma, but not significantly related to the risk of EBVaGC.     

Inhibitory effect of Wendan decoction on atherosclerotic plaque formation in ApoE-/- mice by regulating expression of membrane proteins involved in reverse cholesterol transport

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on reverse cholesterol transport. METHODS Eight-week-old apolipoprotein E gene knockout （ApoE^-/-） mice with high-fat diet and daily drug gavage were randomly divided into model group, simvastatin group, and low-, middle- and high-dose Wendan decoction groups, with 15 mice in each group. The C57BL/6 mice of the same age served as control group. The mice were weighed once every week. After 10 weeks, the mice were anesthetized with chloral hydrate. The serum were collected for lipid level examination. The atherosclerotic plaque buildup in aortic root and whole aorta was observed by HE staining and oil red O staining, respectively. The levels of proteins related to cholesterol transport, ATP-binding cassette transporter A1 （ABCA1） and caveolin-1 in the aorta, and scavenger receptor class B type I （SR-BI） and CD36 in the liver, were quantified by Western blot. RESULTS Wendan decoction at middle dose inhibited the increase in the body weight of ApoE^-/- mice fed with high-fat diet （P<0.05）. Wendan decoction at different doses significantly reduced the serum levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the ApoE^-/- mice （P<0.05 or P<0.01）, but had no effect on serum high-density lipoprotein cholesterol level （P>0.05）. Wendan decoction at different doses inhibited the formation of atherosclerotic plaques in whole aorta of the ApoE^-/- mice （P<0.05 or P<0.01）. Middle- and high-dose Wendan decoction significantly inhibited the formation of atherosclerotic plaques in the aortic root （P<0.05）. Bedsides, Wendan decoction at different doses increased the protein level of ABCA1 and decreased the protein level of caveolin-1 in the aorta of the ApoE^-/- mice （P<0.01）. Middle- and high-dose Wendan decoction increased the liver protein level of SR-BI in the ApoE^-/- mice （P<0.01）. However, Wendan decoction at different doses had no effect on the liver protein level of CD36 in the ApoE^-/- mice （P>0.05）. CONCLUSION Wendan decoction reduces the body weight, serum lipid levels and formation of atherosclerotic plaques in ApoE^-/- mice fed with high-fat diet, and its mechanism is related to up-regulation of ABCA1 protein level in the aorta and SR-BI protein level in the liver as well as down-regulation of caveolin-1 protein level in the aorta.     

Expression of histone chaperone ASF1B in prostate cancer cells and its effect on cell viability in vitro

AIM To investigate the expression of histone chaperone anti-silencing function 1B （ASF1B） in prostate cancer cells and its effect on cell viability in vitro. METHODS Human prostate cancer PC-3 cells were used, and knockdown of ASF1B was conducted by small interfering RNA （siRNA） transfection into the cells. The cells were divided into control group, siRNA negative control vector （mock） group and siRNA-ASF1B group. The viability of the PC-3 cells treated with ASF1B-siRNA for 12, 24 and 48 h was measured by MTT assay. Flow cytometry was used to analyze cell apoptosis and cell cycle distribution. The mRNA expression of apoptosis-related molecules was detected by RT-qPCR, and the expression levels of MAPK/JNK/ERK signaling pathway-related proteins were determined by Western blot. RESULTS The protein level of ASF1B in the normal cells （benign prostatic hyperplasia） was significantly lower than that in the PC-3 cells （P<0.01）. Compared with control group and mock group, the protein expression level of ASF1B in the PC-3 cells transfected with siRNA-ASF1B plasmid and the viability of the PC-3 cells were significantly decreased （P<0.01）. The apoptotic rate of the PC-3 cells transfected with siRNA-ASF1B plasmid was significantly higher than that in control group （P<0.01）, and the cell cycle was arrested at G₁ phase. The mRNA levels of p53, caspase-3, Bax and PARP-1 in the PC-3 cells transfected with siRNA-ASF1B plasmid were up-regulated compared with those in control group and Mack group （P<0.01）. In addition, the protein levels of MAP2K4 and p-JNK in the PC-3 cells in siRNA-ASF1B group were significantly higher than those in mock group （P<0.01）, while the protein level of p-ERK was significantly lower than that in mock group （P<0.01）. CONCLUSION ASF1B silencing induces G₁ arrest and promotes apoptosis of PC-3 cells. Activating MAPK/JNK/ERK signaling pathway may be a possible contributor to the anti-prostate cancer effect of siRNA-ASF1B.     

Dasatinib inhibits the viability and migration of renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation in vitro

AIM To explore the effect of dasatinib on the viability, apoptosis and migration of human renal carcinoma cell lines 786-O and 769-P, as well as the molecular mechanism in vitro. METHODS 786-O cells and 769-P cells were treated with different concentrations （0~2 μmol/L） of dasatinib, and 0 μmol/L dasatinib was used as blank control group. MTT method was used to detect cell viability. Wound healing assay was used to detect the effect of dasatinib on migration. Hoechst 33258 staining was used to observe the effect of dasatinib on apoptosis. Flow cytometry was used to detect the effect of dasatinib on cell cycle. Western blot method was used to detected cell cycle- and apoptosis-related protein levels. RESULTS Dasatinib inhibited viability and migration of 786-O and 769-P cells, and the inhibitory effect of dasatinib increased with the concentration of dasatinib （P<0.05）. The IC₅₀ values of dasatinib against 786-O and 769-P cell lines were （0.958 7±0.028 8） μmol/L and （0.784 3±0.066 0） μmol/L, respectively. After treatment with dasatinib for 24 h, the expression of pro-apoptotic proteins cleaved caspase-3 and cleaved caspase-9 increased significantly （P<0.01）, while the expression of cyclin D1 decreased （P<0.05）. The cycle-related pathway proteins p53 and p21 increased （P<0.05）, while the level of p-AKT was decreased （P<0.05）. CONCLUSION Dasatinib impaired the viability and migration ability of human renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation.     

CYP450 epoxygenase/EET pathway attenuates adipose inflammation of obese mice through HIF-1α

AIM To investigate the effects of cytochrome P450 （CYP450） epoxygenase/epoxyeicosatrienoic acid （EET） pathway on insulin resistance in obese mice, and to explore the possible mechanisms. METHODS High-fat diet-induced obesity model was established in C57BL/6Cnc mice, and the obese mice were randomly divided into 3 groups, including obesity group （treated with saline; n=10）, EET group （treated with 11,12-EET; n=10） and EET inhibitor 14,15-epoxyeicosa-5（Z）-enoic acid （EEZE） group （n=10）. Normal C57BL/6Cnc mice （n=10） treated with saline served as control. Protein expression of CYP2J2 （one of CYP450 epoxygenases） and hypoxia-inducible factor-1α （HIF-1α） was measured by Western blot. Vessel-like structure was detected by immunofluorescence staining. The serum levels of insulin, tumor necrosis factor-α （TNF-α）, interleukin （IL）-1β, IL-6 and monocyte chemoattractant protein-1 （MCP-1） were measured by ELISA. RESULTS In obese mice, homeostasis model assessment of insulin resistance （HOMA-IR） values were increased, the protein level of CYP2J2 was reduced, and the protein level of HIF-1α was increased in adipose tissues as compared with the controls （P<0.05）. The serum levels of MCP-1, IL-1β, IL-6 and TNF-α were also significantly increased in obese mice （P<0.05）. After treatment with 11, 12-EET, the HOMA-IR values were decreased compared with vehicle-treated obese mice, HIF-1α expression levels were decreased in the adipose tissue, and the serum levels of MCP-1, IL-1β, IL-6 and TNF-α were reduced （P<0.05）. Immunohistochemical results of adipose tissue from vehicle-treated obese mice showed a marked decrease in vessel-like structures （CD31-positive） compared with normal control mice （P<0.05）. EET treatment significantly increased the newly formed vessel-like structures in the visceral adipose tissues of obese mice as compared with vehicle-treated obese mice （P<0.05）. CONCLUSION High-fat diet-induced obesity and insulin resistance are closely related to the CYP450 pathway. Exogenous EETs effectively decrease obesity-induced insulin resistance possibly through pro-angiogenesis and attenuation of hypoxia and inflammation.     

Curcumin inhibits Porphyromonas gingivalis LPS-induced inflammatory response in human gingival fibroblasts by up-regulating miR-124

AIM To investigate the effects of curcumin （Cur） on the inflammatory response of human gingival fibroblasts （HGFs） induced by Porphyromonas gingivalis （Pg） lipopolysaccharide （LPS） and the role of microRNA-124 （miR-124） in this process. METHODS The HGFs were divided into control group, LPS group （10 mg/L LPS） and LPS+Cur （20, 40 and 80 μmol/L） groups （10 mg/L LPS+corresponding dose of Cur）. After treatment for 24 h, CCK-8 assay was used to measure the cell viability. ELISA was used to measure the levels of interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the supernatant. The level of miR-124 in the cells was detected by RT-qPCR. The protein levels of nuclear factor kappa B （NF-κB） p-p65 in cytoplasm and nucleus were determined by Western blot, and the nuclear translocation of NF-κB p-p65 was evaluated by laser confocal microscopy. After transfection with mimic-NC or miR-124 mimic, the expression of miR-124 and NF-κB p-p65 protein in the cytoplasm and nucleus of the cells were also detected. RESULTS The cell viability, the level of miR-124 in the cells and NF-κB p-p65 protein level in cytoplasm of LPS group were lower than those in control group （P<0.05）, while the levels of IL-1β and TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were higher than those in control group （P<0.05）. The cell viability, the level of miR-124 in cells and NF-κB p-p65 protein level in the cytoplasm of LPS+Cur （40 and 80 μmol/L） groups were higher than those in LPS group （P<0.05）, while the level of TNF-α in the supernatant and NF-κB p-p65 protein level in the nucleus were lower than those in LPS group （P<0.05）. The level of IL-1β in the supernatant of LPS+80 μmol/L Cur group was lower than that in LPS group （P<0.05）. The levels of miR-124 and NF-κB p-p65 protein level in the cytoplasm of miR-124 mimic group were higher than those in LPS group and mimic-NC group （P<0.05）, while the level of NF-κB p-p65 proteinlevel in the nucleus was lower than that in LPS group and mimic-NC group （P<0.05）. CONCLUSION Curcumin inhibits the inflammatory response of HGFs induced by Pg LPS, which may be achieved by up-regulating miR-124 and then inhibiting the nuclear translocation of NF-κB p-p65.     

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.     

Effects of different active constituents of Gynostemma pentaphyllum on mitochondrial energy metabolism-related proteins in endothelial cells induced by ox-LDL

AIM To investigate the effects of different components of Gynostemma pentaphyllum ［gypenosides (Gps), gypenoside XLIX (GpXLIX) and ginsenoside Rb3 (GRb3)］ on mitochondrial energy metabolism-related proteins in endothelial cells induced by oxidized low-density lipoprotein (ox-LDL). METHODS EA.hy926 cells were divided into control group, model group, Gps group, GpXLIX group and GRb3 group. The cells in control group were cultured only in DMEM complete medium. The cells in model group were treated with 100 mg/L ox-LDL for 48 h. The cells in Gps group, GpXLIX group and GRb3 group were treated with 100 mg/L ox-LDL for 24 h, and then treated with Gps, GpXLIX and GRb3 at 100 mg/L for another 24 h, respectively. The ATP content in each group was detected by ELISA. The expression levels of mitochondrial energy metabolism-related proteins, cytochrome C oxidase subunit 5a （Cox5a）, NADH:ubiquinone oxidoreductase core subunit S1 （Ndufs1）, ATP synthase F1 subunit alpha (ATP5a) and cytochrome C (Cyt C）, were determined by Wes automatic Western blot quantitative analysis system and Western blot. RESULTS Compared with control group, the ATP content in model group was decreased (P<0.01). After drug intervention, the ATP content increased to different degrees in Gps group, GpXLIX group and GRb3 group (P<0.01). The results of Wes automatic Western blot quantitative analysis system were consistent with those of Western blot. These results showed that compared with control group, the protein expression of Cox5a, Ndufs1 and ATP5a in model group was decreased, and the protein expression of Cyt C was increased (P<0.01). After intervention, the protein expression of Cox5a, Ndufs1 and ATP5a was increased and the protein expression of Cyt C was decreased in Gps group, GpXLIX group and GRb3 group (P<0.05 or P<0.01). Among them, the effect of Gps on the protein expression of Cox5a, Ndufs1 and Cyt C was significantly stronger than those of the 2 monomer components, and the effect of GRb3 was found to be superior in the 2 monomer components. The effect of GpXLIX on ATP5a protein was superior to the other 2 components. CONCLUSION Gynostemma total saponins and related active ingredients protect ox-LDL-induced endothelial cells by affecting mitochondrial energy metabolism-related proteins, thereby preventing and treating atherosclerosis.     

Comparison of isolation and primary culture in vitro between human ovarian mural granulosa cells and cumulus cells

AIM To establish a suitable cell model for the study of ovarian function through comparing the isolation and primary culture effect of human ovarian mural granulosa cells （MGCs） and cumulus cells （CCs）. METHODS The follicular fluid of 16 patients who underwent assisted reproductive technique and their cumulus oocyte complexes （n=223） were collected. Density gradient centrifugation was used to isolate the MGCs and the methods of mechanical cutting plus enzyme hydrolysis were used to isolate the CCs. The cell counts and survival rates were analyzed by trypan blue staining and the expression of follicle stimulating hormone receptor （FSHR） was analyzed by flow cytometry to identify the purity. The expression of microtubule-associated protein 1 light chain 3 （LC3）, P62 and Bax at mRNA and protein levels was determined by qPCR and Western blot, respectively. RESULTS There had less isolation time, higher survival rate （P<0.05） and better tractility in vitro of CCs compared with MGCs. The results of flow cytometry showed that the FSHR expression of CCs and MGCs after isolation was （92.23±2.66）% and （81.33±6.57）%, respectively, with significant differences （P<0.05）. The mRNA level of LC3 in CCs was significantly lower than that in MGCs （P<0.01）, and the mRNA level of Bax was significantly higher than that in MGCs （P<0.05）. There was no significant difference in P62 mRNA expression between CCS and MGCs（P>0.05）. The difference of protein expressions of these molecules in the 2 kinds of cells were consistent with that in mRNA. CONCLUSION Mechanical cutting method plus enzyme hydrolysis is a simple way to isolate the CCs, with high purity and good cellular state in vitro, which can be used as a cell model for ovarian function research.     

Effects of oxidative stress and inflammatory response on kidney injury in mice with hyperthyroidism

AIM To explore the effects of oxidative stress and inflammatory response on kidney injury induced by hyperthyroidism in mice. METHODS Forty male Kunming mice were randomly divided into control group （n=20） and L-thyroxine （T4） group （n=20）. The mice in T4 group were intraperitoneally injected with T4 diluent at a dose of 1 mg/kg to induce hyperthyroidism, and those in control group were injected with normal saline of the same volume. After 7 weeks, the mice were weighed and dissected, the kidneys were removed and weighed, and the length of tibia was also measured. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the kidney tissues were detected. The pathological changes of the kidney tissues were observed by HE staining. The levels of 4-hydroxynonenal （4-HNE）-modified proteins, interleukin-1 receptor-associated kinase 1 （IRAK1） and tumor necrosis factor receptor-related factor 6 （TRAF6） were determined by Western blot and immunohistochemistry. RESULTS Compared with control group, the body weight of the mice was decreased, while the kidney size and weight were increased significantly in T4 group. In addition, the ratios of kidney weight/body weight and kidney weight/tibia length were also increased （P<0.05）. In T4 group, the renal tubules were enlarged, and the epithelial cells of renal tubules were swollen and exfoliated, with vacuolar degeneration. Furthermore, reduced SOD activity, and increased MDA content and 4-HNE-modified proteins were found in T4 group, all of which were related to oxidative stress （P<0.05）. The levels of inflammation-related proteins IRAK1 and TRAF6 were significantly increased in T4 group （P<0.05）. CONCLUSION Excessive T4 may lead to kidney hypertrophy and injury in mice, and the mechanism may be related to oxidative stress and inflammatory response.     

Effect of compound of Epimedium, Astragalus and Radix Puerariae on expression of ADAM10 in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin expression knock-down

AIM To explore the effect of compound of Epimedium, Astragalus and Radix Puerariae on the expression of a disintegrin and metalloproteinase 10 （ADAM10） in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin （HAMP） expression knock-down for analyzing the pathogenesis of Alzheimer disease （AD） at cell level. METHODS Hippocampal neuron HT22 cells were cultured in vitro and randomly divided into 7 groups： control group, Aβ group （Aβ_25-35-induced HT22 cells）, RNAi group （HAMP gene was silenced in HT22 cells）, Aβ+RNAi group （HAMP gene expression in Aβ_25-35-induced HT22 cells was silenced）, Aβ+TCM group （Aβ_25-35-induced HT22 cells were treated with Epimedium, Astragalus root and Radix Puerariae effective components）, RNAi+TCM group （HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components） and Aβ+RNAi+TCM group （Aβ_25-35-induced HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components）. The silence efficiency of HAMP siRNA was detected by qPCR and Western blot. The ADAM10 expression in each group was determined by immunofluorescence, qPCR and Western blot. RESULTS The HAMP siRNA-3 sequence had the highest interference efficiency. Compared with control group, the expression levels of ADAM10 in Aβ group, RNAi group and Aβ+RNAi group were decreased （P<0.05）. Compared with Aβ group,the expression levels of ADAM10 in Aβ+RNAi group was also decreased （P<0.05）, and the expression levels of ADAM10 in Aβ+TCM group was increased （P<0.05）. Compared with RNAi group, the expression levels of ADAM10 in Aβ+RNAi group was decreased （P<0.05）, while the expression levels of ADAM10 in RNAi+TCM group was increased （P<0.05）. Compared with Aβ+RNAi group, the expression levels of ADAM10 in Aβ+RNAi+TCM group was increased （P<0.05）. CONCLUSION The effective components of Epimedium, Astragalus and Radix Puerariae compound promotes the expression of ADAM10 in Aβ_25-35-induced HT22 cells, which mechanism may be related to the expression of HAMP.     

SUMO-specific protease 3 aggravates CaPO4-induced mouse abdominal aortic aneurysm formation by promoting macrophage polarization

AIMTo investigate the role of SUMO-specific protease 3 (SENP3) in macrophage polarization and calcium phosphate (CaPO₄)-induced abdominal aortic aneurysm (AAA) formation in mice. METHODS（1） Bone marrow-derived monocytes (BMDMs) in Senp3^flox/flox (wild-type, WT) mice and Senp3^flox/flox; Lyz2-Cre (monocyte-specific SENP3 knockout, i.e. conditioned knockout, cKO) mice were isolated and induced for M1 and M2 polarization. The mRNA and protein expression level of SENP3 were detected by RT-qPCR, Western blot and immunocytofluorescence, and the differential distribution of M1/M2 BMDMs from WT and cKO mice was analyzed. （2） CaPO₄ was administrated to induce AAA model in 8~12-week-old male WT and cKO mice. The AAA incidence, survival rate and maximal aortic diameter were analyzed between the 2 groups. Aortic aneurysm tissues were collected for pathological analysis, and the expression levels of SENP3, interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), IL-6 and matrix metalloproteinases-9 (MMP-9) were measured by RT-qPCR and Western blot. Dihydroethidium staining in situ in frozen sections was used to analyze the production of reactive oxygen species (ROS). （3） To explore the potential mechanisms, Western blot and co-immunoprecipitation were used to verify the de-SUMO modification of mitogen-activated protein kinase kinase 7 (MKK7) induced by SENP3. Besides, BMDMs were transfected with Flag-MKK7 wild type (Flag-MKK7 WT) and SUMO-modified site K18 mutant (Flag-MKK7 K18R mutant), and then M1 polarization of the cells was induced. The protein levels of p-JNK and MMP-9 in the 2 groups were determined by Western blot. RESULTS(1) SENP3 expression was up-regulated in M1 polarized macrophages (P<0.01), but was down-regulated in M2 polarized macrophages (P<0.01). The expression of SENP3 was decreased during the transformation of M1 to M2 in the macrophages (P<0.01), but was significantly up-regulated during the opposite process (P<0.01). Besides, more M1 macrophages and less M2 macrophages after induction were observed in the BMDMs from cKO mice than those from WT mice. (2) SENP3 expression was up-regulated in AAA tissues (P<0.05). The AAA incidence of cKO mice was significantly reduced after CaPO₄ induction (P<0.01), the survival rate was significantly improved (P<0.05), and maximal aortic diameter was significantly reduced in cKO group (P<0.01). The levels of IL-1β, IL-6 and TNFα, and the production of ROS were significantly down-regulated (P<0.01), meanwhile MMP-9 expression was also down-regulated in cKO mice (P<0.05). (3) the SUMO2/3 modification of MKK7 was reduced during M1 polarization, and MKK7 interaction with SENP3 was enhanced. Significantly up-regulated protein level of p-JNK and MMP-9 were verified in the M1 macrophages transfected with Flag-MKK7 K18R mutant (P<0.05). CONCLUSION SENP3 activates the MAPK/JNK pathway via de-SUMOylation of MKK7, regulates the M1/M2 polarization of macrophages and promotes the protein level of MMP-9, thus aggravating AAA formation.     

Effect of abnormal expression of PDK4 on glycolysis and proliferation in prostate cancer cells

AIM To investigate the expression of pyruvate dehydrogenase kinase 4 （PDK4） in prostate cancer tissue and its effect on glycolysis and growth of prostate cancer cells. METHODS Immunohistochemistry was used to compare the expression differences of PDK4 protein in benign prostatic hyperplasia （BPH） and prostate cancer tissues. The expression levels of PDK4 in normal prostatic epithelial cells （RWPE-1） and different prostate cancer cell lines （PC3, LNCaP, DU145 and C4-2） were detected by RT-qPCR and Western blot. Recombinant plasmid carrying PDK4-shRNA was constructed, and the expression of PDK4 in prostate cancer PC3 cells was down-regulated by transfection with PDK4-shRNA. The changes in glycolysis level of PC3 cells before and after transfection were determined by cell glycolysis kit, and the effects of PDK4 on the viability and cell cycle distribution of PC3 cells were detected by CCK-8 assay and flow cytometry. RESULTS In prostate cancer tissues, the expression level of PDK4 protein was significantly higher than that in BPH tissues （P<0.05）, and the analysis of immunohistochemical score showed that prostate cancer tissues with high Gleason score displayed significantly higher PDK4 expression than those with low Gleason score （P<0.05）. Compared with normal prostatic epithelial cells, RT-qPCR and Western blot results indicated that the expression level of PDK4 was also significantly increased in prostate cancer cell lines （P<0.05）. In addition, CCK-8 assay results showed that the viability of prostate cancer PC3 cells was significantly decreased after knockdown of PDK4 expression （P<0.05）. The results of flow cytometry demonstrated that knockdown of PDK4 expression in PC3 cells resulted in a notable increase in G₀/G₁ phase arrest （P<0.05）. CONCLUSION PDK4 is highly expressed in prostate cancer tissues and cell lines, and significantly increases in prostate cancer with high Gleason score. In addition, down-regulation of PDK4 expression significantly inhibits glycolysis and growth of prostate cancer cells, resulting in cell cycle arrest at G₀/G₁ phase.     

Effect of NLRC3 expression knock-down on growth of normal human bronchial epithelial BEAS-2B cells

AIM To investigate the effect of NOD-like receptor family caspase recruitment domain containing 3 (NLRC3) expression knock-down on the viability and apoptosis of normal human bronchial epithelial BEAS-2B cells and its mechanism. METHODS The small interfering RNA (siRNA) fragments of NLRC3 gene were transfected into BEAS-2B cells using Lipofectamine 2000 transfection reagent to knock down the NLRC3 expression. The interference fragment was screened by RT-qPCR. The cell viability was measured by MTT assay. The mitochondrial membrane potential was detected by JC-1 staining. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. The protein expression levels of Bcl-2 and Bax were determined by Western blot. RESULTS The interference segment 3 of NLRC3 gene (siNLRC3-3) displayed the best interference effect on NLRC3 expression in BEAS-2B cells (P<0.01). Knock-down of NLRC3 expression in BEAS-2B cells enhanced the cell viability (P<0.01). Knock-down of NLRC3 increased the mitochondrial membrane potential, and decreased the apoptotic rate (P<0.05). Moreover, knock-down of NLRC3 significantly up-regulated Bcl-2 protein expression and significantly down-regulated Bax protein expression (P<0.01). CONCLUSION Knock-down of NLRC3 expression enhances the viability and inhibits the apoptosis of BEAS-2B cells, which may be related to increase in the expression of Bcl-2 protein and decrease in the expression of Bax protein.