Alleviating effect of exenatide on ectopic lipid accumulation in skeletal muscle of ob/ob mice is independent on reducing body weight

AIM To investigate the alleviating effect of exenatide （Exe）， a glucagon-like peptide-1 （GLP-1） receptor agonist， on the ectopic lipid accumulation in skeletal muscle of ob/ob mice and its mechanism. METHODS Eight-week-old male ob/ob mice and their wild-type （WT） littermates were randomly divided into 3 groups， ob/ob group， ob/ob+Exe group and WT group， and treated with Exe at 24 nmol/kg or the same volume of saline intraperitoneally once daily for 4 weeks. The body weight， fasting blood glucose （FBG） and fat content were measured after the 4-week treatment. The oil red O staining and the quantification of triglyceride （TG） were performed on the skeletal muscle. The serum levels of TG， total cholesterol and free fatty acid （FFA） were also measured by ELISA. The expression levels of AMP-activated protein kinase （AMPK） and lipid metabolism-related proteins were determined by Western blot. Mouse myoblast C2C12 cells were used as an in vitro model to further investigate the effects of Exe. RESULTS As compared with the ob/ob mice treated with saline， 4-week Exe treatment did not reduce body weight， FBG， food intake and fat content in ob/ob mice （P>0.05）. However， serum FFA was decreased （P<0.05）. Oil red O staining and the quantification of TG showed that 4-week Exe treatment significantly attenuated the ectopic lipid accumulation in the skeletal muscle of ob/ob mice （P<0.05）. The results of Western blot showed that the levels of phosphorylated AMPK （p-AMPK） and lipolysis-related proteins were up-regulated， while the lipid synthesis-related proteins were down-regulated by Exe （P<0.05）. Treatment with Exe alleviated the lipid accumulation in the C2C12 cells induced by sodium palmate （P<0.05）， and the effects of Exe on the levels of p-AMPK and lipid metabolism-related proteins in the C2C12 cells were consistent with those in the ob/ob mice （P<0.05）. Treatment with Exe also up-regulated the protein expression of glucose transporter 4 and improved the ability of glucose uptake in the C2C12 cells （P<0.05）. CONCLUSION Short-term Exe treatment attenuates the ectopic lipid accumulation in skeletal muscle of ob/ob mice by up-regulating lipolysis-related proteins and down-regulating lipid synthesis-related proteins， which is independent on body weight loss.     

Lipophagy inhibits foam cell formation by reducing cholesterol content

AIM To observe the changes of lipophagy during foam cells formation， and to determine the effect of lipophagy on the lipid content and cholesterol outflow of foam cells. METHODS Human THP-1 monocytes were induced by phorbol-12-myristate-13-acetate for 48 h to differentiate into macrophages， and then were incubated with 50 mg/L oxidized low-density lipoprotein （oxLDL） to form foam cells. Lipids in foam cell were stained by oil red O， and the lipid content was determined. The total cholesterol （TC） and free cholesterol （FC） levels in foam cells were measured by cholesterol testing kit. Cholesteryl ester （CE） and CE/TC ratio were calculated. The cholesterol efflux rate was detected by cholesterol efflux assay kit. The expression of autophagy-related proteins， including autophagy-related protein 5 （Atg5）， microtubule-associated protein 1 light chain 3 （LC3） and P62， were detected by Western blot. The colocalization of lipid droplets （LD） and LC3 was detected by immunofluorescence staining. The autophagy inducer rapamycin （Rap） or blocker 3-methyladenine （3MA） was used to intervene foam cells， and the expression of Atg5， LC3 and P62， the co-expression of LD and LC3， the cholesterol content and the cholesterol efflux rate were determined. RESULTS Formation of foam cells was observed at 24 h after stimulation with oxLDL at 50 mg/L， as indicated by intracellular CE/TC ratio exceeding 50%.Cholesterol efflux assay revealed that the cholesterol efflux rate increased within 24 h during foam cell formation but decreased after 48 h （P<0.05）. Western blot results displayed that the expression of Atg5 and LC3-II/LC3-I ratio were increased within 24 h of foam cell formation， but was deceased after 48 h （P<0.05）. The expression of P62 was decreased within 24 h but was increased at 48 h （P<0.05）. The colocalization of LD and LC3 was increased at 24 h but was decreased at 48 h after oxLDL stimulation. Treatment with Rap up-regulated the expression of Atg5 and LC3-II/LC3-I ratio， reduced the level of P62， increased the colocalization of LD and LC3， promoted the cholesterol efflux， anf reduced cholesterol content in foam cells （P<0.05）. On the contrary， 3MA inhibited the expression of Atg5， reduced LC3-II/LC3-I ratio， elevated the level of P62， decreased the colocalization of LD and LC3， reduced the outflow of cholesterol， increased the content of TC and CE， and elevated CE/TC ratio in foam cells （P<0.05）. CONCLUSION Lipophagy is enhanced at 24 h but decreased at 48 h during foam cell formation. Lipophagy inhibited foam cell formation by reducing cholesterol content and increasing cholesterol efflux.     

Inhibitory effect of Wendan decoction on formation of foam cells induced by ox-LDL

AIM To explore the anti-atherosclerotic mechanism of Wendan decoction based on formation of foam cells. METHODS The optimal concentrations of Wendan decoction without cytotoxity to cells were selected by MTT assay. After Wendan decoction treatment， the formation of foam cells was examined by oil red O staining. The cholesterol efflux， cholesterol level， free cholesterol level and cholesterol esterification rate were analyzed using cholesterol efflux assay， total cholesterol assay and free cholesterol assay. The expression levels of macrophage membrane proteins， including CD36， scavenger receptor class A （SR-A）， ATP-binding cassette transporter A1 （ABCA1） and scavenger receptor class B type I （SR-BI）， were quantified by Western blot. RESULTS The optimal concentrations of Wendan decoction without cytotoxity to the cells were 0~6 g/L. Wendan decoction at the concentrations of 1.5， 3 and 6 g/L were selected for the experiments. Wendan decoction at these concentrations inhibited the formation of foam cells induced by oxidized low-density lipoprotein （ox-LDL）， and reduced the accumulation of intracellular lipids in a concentration-dependent manner （P<0.05 or P<0.01）. Wendan decoction also reduced intracellular total cholesterol level， cholesterol ester level and cholesterol esterification rate （P<0.05 or P<0.01）， promoted efflux of intracellular cholesterol （P<0.01）， and decreased the protein level of CD36 in THP-1 cell-derived macrophages （P<0.01） in a concentration-dependent manner. Wendan decoction at the concentration of 6 g/L significantly reduced the protein level of SR-A in THP-1 cell-derived macrophages （P<0.05）. At the concentrations of 3 and 6 g/L， Wendan decoction significantly increased the protein levels of ABCA1 and SR-BI in THP-1 cell-derived macrophages （P<0.05 or P<0.01）. CONCLUSION Wendan decoction significantly inhibits ox-LDL-induced formation of foam cells by reducing cholesterol deposition and promoting cholesterol efflux， and its mechanism may be related to the down-regulation of CD36 and SR-A and the up-regulation of ABCA1 and SR-BI.     

RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice， and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group， and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group， ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method， and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1， p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method， and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG， TC and LDL-C， and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner， inhibited the intimal plaque formation and vascular medial proliferation， and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG， TC， HDL-C and LDL-C， and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group， ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs， while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）， but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice， and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Research progress of organic solute transporter α/β in bile acid metabolism

The organic solute transporter α/β （OSTα/β） is a recently discovered transporter that controls bile acid secretion into portal blood stream in the basal lateral membrane of intestinal epithelial cells. OSTα/β is a compound composed of 2 subunits， OSTα and OSTβ. Only when the 2 subunits are expressed at the same time， they exist stably and function properly. It is responsible for the transmembrane transport of organic solutes such as bile acids in a way of easy diffusion. OSTα/β is regulated by bile acid receptor， also named as farnesoid X receptor （FXR）. Studies showed that the bile acid synthesis in OSTα deficient mice is decreased， while the bile acid content in the urine is increased. It is worth mentioning that the single gene mutation leads to OSTβ deficiency in the patients with clinical symptoms such as chronic diarrhea and cholestatic liver disease. This paper reviews the structure， function and role of OSTα/β in enterohepatic circulation and the diseases caused by loss of OSTα/β.     

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.     

Estrogen treatment enhances mesenteric lymphatic contractility in rats after hemorrhagic shock

AIM To investigate the effects of 17β-estradiol （E2） treatment on the mesenteric lymphatic microcirculation and isolated lymphatic contractility in rats after hemorrhagic shock, and to explore the relationship between contractility and the difference between intra- and extracellular calcium ion concentrations （［Ca²⁺］） of lymphatic smooth muscle cells （LSMCs）. METHODS Male Wistar rats were divided into sham group, shock group and shock+E2 group. The rats were subjected to hemorrhage ［（40±2） mmHg for 90 min］ and resuscitation with or without subcutaneous injection of E2 （2 mg/kg）. After resuscitation for 3 h, the mesenteric lymphatic microcirculation in vivo was observed. Moreover, the isolated mesenteric microlymphatic rings were prepared for the observations of lymphatic contractility evaluated by the indexes including end-systolic diameter, end-diastolic diameter, contraction frequency （CF） and passive diameter. Meanwhile, the difference between intra- and extracellular ［Ca²⁺］ of LSMCs was recorded during lymphatic contraction. RESULTS Treatment with E2 significantly enhanced the CF, total contractile fraction and lymphatic dynamics index in vivo in the rats after hemorrhagic shock, and increased the CF, the fractional pump flow and the difference between intra- and extracellular ［Ca²⁺］ of LSMCs in isolated lymphatics from the shocked rats （P<0.05）. CONCLUSION Estrogen treatment enhances lymphatic contractility in rats after hemorrhagic shock, which is related to enhancement of difference between intra- and extracellular ［Ca²⁺］ of LSMCs.     

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 miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.     

PPARγ affects epithelial-mesenchymal transition in renal tubular epithelial cells under high glucose by regulating AKT/FAK signaling pathway through PTEN

AIM To investigate the role of peroxisome proliferator-activited receptor γ （PPARγ） in the regulation of PTEN/AKT/FAK signaling pathway and epithelial-mesenchymal transition （EMT） in renal tubular epithelial cells grown in high-glucose environment. METHODS Renal tubular epithelial cells （NRK52E cells） cultured in high glucose were used as an in vitro model system. PPARγ was over-expressed or knocked down in these cells， and its effect on PTEN expression was determined by RT-qPCR， immunofluorescence and Western blot. The changes of EMT-related proteins were also measured. The PPARγ inhibitor GW9662 and the PPARγ agonist rosiglitazone were used along with PTEN over-expression or knockdown to determine whether the effects of PPARγ were mediated through PTEN. RESULTS PPARγ over-expression resulted in the increased expression of PTEN at mRNA and protein levels， the up-regulation of E-cadherin， and the down-regulation of vimentin and α-SMA. Knockdown of PPARγ expression reduced the mRNA and protein levels of PTEN， down-regulated E-cadherin， and up-regulated vimentin and α-SMA （P<0.05）. Treatment of the NRK-52E cells with GW9662 decreased PTEN expression and increased the protein levels of p-AKT （Thr308）， FAK and p-FAK （Tyr397）. These effects were rescued by PTEN over-expression. Treatment of the NRK-52E cells with rosiglitazone increased PTEN expression and decreased the protein levels of p-AKT （Thr308）， FAK and p-FAK （Tyr397）. These effects were rescued by PTEN knockdown. These changes were all statistically significant （P<0.05）. CONCLUSION PPARγ regulates the mRNA and protein expression of PTEN in renal tubular epithelial NRK52E cells， and affects EMT in renal tubular epithelial cells. The regulation of AKT/FAK signaling pathway by PPARγ is primarily mediated by PTEN.     

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.     

Mast cells change cellular cholesterol content and inhibit cholesterol efflux from THP-1 macrophage-derived foam cells

AIM:Mast cells (MC) are present in the arterial intima,the site of atherogenesis. The present studies explore the effect of MC on cholesterol content,distribution and efflux in THP-1 macrophage-derived foam cells (THP-1FCs）. METHODS:THP-1FCs were incubated with high-density lipoproteins 3 (HDL₃) in the absence or presence of mast cell granules (MCGs) harvested from compound 48/80-stimulated rat peritoneal MC. The intracellular cholesterol level,cholesterol effluxing capacity,ATP-binding cassette transporter A1(ABCA1) mRNA and HDL₃ treated with MCGs were detected to characterize the role of MC on intracellular cholesterol. RESULTS:MCGs had high levels of cellular total cholesterol(TC),free cholesterol(FC) but not esterifed cholesterol(EC) compared to control group where the TC concentrations ranged from 527.3 mg/g to 917.9 mg/g cellular protein with EC accounting for 7.6% of the cholesterol. Cholesterol efflux was 14% less in MCGs group compared to control group. ABCA1 mRNA expression in MCG-treated THP-1FCs remained unchanged in 20 hours. In contrast,treatment of HDL₃ with MCGs resulted in rapid degradation of the main HDL₃ apoliproteins,apoA-Ⅰ. SDS-PAGE revealed that a minor polypeptide band with about 26 kD molecular mass appeared below the apoA-Ⅰband. Densitometric analysis of the gel demonstrated that ≈ 28% of apoA-Ⅰhad been degraded by the MCGs. CONCLUSION:These results indicate that MC decreases cholesterol efflux,increases cellular accumulation in TC and FC by depleting HDL₃ and apoA-Ⅰ,but not by inhibiting ABCA1 mRNA expression.     

Effect of Yangjing-Zhongyu decoction on in vitro maturation and expression of PDGFA and PDGFRα of mouse oocytes

AIMTo investigate the effects of Yangjing-Zhongyu decoction on the in vitro maturation and expression of platelet-derived growth factor subunit A (PDGFA) and platelet-derived growth factor receptor α (PDGFRα) of mouse oocytes. METHODSThe SPF female KM mice were given Yangjing-Zhongyu decoction, and the blood was collected to prepare serum. The serum containing Yangjing-Zhongyu decoction was used to culture immature oocyte-granulosa cell complexes. After the in vitro culture, the oocyte maturation rate, fertilization rate and cleavage rate were observed and calculated, and the expression of PDGFA and PDGFRα in the oocytes at protein and mRNA levels was determined by West?ern blot and real-time PCR. RESULTSYangjing-Zhongyu decoction increased the in vitro maturation rate and fertil?ization rate of the oocytes (P<0.05 or P<0.01), and down-regulated the protein and mRNA expression of PDGFA and PDGFRα (P<0.01). CONCLUSION Yangjing-Zhongyu decoction may promote the in vitro maturation of mouse oocytes by down-regulating the expression of PDGFA and PDGFRα.     

Nicotinic acid promotes lysosomal free cholesterol efflux via LXRα/NPC1 pathway in macrophages

AIM To explore the effects of nicotinic acid (NA) on lysosomal free cholesterol efflux in macrophages and its underlying mechanism. METHODS Macrophages induced from human monocytic leukemia cell line THP-1 by phorbol myristate acetate served as the cell model. Laser scanning confocal microscopy was applied to observe the effects of NA on lysosomal free cholesterol efflux in macrophages loaded with oxidized low-density lipoprotein (oxLDL). The influences of nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19, Ca²⁺ chelator BAPTA, liver X receptor α (LXRα) siRNA and Niemann-Pick C1 protein (NPC1) siRNA on NA effects were also evaluated. RT-qPCR and Western blot were conducted to evaluate the influence of NA, Ned-19 and BAPTA on LXRα mRNA and NPC1 protein expression. RESULTS NA dose-dependently promoted lysosomal free cholesterol efflux in macrophages. This effect was markedly inhibited by Ned-19 and BAPTA. NA increased NPC1 protein and LXRα mRNA expression. These effects were also attenuated by Ned-19 and BAPTA remarkably. LXRα siRNA significantly inhibited the promoting effect of NA on NPC1 protein expression. Silencing of LXRα and NPC1 with siRNA remarkably abolished the effect of NA on lysosomal free cholesterol efflux. CONCLUSION NA promotes lysosomal free cholesterol efflux in macrophages. This effect may be mediated by the increased production of NAADP, which subsequently promotes Ca²⁺ release through lysosomal transient receptor potential mucolipin 1 (TRPML1) channel and finally up-regulates NPC1 protein expression via LXRα.     

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 simvastatin on proliferation of esophageal cancer cells through NF-κB p65 pathway

AIM To investigate the effects of simvastatin （SIM） on the proliferation of esophageal cancer Eca109 cells through NF-κB p65 pathway. METHODS Esophageal cancer Eca109 cells were cultured in vitro and treated with SIM at different concentrations （2 ， 4， 8， 16 and 32 μmol/L）. The proliferation of Eca109 cells was measured by MTT assay and plate colony formation assay. The proliferation-related protein levels of cyclin D1， c-Myc， NF-κB p65 （p65） and IκB-α in the esophageal cancer Eca109 cells were determined by Western blot. ELISA was used to detect the levels of tumor necrosis factor α （TNF-α） and interleukin-6 （IL-6） in the culture supernatant of Eca109 cells. RESULTS Compared with control group， the inhibitory effects of SIM at 2 ， 4 ， 8 and 16 μmol/L on the viability of Eca109 cells were increased in a concentration-dependent and time-dependent manners （P<0.05）. Simultaneously， the inhibitory rates of SIM at 16 and 32 μmol/L on the viability of Eca109 cells showed no significant difference （P>0.05）. The inhibitory rates of SIM at 16 μmol/L on the viability of esophageal cancer cells was 50.61% at 48 h， which was closed to half of the inhibitory dose IC₅₀， and it was used as the optimum concentration and time for follow-up experiments. Compared with control group， the plate colony formation rate of Eca109 cells， the protein levels of cyclinD1， c-Myc， nuclear p65， TNF-α and IL-6 in 8 and 16 μmol/L groups were decreased， while the levels of cytosolic p65 and IκB-α proteins were increased （P<0.05）. No significant difference of plate colony formation rate in Eca109 cells， and the protein levels of cyclin D1， c-Myc， nuclear and cytoplasmic p65， IκB-α， TNF-α and IL-6 between 16 μmol/L SIM group and 32 μmol/L SIM group was observed （P>0.05）. CONCLUSION Simvastatin inhibits the proliferation of esophageal cancer Eca109 cells， and its mechanism may be related to up-regulation of IκB-α, down-regulation of cyclinD1 and c-myc, inhibition of p65 nuclear translocation, and the expression of TNF-α and IL-6 downstream of NF-κB p65 pathway.     

Protective effect of curcumin on MRL/lpr mice with lupus nephritis and its mechanism

AIM To observe the effect of curcumin （Cur） on lupus nephritis （LN） and its possible mechanism. METHODS Thirty 10-week-old MRL/lpr lupus mice were randomly divided into MRL/lpr group， Cur-L and Cur-H group with 10 mice in each group， and C57BL/6 mice （n=10） served as normal control （NC） group. The mice in Cur-L group and Cur-H group were given intragastric administration of Cur at 100 and 200 mg·kg^-1·d^-1 for 12 weeks， respectively， and the same volume of normal saline was given to the mice in NC group and MRL/lpr group. The urine protein was detected， and the morphological changes of the renal tissue were observed by HE staining after treatment. The levels of serum creatinine （SCr）， blood urea nitrogen （BUN） and serum anti-double-stranded DNA （dsDNA）， and interleukin-1β （IL-1β）， IL-6 and tumor necrosis factor-α （TNF-α） levels in serum and renal tissues were detected. The protein levels of p-IκB， NF-κB， NLRP3 and caspase-1 in the renal tissues were determined by Western blot. RESULTS Compared with MRL/lpr group， the content of urine protein in Cur groups was significantly reduced， and the renal injury was relieved. The SCr， BUN， serum anti-dsDNA， and the serum and renal levels of IL-1， IL-6 and TNF-α were all significantly reduced， and the protein levels of p-IκB， NF-κB， NLRP and caspase-1 in the renal tissue were significantly decreased （P<0.05）. CONCLUSION Cur has a certain protective effect on the kidney of MRL/lpr mice， and its mechanism may be related to the inhibition of NF-κB and NLRP3 signaling pathways.