Objective To elucidate the regulatory role and the underlying molecular mechanisms of serine/threonine protein kinase 1(AKT1)in the lens epithelium of patients with age-related cataract(ARC).Methods 1)Differentially expressed genes in ARC were screened using bioinformatics analysis(Genecard and GEO database GSE213546),and key genes were identified through functional enrichment analysis(KEGG and GO).2)An oxidative stress model of lens epithelial cells was established by treating HLE-B3 cells with 200 μmol/L H2O2 for 24 h.RT-qPCR and Western blot were performed to assess AKT1 gene and protein expression changes.3)Model cells were randomly divided into a si-NC group transfected with si-NC plasmids and 3 parallel si-AKT1 groups transfected with 3 types of si-AKT1 plasmids.A control+si-NC group was also set up,in which HLE-B3 cells not treated with H2O2 were transfected with si-NC empty plasmids.The AKT1 gene and protein expression levels in the si-NC and si-AKT1 groups were determined by RT-qPCR and Western blot.Two si-AKT1 parallel groups demonstrating the most significant changes in expression levels were selected for further experiments.The protein expression levels of AMPK and phosphorylated AMPK(p-AMPK)in the si-NC group,the two selected si-AKT1 parallel groups,and the control+si-NC group were determined by Western blot.A si-AKT1 parallel group demonstrating significant changes in p-AMPK/AMPK values was selected and treated with Acadesine(AICAR),an AMPK agonist,to verify the role of the AMPK pathway.Western blot was performed to determine Bcl-2 and Bax protein levels in the si-NC group,the control+si-NC group,and the si-AKT1 group before and after the administration of AICAR.Flow cytometry was performed to measure apoptosis and reactive oxygen species(ROS)levels,while ELISA kits were used to assess the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and reduced glutathione(GSH).Results 1)Through bioinformatics analysis,78 differentially expressed genes were identified,with AKT1 significantly upregulated in ARC samples(P<0.05)and enriched in the AMPK pathway.2)Compared with cells not treated with H2O2,AKT1 mRNA and protein expression increased in the oxidative stress model cells.3)The p-AMPK/AMPK ratio was higher in the si-NC group than that in the control+si-NC group.In contrast,AKT1 knockdown suppressed AMPK pathway activity,with all the si-AKT1 groups showing a significantly decreased p-AMPK/AMPK ratio compared to that of the si-NC group(P<0.05).Compared with the control+si-NC group,the si-NC group exhibited elevated ROS and MDA levels,increased apoptosis rate,reduced SOD and GSH levels,downregulated Bcl-2,and upregulated Bax(all P<0.05).Compared to those in the si-NC group,these indicators were improved in the si-AKT1 group(all P<0.05).However,compared with the findings before AICAR treatment,these effects were antagonized after AICAR treatment in the si-AKT1 group(all P<0.05).Conclusion The oxidative stress-related gene AKT1 may be a key pathogenic factor in cataract,and AKT1 induces oxidative stress and apoptosis in lens epithelial cells by modulating AMPK pathway activity.

S100A9 is an important alarmin in vivo,which plays a role in regulating inflammation and tumorigenesis.Recently,many studies have also explored the biological function and related mechanism of S100A9 in renal diseases,including acute kidney injury,chronic kidney disease,renal stone,renal transplantation,renal tumor,renal cyst,and urinary infection.They pointed out the potential role of S100A9 as a new diagnostic and therapeutic biomarker for renal diseases.In the current study,we conduct a review of these findings and summarize possible future study directions,which aims to help people understand the impact of S100A9 on renal diseases.

OBJECTIVETo establish a diet-induced obesity model in zebrafish larvae.

METHODSAt 7 days post-fertilization (dpf), 200 zebrafish larvae with normal development were randomly allocated to two groups with the feeding quantity of 30 mg per day (normal feeding group) or 180 mg per day (overfed group) for 20 days. The weight, length, BMI, triglyceride (TG) and total cholesterol (TCH) of each group were measured. Whole-mount Oil Red O staining, frozen Oil Red O staining and hematoxylin-eosin (HE) staining were used to estimate the rate of hepatic steatosis and liver histology of the zebrafish. The dynamic change of hepatic lipid droplets and distribution of adipose tissue were observed with Nile Red staining in overfed zebrafish in vivo.

RESULTSThe weight, length, BMI and TG of overfed zebrafish were significantly increased compared with those in normal feeding group. Whole-mount Oil Red O staining showed that the percent of hepatic steatosis in overfed group (89.4%) was markedly higher than that in normal feeding group (20.7%). Macrovesicular steatosis was observed in the liver of the overfed larvae. Nile Red staining visualized hepatic lipid droplets and the distribution of larval adipose tissue, which increased with feeding time in the overfed zebrafish. Starving larvae showed depletion of fat and hepatic lipid, and adipose tissue was induced after refeeding.

CONCLUSIONSWe successfully established an diet-induced obesity model in zebrafish larva, in which Nile Red staining allows in vivo observation of the adipocytes and hepatic lipid droplets.

Adipose Tissue ; Animals ; Cholesterol ; Diet ; adverse effects ; Disease Models, Animal ; Fatty Liver ; Larva ; Lipids ; Obesity ; pathology ; Triglycerides ; Zebrafish

Background and objective Artesunate, an anti-malarial drug, elicits an inhibitory effect on pulmonary carcinoma. However, the mechanisms of artesunate activity on pulmonary carcinoma have not been completely elucidated. hTe aim of this study is to investigate the effect of artesunate on the invasion of human lung adenocarcinoma A549 cells. Methods hTe inhibitory effect of artesunate on the proliferation and invasion of A549 cells was determined in vitro by MTT assay and transwell chamber invasion assay, respectively. A nude mouse model of human lung A549 cell xenogratf tumor was established. hTe inhibitory effect of artesunate on the tumor of the mouse model as well as ICAM-1 and MMP-9 protein expressions were determined by Western blot. Results A low dose of artesunate ranging between 1.25μg/L and 5μg/L did not signiifcantly inhibit the proliferation of A549 cells in vitro. By contrast, 1.25μg/L artesunate inhibited the invasion of A549 cells in vitro as determined by transwell chamber invasion assay (96.33±6.41 vs 75.43±4.37, P<0.05). Although 10 mg/kg artesunate did not signiifcantly inhibit A549 xenogratf tumor proliferation (P>0.05), artesunate decreased the ICAM-1 and MMP-9 protein levels in the mouse model (P<0.05). Conclusion Artesunate could inhibit the invasion of human lung adenocarcinoma A549 cells by possibly downregulating ICAM-1 and MMP-9 expressions.

Objectives To study the effect of mesenchymal stem cells on the aging rat kidney and to explore the underlying mechanism. Methods Rat models of senile kidney were built with hypodermic injection of D-galactose daily. Injections of MSCs of 3 × 10<'6>were given to each rat through vena caudalis and CFSE was used as a tracing label to detect the distribution of MSCs in vivo. After 24 h, rats were dissected and their kidneys were frozen for section. MSCs were observed with Fluophot and quantitative analysis of the various parameters of kidney was performed under a light microscope with B12000 image analysis system.The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and kidney were measured wtih hydroxylamine and chromatometry. The expression of VEGF and P16 mRNA in kidney tissue was detected with real-time PCR and Western blotting. Results MSCs was found homing in the rat kidney,and the glomerular size, sklerosis-rate and the average cell count of glomerulus in the treated group were different from those of the model group (P<0.05). In the treated group, the activity of SOD was significantly higher and the content of MDA was lower in serum and kidney than that in the model control group (P<0.05). The expression of VEGF mRNA and protein in the kidneys of MSCs group increased significantly as compared with the model group (P<0.05). The expression of P16 mRNA and protein in the kidney of MSCs group decreased significantly compared with the model group (P<0.05). Conclusion MSCs can increase the expression of VEGF while decrease the expression of P16, so as to play a key role in the anti-aging on rat kidney.

Objective:To investigate the inducing effects of sunitinib malate on expression of NKG2D ligands in nasopharyngeal carcinoma cell ABCG2high CNE2/DDP.Methods:ABCG2highCNE2/DDP cells and Allo-NK cells were isolated by magnetic activated cell sorting(MACS).Flow cytometry was used to evaluate the purity of isolated cells and the expression of NKG2D-ligands on target cells before and after incubation with sunitinib malate.Then the cytotoxic sensitivity of treated and un-treated ABCG2high CNE2/DDP cells to Allo-NK cells were measured by LDH releasing assay.Results:The positive rate of ABCG2 in ABCG2highCNE2/DDP cells was(91.40?2.32)%.More than 90% of isolated Allo-NK cells were proven to be CD3-CD16+CD56+ cells.The expression of MICA,MICB,ULBP1,ULBP2 and ULBP3 on ABCG2high CNE2/DDP cells incubated with sunitinib malate increased from(2.92?0.33)%,(4.27?0.33)%,(5.80?0.62)%,(11.10?3.15)%,and(7.75?1.14)% to(89.12?4.56)%,(66.10?2.22)%,(67.56?4.19)%,(69.37?8.83)%,and(63.28?3.31)%,respectively.At the E ∶T ratios of 10 ∶1 and 20 ∶1,the cytotoxic sensitivities of ABCG2high CNE2/DDP cells to Allo-NK cells increased from(15.32?13.86)% and(27.26?6.81)% to(41.12?4.12)% and(57.25?2.37)%,respectively,after treatment with sunitinib malate,with significantly difference found in the cytotoxic sensitivities of target cells in each group before and after sunitinib malate treatment(F=15.58,P=0.000).Conclusion:Sunitinib malate can up-regulate expression of NKG2D-ligands(MICA/B,ULBP1-3)in ABCG2high nasopharyngeal carcinoma cells,which results in higher cytotoxic sensitivity to Allo-NK cells.

Objective To investigate the mechanisms and effects of Sorafenib on cytotoxic sensitivity of allo-reactive natural killer(Allo-NK) cells against human multi-drug resistant nasopharyngeal carcinoma CNE2/DDP cells which expressing highly ATP-binding cassette superfamily G member 2(ABCG2)(abbr.as ABCG2HighCNE2/DDP cells).Methods ABCG2HighCNE2/DDP and Allo-NK cells were isolated by magnetic bead technique.The target cells were divided into 3 groups: a) treated group(ABCG2HighCNE2/DDP cells incubated with 10 ng/ml sorafenib for 4h);b) untreated group(conventionally cultured ABCG2HighCNE2/DDP cells);and c) control group(conventionally cultured K562 cells).Expression rates of ABCG2 in treated and untreated groups,and of five NKG2D-ligands(MICA,MICB,ULBP1,ULBP2,ULBP3) were evaluated by flow cytometry.The cytotoxic effects of NK cells against different groups of target cells were detected with LDH releasing assay.Results Expression rate of ABCG2 in isolated CNE2/DDP cells was 91.40%?2.32%.The purity of sorted CD3-CD16+CD56+ Allo-NK cells was 90% and higher.The expression rates of NKG2D-ligands(MICA,MICB,ULBP1,ULBP2 and ULBP3) in untreated group were 2.92%?0.33%,4.27%?0.33%,5.80%?0.62%,11.10%?3.15% and 7.75%?1.14%,respectively,which were remarkablely higher than that in treated group(10.38%?1.23%,10.68%?1.26%,11.62%?1.22%,43.24%?4.42% and 11.91%?0.88%,respectively,P