Objective:To investigate the regulatory effect of WNT1-inducible signaling pathway protein 2(WISP2)on macrophage polarization in palmitic acid(PA)and lipopolysaccharide(LPS)-induced inflammation.Methods:The macrophage cell line RAW264.7 was treated with different concentrations of WISP2 protein, and cell viability was determined by means of luminescence assay using Cell-Titer Glo to determine the concentration of WISP2.The cells were divided into control group, palmitic acid group, palmitic acid combined with different concentrations of WISP2 group(10 μg/L and 100 μg/L)and lipopolysaccharide group, lipopolysaccharide combined with different concentrations of WISP2 group(10 μg/L and 100 μg/L). mRNA expression of M1 and M2 macrophages phenotype of each group were detected by real-time quantitative polymerase chain reaction.The protein expression of important inflammatory factors, TNF-α and IL-6, were evaluated by ELISA.Results:Compared with the control group, both 10 μg/L and 100 μg/L WISP2 groups had no effect on the activity of RAW264.7 cells, but significantly up-regulated the expression of various inflammatory factors, including Tnfα(1.877±0.039, 2.202±0.034, F=309.7, P<0.001), Il6(1.418±0.056, 1.506±0.059, F=81.39, P<0.001), Mcp1(1.620±0.014, 1.982±0.125, F=71.45, P<0.001), Ccl3(1.892±0.118, 1.942±0.132, F=32.93, P<0.001), and iNos(1.691±0.201, 1.548±0.090, F=13.60, P<0.05). mRNA in macrophages, and significantly down-regulated the expression of anti-inflammatory factors, including Tgfβ(1.376±0.025, 2.152±0.107, F=1.846, P<0.05), CD206(2.123±0.031, 3.139±1.663, F=8.037, P<0.05), Il4(2.098±0.464, 2.494±0.141, F=48.68, P<0.01), and Il10(1.303±0.216, 1.574±0.274, F=5.774, P<0.05)mRNA, causing M1 type macrophage polarization.Compared with the control group, 100 μmol/L palmitic acid could mildly but significantly increase the expression of inflammatory factors such as TNF-α and IL-6 at the transcriptional and protein levels.Compared with palmitic acid stimulation alone, the combination of palmitic acid and WISP2 further promoted the protein expression of macrophage inflammatory factors TNF-α[(589.4±17.0)ng/L, (692.6±83.4)ng/L, F=56.38, P<0.05], IL-6[(15.13±1.14)ng/L, (13.33±1.22)ng/L, F=23.32, P<0.001]and the mRNA expression of chemokines Mcp1(160±9.796, 140±18.91, F=141.1, P<0.0001)and C cl3(17.76±1.92, 14.41±1.27, F=125.2, P<0.0001). Compared with the control group, 100 μg/L lipopolysaccharide strongly stimulated the expression of inflammatory factors such as TNF-α[(3444±423)ng/L, F=71.20, P<0.0001]and IL-6[(497.0±41.2)ng/L, F=63.50, P<0.0001]in macrophages at the protein level.Compared with lipopolysaccharide stimulation alone, the combination of lipopolysaccharide and WISP2 further significantly up-regulated the mRNA expression of chemokines Mcp1(106.8±8.7, 118.7±4.6, F=251.5, P<0.0001)and Ccl3(35.3±12.5, 116.4±4.5, F=160.1, P<0.0001). Conclusions:The adipokine WISP2 can promote M1 macrophage polarization in palmitic acid and lipopolysaccharide-induced inflammation, and it had distinct regulation in macrophage polarization under different inflammatory response conditions.

Objective:To investigate the effects of various concentrations of recombinant human WISP2 protein(WISP2)on lipid metabolism in HepG2 cells.Methods:HepG2 cells were treated with different concentrations(0, 0.4, 1 and 2 μg/L)of recombinant human WISP2 for 48 hours.Cell viability was detected by Cell-Titer, and enzymatic hydrolysis methods were used to measure intracellular triacylglycerol(TG)and total cholesterol(TC)levels.The mRNA expression was detected by quantitative real-time reverse transcription-PCR(RT-qPCR)and protein expression in HepG2 cells was detected by western blot.Results:Compared with the control group, the WISP2 groups treated with various concentration did not significantly reduce the viability of HepG2 cells.TG and TC in HepG2 cells were significantly increased by recombinant human WISP2 treatment(all P<0.05).The concentrations of TG in the 0.4, 1 and 2 μg/L recombinant human WISP2-treated groups were 1.254±0.039, 1.216±0.028 and 1.174±0.014)times the concentration in the untreated group, respectively( F=6.791, P=0.006).The concentration of TC in the untreated group was 1.264±0.057, 1.394±0.101 and 1.392±0.077), respectively, times the concentration in each of the treated groups( F=7.045, P=0.005).Further experiments found that the mRNA expression of sterol regulatory element binding protein 1(SREBP1), 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGCR), acetyl-CoA carboxylase(ACC), type 2 diacylglycerol acyltransferase(DGAT2)and the protein expression of SREBP1, ACC and fatty acid synthase(FAS)were significantly increased in the recombinant human WISP2-treated groups, compared with the control group(all P<0.05).However, the expression of lipid transporters such as the low-density lipoprotein receptor(LDLR), ApoB and ApoE and adipose triglyceride lipase(ATGL), a key lipolysis protein, was not significantly affected. Conclusions:Human recombinant WISP2 protein increases lipid levels in hepatocytes and the key underlying mechanisms may be through promoting lipid synthesis.

Objective:To investigate the effects and potential mechanisms of resveratrol on obesity in elderly mice.Methods:In this study, 3 groups were randomly formed for 32-week-old mice and for 48-week-old mice.The normal diet group received regular chow and 0.3 ml saline by gavage once a day, the high-fat diet group received a high-fat diet(containing 21% fat and 1.25% cholesterol)and 0.3 ml saline once a day, and the high-fat diet plus resveratrol group received a high-fat diet and resveratrol(22.4 mg/kg, dispersed in 0.3 ml saline)by gavage once a day.After 12 weeks, body weight and adipose tissues were measured.Plasma leptin concentrations were determined by an enzyme-linked immunosorbent assay(ELISA), and values for hypertrophic obesity-related indexes of mice were obtained by quantitative real-time PCR.Results:The body weight and the proportion of subcutaneous fat tissues were lower in the high-fat diet plus resveratrol group than in the high-fat diet group[(34.43±3.23)g vs.(53.16±2.16)g, (3.21±1.58)% vs.(4.86±0.64)%, P<0.01], and were similar to those in the normal diet group.Resveratrol had a more obvious inhibitory effect on leptin in elderly mice than in middle-aged mice.In elderly mice, the plasma leptin concentration was lower in the high-fat diet plus resveratrol group than in the high-fat diet group[(0.015±0.009)g/L vs.(0.100±0.027)g/L]and the normal diet group( F=19.85, P=0.001), and it was similar to that in the middle-aged mice on a normal diet.Resveratrol significantly increased the expression of peroxisome proliferator-activated receptor gamma(PPARγ)and glucose transporter 4(GLUT4)and reduced the expression of tumor necrosis factor-α(TNF-α)( F=10.79, 9.31 and 7.02, P=0.003, 0.006 and 0.010). Conclusions:Resveratrol can significantly improve hypertrophic obesity in elderly mice, and the inhibition of leptin secretion and up-regulation of PPARγ may be the key mechanisms.

Aim Toinvestigatetheeffectofginkgolide B on apoptosis in high glucose-treated endothelial cells.Methods Humanumbilicalveinendothelial cells(HUVECs)were used in the present study.The level of transmigration of HUVECs was analyzed by Tr-answell experiment.Apoptosis was detected by flow cy-tometry.Reactive Oxygen Species (ROS ) was meas-ured by immunofluorescence kit.The protein expres-sionwasanalyzedbyWesternblot.Result Highglu-cose treatment resulted in a reduction in transmigration of HUVECs and ginkgolide B recovered the phenome-non in glucose-treated endothelial cells.The level of ROS generation was increased in high glucose-treated group,whereas ginkgolide B inhibited ROS genera-tion.Immunofluorescence data showed high glucose in-creased apoptosis,whereas ginkgolide B inhibited ap-optosis in high glucose-treated HUVECs.Moreover, the expressions of Bax and caspase-3 were increased and Bcl-2 was reduced in high glucose-treated group. In contrast,ginkgolide B abolished the expressions of Bax and caspase-3 and increased Bcl-2 expression. Moreover,high glucose enhanced the expression and phosphorylation of p53,while ginkgolide B suppressed the expression and phosphorylation of p53 induced by highglucose.Conclusions GinkgolideBcaninhibit apoptosis and improve transmigration function in high glucose-treated HUVECs.Ginkgolide B has protection against high glucose-induced endothelial cell injury.

Objective To investigate the effect and its significance of low-dose insulin on Nmethyl-D-aspartate receptor (NMDAR) level and its downstream signaling of c-Jun N-terminal kinase (JNK) and the extracellular signal-regulated protein kinase (ERK,p44/42MAPK) in streptozotocininduced diabetic rats.Methods Ten-week-old male SD rats were randomly divided into 3 groups:control group,diabetic group and insulin-treated diabetes group.Diabetes was induced by streptozocin (STZ,60mg/kg) injected intraperitoneally.Two weeks after STZ injection,insulin glargine (92u/day for 8 weeks) was subcutaneously injected in the insulin-treated diabetes group.Then,the rat lumbar spinal cords were collected.The protein levels of phospho-Insulin receptor substrate 1 (P-IRS1),phospho-NMDAR NR1 subunit (P-NR1),P-JNK and P-p44/42MAPK were evaluated by Western blotting.One week before the terminal of the study,paw thermal response latency was measured in all groups.Results Blood glucose levels were tremendously high in both the diabetic group and insulintreated diabetes group.Compared with the control group,paw thermal response latency was markedly shortened in the diabetic group (P＜ 0.001) and the insulin-treated diabetes group (P＜ 0.001),and the alteration was more pronounced in the diabetic group (P＜0.05).Compared with the control group,the protein levels of P-IRS1,P-NR1,P-JNK and P-p44/42MAPK were increased by 79.2％,35.1％,47.6 ％,64.3 ％ and 87.6 ％,respectively in diabetic group and 49.4 ％,19.1％,16.5 ％,31.8％ and 39.9％,respectively in insulin-treated diabetes group (all P＜0.001 or 0.05).In comparison with diabetic group,the increased amplitudes of above 4 parameters were decreased by 29.8％,16.0％,31.2％,32.5％ and 47.7％ respectively in the insulin-treated diabetes group (all P＜0.05).Conclusions NMDAR and its downstream signals,such as JNK and p42/44MARK,are involved in the pathogenesis of painful diabetic neuropathy.Although it could not efficiently control the blood glucose level,low-dose insulin treatment may partly inhibit the occurrence of thermal hyperalgesia through inhibiting NMDAR signal pathway.

Aim To investigate the effect of ginkgolide B on TLR4 expression in glucose-treated endothelial cells.Methods Human umbilical vein endothelial cells (HUVECs)were stimulated by high concentra-tion of glucose.TLR4,inflammatory protein expression and Akt phosphorylation were analyzed by Western blot.Transcription factor NF-κB nuclear translocation was analyzed by immunofluorescence.Results The expression of TLR4 and PAF receptor was increased in high glucose-treated HUVECs. In contrast, both ginkgolide B and CV3988 dose-dependently decreased TLR4 and PAF receptor expression in high glucose-treated cells,respectively.Ginkgolide B decreased in-flammatory protein ICAM-1 ,VCAM-1 expression.Mo-reover,ginkgolide B potently abolished Akt phospho-rylation and NF-κB p65 nuclear translocation.Conclu-sion Ginkgolide B can reduce TLR4,PAF receptor, ICAM-1 and VCAM-1 expression in high dose of glu-cose-treated HUVECs,the mechanism might be linked to inhibition of Akt phosphorylation and NF-κB activa-tion.

Aim To investigate the effect of resveratrol on ROS level and PECAM-1 expression in ox-LDL-stimulated platelets. Methods The expression of PE-CAM-1 , Sirt1 and p38 MAPK phosphorylation in ox-LDL-stimulated platelets was determined by Western blot. The level of ROS was measured by immunofluo-rescence kit. Results ox-LDL induced platelet aggre-gation by 14%, whereas resveratrol inhibited platelet aggregation by 50%. Resveratrol decreased ROS level by 3 . 2 fold and completely suppressed PECAM-1 expression in ox-LDL-treated platelets. Resveratrol re-covered Sirt1 expression in ox-LDL-treated platelets. EX527 ( a Sirt1 inhibitor ) increased ROS level and PECAM-1 expression in ox-LDL-stimulated platelets. Meanwhile, resveratrol also suppressed p38MAPK phosphorylation induced by ox-LDL. Conclusion Resveratrol can inhibit platelet aggregation, decrease ROS production and PECAM-1 expression in ox-LDL-stimulated platelets. The mechanism maybe associated with recovery of Sirt1 expression. Moreover, resveratrol can decrease PECAM-1 expression, which may be linked to abolishing p38MAPK phosphorylation.

Aim To investigate the effect of ginkgolide B on junctional proteins in ox-LDL-stimulated human umbilical vein endothelial cells ( HUVECs) . Methods After incubation with ginkgolide B ( 0 . 2 ,0 . 4 ,0 . 6 g · L-1 ) for 1 h, HUVECs were treated with ox-LDL (0. 1 g·L-1 ) for 4 h. The expressions of JAM-A and Cx43 were analyzed with Western blot and immunofluo-rescence. The effect of ginkgolide B on vascular per-meability was analyzed by Transwell experiments. Re-sults JAM-A and Cx43 expressions increased by 22%and 24% in ox-LDL-treated HUVECs, respectively. Whereas ginkgolide B significantly decreased JAM-A and Cx43 expressions. LY294002, a specific inhibitor of PI3K, suppressed JAM-A and Cx43 expressions in ox-LDL-stimulated cells. Ginkgolide B potently re-duced monocyte migration in ox-LDL-treated cells. Conclusion Ginkgolide B significantly suppresses JAM-A and Cx43 expressions, and reduces monocyte migration in ox-LDL-stimulated cells. This demon-strates that ginkgolide B can improve vascular permea-bility. The mechanism might be associated with the in-hibition of PI3K/Akt signaling pathway.

Objective To study the effects of type 2 diabetes (T2DM) with hypertension on cognitive function in those community-based elderly who were aged 60 and over,in Beijing.Methods 82 patients with T2DM,142 patients with both T2DM and hypertension and 277 normal controls were investigated in this study.Both methods as:the Montreal Cognitive Assessment (MoCA) and Mini-Mental Status Examination (MMSE) were used to determine cognitive change.Results The total MMSE scores showed significant decrease between T2DM with hypertension and controls [(28.42± 1.52) vs.(28.88± 1.47),P＜0.05].The MoCA score of the total scores [(25.20± 3.91) vs.(26.50 ± 3.29),P＜0.05],sub-scores of visuospatial,executive [(3.60± 1.56) vs.(3.96± 1.18),P＜0.05] and language [(2.10± 0.80) vs.(2.37± 0.80),P＜0.05] significantly decreased in T2DM patients with hypertension and in the normal controls.Data from the Multiple logistic regression analysis showed that older age and less education were risk factors for cognitive impairment.Conclusion T2DM and hypertension damaged the cognitive function of patients.

Objective To investigate the molecular mechanisms of protective effects of thioredoxin (Trx) on human vascular endothelial cells in atherosclerosis.Methods The cell models of Trx-overexpressing cells (Ad Trx) and the control cells (Ad-con) were established by adenovirus vector gene transfer technology in human umbilical vein endothelial cells (HUVECs).The oxidized low density lipoprotein,a risk factor of atherosclerosis,was used as a stimulator.Western blot and indirect immunofluorescence were used to detect the protein expression levels and the cellular localization of Trx,adhesion molecules (ICAM-1,VCAM-1) and the upstream signal pathways.Trx activity was detected by insulin disulfide reduction assay,and cellular reactive oxygen species (ROS)production was detected by fluorescent probe DCFH-DA.Results As compared with control group,Trx protein expression level was enhanced in Ad-trx group and the Trx activity in Ad-Trx group was upregulated by (26.2 ±3.3)％.The result of ROS detection showed that overexpression of Trx significantly inhibited the cellular ROS generation.As compared with control group,overexpression of Trx obviously inhibited the adhesion molecules expression but markedly promoted the phosphorylation of Smad3 in endothelial cells with or without oxLDL stimulation (P＜0.05).Pretreatment of cells with SIS3,a specific inhibitor of Smad3 phosphorylation,reversed Trx-induced inhibition of adhesion molecules expression.Further studies showed that pretreatment of cells with SIS3 enhanced oxLDL-induced AP-1 subunit c-fos nuclear expression.Conclusions The enhancement of Smad3 phosphorylation and c-Fos nuclear expression are mainly responsible for the Trx-induced downregulation of adhesion molecules.