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Objective @# To investigate the influences of circ_WBSCR17 on high glucose-induced fibrosis and inflammation in human mesangial cells by regulating the miR-30a-5p /JAK1 axis．@*Methods @#Human mesangial cells HMCL were grouped into : NG group (5．5 mmol / L glucose-treated HMCL cells) ，HG group (30 mmol / L glucose- treated cells) ，si-NC group (30 mmol / L glucose + transfected with si-NC) ，si-circ_WBSCR17 group (30 mmol / L glucose + transfected with si-circ _ WBSCR17 ) ，si-circ _ WBSCR17 + inhibitor-NC group ( 30 mmol / L glucose + co-transfected with si-circ_WBSCR17 and inhibitor-NC) ，and si-circ_WBSCR17 + miR-30a-5p inhibitor group (30 mmol / L glucose + co-transfected with si-circ_WBSCR17 and miR-30a-5p inhibitor) ; RT-qPCR was performed to detect the expression of circ_WBSCR17 and miR-30a-5p in cells ; CCK-8 assay was performed to detect cell prolifer- ation ; flow cytometry was performed to detect apoptosis ; ELISA was performed to detect the expression levels of tumor necrosis factor-α (TNF-α) ，interleukin (IL) -6 and IL-8 ; Western blot was performed to detect the expression of JAK1，proliferating cell nuclear antigen ( PCNA) ，Bax，transforming growth factor-β1 ( TGF-β1 ) ，fibronectin (FN) ，collagen IV，and α-smooth muscle actin ( α-SMA) ; distribution of WBSCR17 was detected by fluorescence in situ hybridization (FISH) ; dual-luciferase reporter gene experiment was performed to verify the relationship between circ _ WBSCR17 and miR-30a-5p，miR-30a-5p and JAK1，respectively. @*Results @#Compared with the NG group，the HMCL cell proliferation ability of the HG group decreased，the levels of TNF-α , IL-6 and IL-8，the pro- tein expressions of p-JAK1 /JAK1，p-STAT1 / STAT1，p-STAT3 / STAT3，TGF-β1，FN，collagenIV and α-SMA，and the apoptosis ability increased (P＜0. 05) ; compared with HG group and si-NC group，the expression of miR-30a- 5p，OD450 value and PCNA expression in HMCL cells of si-circ_WBSCR17 group increased，the levels of TNF-α , IL- 6 and IL-8，the expressions of circ_WBSCR17，p-JAK1 /JAK1，p-STAT1 / STAT1，p-STAT3 / STAT3，Bax，TGF-β1， FN，collagenIV and α-SMA decreased ( P ＜0. 05 ) ; inhibition of miR-30a-5p attenuated the promoting effect of knockdown of circ_WBSCR17 on proliferation of HMCL cells，and enhanced apoptosis，cellular fibrosis and inflammatory responses ; FISH experiment confirmed that WBSCR17 was mainly distributed in the cytoplasm ; dual-luciferase reporter gene experiment confirmed that circ_WBSCR17，JAK1 and miR-30a-5p had a targeted regulatory rela- tionship．@*Conclusion @#Knockdown of circ_WBSCR17 can reduce high glucose-induced fibrosis and inflammation in human mesangial cells by regulating the miR-30a-5p /JAK1 axis.
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In order to ensure the normal teaching order during the prevention and control of the COVID-19 epidemic, the second semester of the 2019-2020 academic year in Central South University was devoted to the online teaching. In response to the school's call, the diagnostics teaching team has applied the Tencent classroom software, WeChat mini programs, analog teaching software and digital curriculum platform to carry out online teaching activities. On the basis of summarizing the previous online teaching experience, we have made a preliminary discussion and reflection on the online teaching, which will provide ideas and directions for the reform of medical education.
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Objective To investigate the role of calcineurin(CaN)in inflammatory pain in rats.Methods Seventy-five male Harlan-Sprague-Dawley rats,weighting of 200-300 g were randomly divided into 3 groups (n=25): group control (group C),group CFA (complete Freunds adjuvant) (group F) and group CaN+CFA (group NF).100 μl CFA were injected on the right hind claw preparaing for inflammatory pain models in groups F and NF,100 μl saline were injected on the right hind claw in group C.CaN 10 U was intracerebroventricular injected 1 d before CFA injection in group NF.Paw withdrawal thermal latency (PWTL) were measured in 30 min prior to (T0),0.5 h (T1),1 h (T2),2 h (T3) and 4 h (T4) after injection.The expression of CaN and nuclear factor kappa B (NF-κB),IL-1β,TNF-α and IL-10 in spinal cord were measured at each time point.Results The PWTL was significantly shorter at T2-T4 in group F,at T3,T4 in group NF than that at T0and in group C (P<0.05);The PWTL at T2-T4 in group NF was significantly longer than that in group F (P<0.05).CaN protein expression in spinal cord at T1-T4 in group F,at T2-T4 in group NF was significantly lower than that of T0 and in the group C,NF-κB p65 protein expression was significantly higher than that of T0 and in the group C (P<0.05).CaN gene and IL-10 protein content at T2-T4 in groups F and NF were significantly lower than that of group C and at T0,NF-κB gene and IL-1β,TNF-α protein content was significantly higher than that of group C and at T0 (P<0.05).CaN protein and CaN gene expression,IL-10 protein content in spinal cord tissue at T1-T4in group NF was significantly higher than that of group F,NF-κB p65 protein and NF-κB gene expression and contents of IL-1β,TNF-α protein were significantly lower than that of group F (P<0.05).Conclusion CaN adjusts pro-inflammatory and anti-inflammatory cytokines by reducing NF-κB and inhibiting the process of inflammatory pain in rats.
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Objective To evaluate the role of spinal CX3C chemokine receptor 1 (CX3CR1) in inflammatory pain and the relationship with calmodulin (CaM)-calmodulin-dependent protein kinaseⅡ(CaMKⅡ) signaling pathways in mice.Methods Ninety-six pathogen-free healthy male C57BL6 mice,weighing 25-27 g,were divided into 3 groups using a random number table:control group (group C,n=30),inflammatory pain group (group IP,n=36) and CX3CR1 antagonist group (group CA,n=30).Inflammatory pain was induced by injecting complete Freund′s adjuvant (CFA) 50 μl into the plantar surface of right hind paws in IP and CA groups,while the equal volume of normal saline was given instead in group C.In group CA,CX3CR1 antagonist (diluted to 1 μg/5 μl in phosphate buffer solution) was intrathecally injected at 1 h before CFA injection.The thermal paw withdrawal latency (TWL) was measured at 30 min before CFA injection (T0) and 30 min,1 h,2 h and 4 h after CFA injection (T2-4).The animals were then sacrificed,and the spinal cord was removed for determination of the expression of phosphorylated CaMKⅡ (p-CaMKⅡ),phosphorylated cyclic adenosine monophosphate response element-binding protein (p-CREB) and c-fos (by Western blot) and expression of CaMKⅡ,CREB and c-fos mRNA (using real-time polymerase chain reaction).Immunofluorescence was used to determine that p-CAMKⅡ was expressed in microglia.Results Compared with group C,the TWL was significantly shortened at T2-4,and the expression of p-CaMKⅡ,p-CREB and c-fos protein and mRNA was up-regulated at T1-4 in IP and CA groups (P<0.05).Compared with group IP,the TWL was significantly prolonged at T2-4,and the expression of p-CaMKⅡ,p-CREB and c-fos protein and mRNA was down-regulated at T1-4 in group CA (P<0.05).p-CaMKⅡ was co-expressed with the microglial specific biomarker.Conclusion CX3CR1 is involved in the development and maintenance of inflammatory pain through activating CaM-CaMKⅡsignaling pathways in mice.
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Objective To investigate the role of CaM/CaMK-Ⅱ signaling pathways in inflammatory pain in mice.Methods Sixty male C57BL6 mice,weighing 25-27 g,were randomly divided into 3 groups (n =20):control group (group C),complete freunds adjuvant (CFA) group (group F) and KN-93+CFA group (group KF).Saline 50 μl were injected into the right side of the claw in group C.CFA 50 μl were injected into the right claw foot for the preparation of inflammatory pain models in group F.KN-93 45 nmol was injected i.c.v.30 min before CFA injection in group KF.The thermal withdrawal latency (TWL) were measured 30 min before injection,1 h and 4 h after injection.The protein expressions of CaMK-Ⅱ,c-fos and CREB in the spinal cord were measured at above time by Western blot.Results Compared with group C,TWL were lower in groups F and KF 1 h and 4 h after injection (P＜0.05).Compared with groups F,TWL in group KF were higher 1 h and 4 h after injection (P＜0.05).Compared with group C,the protein expressions of p-CaMK-Ⅱ,p-CREB,e-fos and mRNA expression of CaMK-Ⅱ,CREB,c-fos were higher in group F and KF 1 h and 4 h after injection (P＜0.05).Compared with group F,the protein expression of p-CaMK-Ⅱ,p-CREB,c-fos and mRNA expressions of CaMK-Ⅱ,CREB,c-fos in group KF were lower 1 h and 4 h after injection (P＜0.05).Conclusion CaM/CaMK-Ⅱ signaling pathways involved in inflammatory pain in mice.
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Objective To investigate the effects of early estrogen replacement therapy(ERT) with different doses on aortic endothelial senescence and its possible mechanism. Methods Twenty- eight healthy New Zealand white female rabbits were randomized into four groups: group A(n=7), in sham operation;group B(n=7), ovariectomized;group C(n=7), ovariectomized and in low-dose ERT(estradiol benzoate 200 μg,im, QOD) ;and group D(n=7), ovariectomized and in high-dose ERT (estradiol benzoate 1000 μg,im,QOD). All rabbits were fed with high fat diet and the rabbits in group C and group D were given ERT from the 7th day after the operation. Blood samples taken via ear central artery before the operation and after the high fat diet for 4 and 12 weeks were used to determine the concentrations of serum estradiol(E2), total cholesterol(TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), trigtyceride (TG) and asymmetric dimethylarginine(ADMA). After 12 weeks, the aorta was separated for histopathologic analysis and the areas of senescent endothelium and atherosclerotic plaque were calculated.Results (1)After ovariectomized, the concentration of serum estradiol(E2) was significantly lower in group B than in other groups (all P<0.05). Twelve weeks later, the level of E2in was higher group D than in group A and C(both P<0. 05), but there was no significant difference between group C and group A (P< 0. 05). (2)After the high-fat diet for 4 and 12 weeks, there was a notable increase of TC, LDL-C, TG and HDL-C levels in rabbits of each group(all P<0. 01). After 12 weeks, the levels of TC and LDL- C were remarkably higher in group B than in other groups(all P<0. 01), however, the levels of TG and HDL-C were lower in group B than in other groups(all P<0. 01). The concentrations of TC and LDL-C in group C and group D were lower than those in group A (all P<0.01), but the differences of the levels of TG and HDL-C in group C and group D were not significant in comparison with group A (all P>0. 05). There were no significant differences in the lipid levels between group C and D (all P> 0. 05). (3)After 12-week high fat diet, there were a striking increases of serum ADMA level in all four groups (all P>0. 05). The level of ADMA was higher in group B than in other groups (P< 0. 05) andlower in group C and D than that in group A (both P<0. 05). There was no significant difference between group C and D (P<0. 05). (4)12 weeks later, the areas of senescent endothelium and atherosclerotic plaque in group B were significantly larger than those in other groups (all P< 0. 01), and the areas were smaller in group C and D than those in group A (all P<0. 01), there was no significant difference between group C and group D(both P>0.05). (5)Linear correlation analysis showed that there was an obvious positive correlation between the areas of senescent endothelium and atherosclerotic plaque(r=0. 962, P<0. 01), and both of them were positively correlated with the levels of serum AMDA (r=0. 812,0. 824,0. 755,0. 797,0. 749,0. 727), TC (r=0. 812,0. 824) and LDL-C(r=0. 755,0. 797) (all P<0. 01) and negatively correlated with the concentration of serum E2 (r=-0. 762,-0. 743, both P<0. 01).Conclusions Early ERT can improve serum lipid metabolism, reduce ADMA level, inhibit vascular endothelial senescence and attenuate atherogenesis. The delaying effect of estrogen on endothelial senescence may be due to its improving lipid metabolism and lowering ADMA level.