Liraglutide protects nucleus pulposus cells by inhibiting the activation of NLRP3 inflammasome in high glucose environment / 中国糖尿病杂志

Objective To evaluate the protective effect of human nucleus pulposus cell by Liraglutide(Lir)under high-glucose environment via inhibition of NOD-like receptor family,pyrin domain containing 3(NLRP3)inflammasome activation and its mechanism.Methods Human nucleus pulposus cell lines were cultured and the third-generation nucleus pulposus cells were randomly divided into control group(Con group),high glucose group(HG group),and Liraglutide interference group(Lir group),and cultured for 48 h.ELISA was used to detect interleukin IL-1β;flow cytometry was used to testreactive oxygen species(ROS),and Western blot was used to evaluate the protein expressions of NLRP3,Pro-caspase-1,and Caspase-1.Results Compared with Con group,IL-1β,ROS,NLRP3,Pro-caspase-1,Caspase-1 protein expression and cell apoptosis were increased in HG and Lir groups(P<0.05).Compared with HG group,IL-1β,ROS,NLRP3,Pro-caspase-1,Caspase-1 protein expression and cell apoptosis were significantly decreased in Lir group(P<0.05).Conclusion Liraglutide protects human NPCs by inhibiting the activation of NLRP3 inflammasome,reducing IL-1β secretion,and inhibiting cell apoptosis.

Rubescensine A reduces podocyte damage induced by high glucose through the autophagy pathway mediated by AMPK/SIRT1 pathway / 中国糖尿病杂志

Objective To investigate the mechanism that Rubescensine A reduces the podocyte damage induced by high glucose(HG)through the autophagy pathway mediated by AMP activated protein kinase/silent information regulator 1(AMPK/SIRT1)pathway.Methods Human glomerular podocytes were cultured in vitro,and randomly divided into Control group(Con),HG group,hydroxychloroquine(HCQ)group,and Rapamycin(RAP)group.CCK-8 was used to detect cell viability.Western blotting was used to detect cell apoptosis and podocyte injury related protein expression in each group.The podocyte model induced by high glucose(HG)was treated with Rubescensine A(Rub A)at different concentrations and the optimal concentration was selected.Then,human glomerular podocytes were randomly divided into Con group,HG group,Rub A group,Compound C group,and Rub A+Compound C group.The expression of autophagy,AMPK/SIRT1 pathway related proteins were detected in each group.Results Compared with Con group,the podocyte viability and the protein expressions of Synaptopodin and Bcl-2 was significantly reduced(P<0.05),while the protein expressions of Desmin and Bax were significantly increased in HG group(P<0.05).Compared with the HG group,all indicators were relieved in RAP group.However,the levels of all indicators were worsened in HCQ group.Compared with Con group,the expression levels of Desminand Bax proteins in podocytes were significantly increased(P<0.05),and the podocyte viability,number of autophagosomes,the expression levels of Synaptopodin,Bcl-2,microtubule associated protein light chain 3(LC3)II/I,Beclin-1,p-AMPK/AMPK and SIRT1 proteins were significantly reduced in HG group(P<0.05).Compared with HG group and Rub A+Compound C group,the above indicators were improved in Rub A group.Compound C group reversed the protective effect of Rub A.Conclusion Rubescensine A can promote autophagy by activating AMPK/SIRT1 pathway,thereby reduce podocyte damage induced by high glucose.

Effect of circLRP6 on high glucose-induced renal tubular epithelial cell injury via miR-31-5p/HMGA1 axis regulation / 中国医科大学学报

Objective To analyze the effect of circLRP6 on high glucose-induced renal tubular epithelial cell injury via miR-31-5p/high mobility group protein A1(HMGA1)axis regulation.Methods Human renal tubular epithelial HK-2 cells were cultured in vitro and divided into eight groups:control,high glucose,high glucose+si-NC,high glucose+si-circLRP6,high glucose+si-circLRP6+miR-NC,high glucose+si-circLRP6+miR-31-5p inhibitor,high glucose+si-circLRP6+miR-31-5p inhibitor+si-NC,and high glucose+si-circ-LRP6+ miR-31-5p inhibitor+si-HMGA1.The circLRP6,miR-31-5p,and HMGA1 mRNA levels were determined using real-time quantitative PCR.Cell supernatant IL-6 and tumor necrosis factor-α(TNF-α)levels,lactate dehydrogenase(LDH)activity,and malondialdehyde(MDA)content were also determined.Furthermore,flow cytometry was used to observe cell apoptosis.HMGA1,Bax,and Bcl-2 protein expression was detected by Western blotting.Finally,dual luciferase assay was used to report the targeting relationship of miR-31-5p with circLRP6 and HMGA1.Results Compared with the high glucose group,the HK-2 cell proliferation inhibition rate;cell superserum IL-6,TNF-α,LDH,and MDA levels;apoptosis rate;and Bax protein expression in the high glucose+si-circLRP6 group decreased significantly,whereas Bcl-2 protein expression increased significantly(all P＜0.05).Consequently,miR-31-5p downregulation possibly weakened the protective effect of si-circLRP6 on high glucose-induced renal tubular epithelial cell injury.HMGA1 expression inhibition reversed the effect of the si-circLRP6+miR-31-5p inhibitor on high glucose-induced renal tubular epithelial cell injury.Finally,miR-31-5p exhibited a targeting relationship with circLRP6 and HMGA1.Conclusion Si-circLRP6 protects high glucose-induced renal tubular epithelial cell injury via miR-31-5p upregulation and HMGA1 expression inhibition.

microRNA-26a inhibits extracellular matrix synthesis in high glucose-induced renal tubular epithelial cells by regulating ferroptosis / 安徽医科大学学报

Objective To investigate the effect and possible mechanism of microRNA-26a(miR-26a)on the syn-thesis of extracellular matrix(ECM)induced by high glucose(HG)in renal tubular epithelial cells(RTECs).Methods A model of diabetic kidney disease(DKD)was constructed by inducing RTECs with HG.MiR-26a was overexpressed in HG-induced RTECs,and RT-qPCR and Western blot were used to assess the effects of miR-26a on ECM synthesis and ferroptosis-related markers in HG-treated RTECs.Ferrostatin(Fer-1)was used to inhibit ferroptosis in the DKD model,and its impact on ECM synthesis was evaluated.RT-qPCR and Western blot were performed to measure ferroptosis-related markers,and fluorescence microscopy was used to observe the intensity of reactive oxygen species(ROS).Results Compared with the control group,the expression of miR-26a decreased in HG-treated cells,while the expression levels of ECM synthesis-related indexes fibronectin and collagen Ⅰ in-creased.After overexpressing miR-26a,the HG+miR-26a group showed a significant increase in miR-26a expres-sion and a decrease in fibronectin and collagen Ⅰ expression compared to the HG group.In terms of ferroptosis,the protein and mRNA expression of SLC7A11 and GPX4 significantly decreased,the expression of TFR-1 and AC-SL4 significantly increased,and the fluorescence intensity of ROS was significantly enhanced in the HG group com-pared with the control group.Inhibition of ferroptosis in the HG+Fer-1 group resulted in significant changes in fer-roptosis and ECM synthesis-related indicators expression levels compared to the HG group.Furthermore,re-expres-sion of miR-26a in the HG+miR-26a led to significant changes in ferroptosis-related indicators expression levels and decreased ROS fluorescence intensity compared to the HG group.Conclusions In HG-induced RTECs,miR-26a inhibits the occurrence of ferroptosis,thus reducing ECM synthesis.

Expression and role of triggering receptor expressed on myeloid cells 2 in high glucose-treated microglia / 基础医学与临床

Objective To explore the expression of triggering receptor expressed on myeloid cells 2(TREM2)in high-glucose microglia and to investigate the role of TREM2 in the proliferation,migration and phagocytosis of high-glucose microglia.Methods Microglia cells were divided into control group and high-glucose treatment group(67.5 mmol/L glucose,24 h).The microglia cells were counted and the expression of Iba1 and TREM2 was de-tected.TREM2 siRNA was transfected to detect the proliferation and migration of microglia.The amyloid β-peptide(Aβ)with a fluorescent tag was added to observe the phagocytosis of Aβ by microglia.Results Compared to normal microglia,the number of microglia significantly decreased after high-glucose treatment(P＜0.001),while the ex-pression of TREM2 and Iba1 markedly increased(P＜0.001).High glucose and TREM2 did not affect the prolifer-ation of microglia.Compared to the normal group,the migration of microglia significantly decreased after high-glu-cose treatment(P＜0.05)and TREM2 did not affect the migration ability of high-glucose microglia.Compared to the normal group,the phagocytosis of Aβ by microglia significantly decreased in the high-glucose treated group(P＜0.001).Furthermore,TREM2 knockdown further decreased the phagocytosis of Aβ by high-glucose microglia(P＜0.001).Conclusions The expression of TREM2 in microglia significantly increases after high-glucose treat-ment,which significantly affects phagocytosis of Aβ by microglia.

Effect of glycyrrhizin on inflammatory factors and fibrotic factors in high glucose-induced glomerular mesangial cells / 解放军医学杂志

Objective To investigate the effect of glycyrrhizic acid(GA)on the inflammatory and fibrotic factors in high glucose-induced glomerular mesangial cells(SV40 MES13).Methods Cultured mouse SV40 MES13 were divided into normal group(NG,5.6 mmol/L glucose),high glucose group(30 mmol/L glucose)and HG+GA group(30 mmol/L glucose+200 μmol/L GA).The expression levels of inflammatory cytokines interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),IL-6,IL-8 and α-smooth muscle actin(α-SMA)in different groups were detected by Western blotting.The fluorescence intensity of IL-1β,TNF-α and α-SMA in different groups were detected by immunofluorescence.The levels of IL-1β,TNF-α,IL-6 and IL-8 in the culture supernatant of different populations were detected by enzyme-linked immunosorbent assay(ELISA).Results The protein expressions of IL-1β,TNF-α,IL-6,IL-8 and α-SMA in HG group were significantly higher than those in NG group(P<0.01);Compared with HG group,the protein expression levels of IL-1β,TNF-α,IL-6,IL-8 and α-SMA decreased significantly in HG+GA group(P<0.05).The fluorescence intensity of inflammatory cytokines IL-1β,TNF-α and α-SMA increased in HG group than those in NG group(P<0.05);While compared with the HG group,the fluorescence intensity of IL-1β,TNF-α and α-SMA in HG+GA group decreased markedly(P<0.05).The experimental results of ELISA showed that compared with NG group,the levels of IL-1β,IL-6,TNF-α and IL-8 in cell supernatent increased in HG group(P<0.01);while the levels of IL-1β,TNF-α,IL-6,IL-8 in HG+GA group significantly lower than those in HG group(P<0.05).Conclusion Glycyrrhizic acid has certain inhibitory effect on high glucose-induced inflammatory factors and fibrotic factors in glomerular mesangial cells,which may play an important role in prevention of diabetic nephropathy.

Polycaprolactone-polydopamine-AOPDM1 scaffold promotes bone formation in a high-glucose environment / 中国组织工程研究

BACKGROUND:Oral and maxillofacial bone tissue defects can seriously affect the physical and mental health of patients.When bone defects occur in diabetic patients,bone metabolism disorders caused by abnormal blood sugar make it more difficult to repair and treat. OBJECTIVE:To attempt to apply AOPDM1,a polypeptide with potential bioactivity to the osteogenic treatment of diabetic patients. METHODS:In normal or high-glucose environment,different concentrations of AOPDM1 were used to interfere with mouse bone marrow mesenchymal stem cells,and cell proliferation,alkaline phosphatase activity,mineralization nodules formation and osteogenic differentiation gene expression were detected.The polycaprolactone scaffold was prepared by electrospinning technology,and the scaffold was modified by polydopamine to prepare the polycaprolactone-polydopamine composite scaffold.Finally,the scaffolds were placed in AOPDM1 solution to prepare polycaprolactone-polydopamine-AOPDM1 scaffolds.The water contact angle and mechanical properties of the scaffolds were tested in the three groups.In normal or high-glucose environment,the three groups of scaffolds were co-cultured with mouse bone marrow mesenchymal stem cells,respectively,and cell adhesion,alkaline phosphatase activity and osteopontin expression were detected. RESULTS AND CONCLUSION:(1)Compared with normal environment,high-glucose environment inhibited the proliferation of bone marrow mesenchymal stem cells.In the same environment,AOPDM1 could promote the proliferation of mouse bone marrow mesenchymal stem cells.When AOPDM1 concentration was the same,alkaline phosphatase activity,mineralization ability and mRNA expression of type Ⅰ collagen,osteopontin,alkaline phosphatase,and Runx2 of bone marrow mesenchymal stem cells were decreased in high-glucose environment compared with normal environment.Under the same environment,AOPDM1 could improve the alkaline phosphatase activity,mineralization ability,and mRNA expression of type Ⅰ collagen,osteopontin,alkaline phosphatase and Runx2 of bone marrow mesenchymal stem cells.(2)The hydrophilicity of polycaprolactone-polydopamine scaffold and polycaprolactone-polydopamine-AOPDM1 scaffold was higher than that of polycaprolactone scaffold(P<0.001),and there was no significant difference in tensile strength and elastic modulus among the three groups(P>0.05).Compared with the other two groups of scaffolds,the cells on the polycaprolactone-polydopamine-AOPDM1 scaffold had better adhesion morphology.When the scaffolds were identical,compared with normal environment,high-glucose environment inhibited alkaline phosphatase activity and osteopontin expression of bone marrow mesenchymal stem cells.When the environment was the same,the alkaline phosphatase activity and osteopontin expression of bone marrow mesenchymal stem cells on the polycaprolactone-polydopamine-AOPDM1 scaffold were higher than those on the other two scaffolds.(3)The above results prove that polycaprolactone-polydopamine-AOPDM composite scaffold can promote the osteogenic properties of bone marrow mesenchymal stem cells in high-glucose environment.

Berberine promotes osteogenic differentiation of bone marrow mesenchymal stem cells in a high-glucose environment / 中国组织工程研究

BACKGROUND:The implant osseointegration rate of patients with diabetes is low,and the failure rate is high,which seriously affects the quality of life.It is urgent to improve the implant osseointegration of patients with diabetes by effective means to elevate the success rate.Exploring the effect of berberine on the osteogenic differentiation of bone marrow mesenchymal stem cells under a high-glucose environment and its specific mechanism will provide effective theoretical support for solving the above problems. OBJECTIVE:To explore the effect of natural extract berberine on the osteogenic differentiation of rat bone marrow mesenchymal stem cells under the high-glucose microenvironment. METHODS:Bone marrow mesenchymal stem cells of SD rats were cultured by the whole bone marrow adherence method.CCK-8 assay was used to detect the effects of different concentrations of berberine on the proliferation of bone marrow mesenchymal stem cells under the high-glucose environment and to screen out the optimal berberine concentration.The expressions of Runx2 and Osx were detected by alkaline phosphatase activity,alicarin red staining and PCR to determine the effect of berberine on osteogenic differentiation of bone marrow mesymal stem cells under the high-glucose environment.To further explore the underlying mechanism,we introduced the AMPK-specific inhibitor Dorsomorphin and used a DCFH-DA reactive oxygen species fluorescent probe to examine reactive oxygen species levels.The p-AMPK expression was also determined by western blot assay. RESULTS AND CONCLUSION:(1)10 μmol/L was the optimal concentration of berberine to promote bone marrow mesenchymal stem cell proliferation.(2)Alberberine promoted alkaline phosphatase viability of bone marrow mesenchymal stem cells and mineralized nodule formation in a high-glucose microenvironment.(3)Alberberine promoted the expression of Runx2 and OSx in a high-glucose microenvironment.(4)Alberensine effectively inhibited the reactive oxygen species level of bone marrow mesenchymal stem cells in a high-glucose environment.(5)The effects of berberine on promoting bone marrow mesenchymal stem cell osteogenesis and inhibition of reactive oxygen species were reversed by the AMPK inhibitor.(6)Berberine activated AMPK and promoted p-AMPK expression.(7)The above results indicate that berberine(10 μmol/L)promotes the osteogenic differentiation of bone marrow mesenchymal stem cells in a high-glucose environment by activating AMPK and reducing intracellular reactive oxygen species levels.

Effect of Icariin Regulating mTOR/Akt/CREB Pathway on High Glucose Induced Autophagy and Apoptosis in Podocytes / 医药导报

Objective To investigate the effects of icariin on high glucose-induced autophagy and apoptosis of podocytes,and the regulating effects on mammalian target of rapamycin(mTOR)/serine-threonine kinase(Akt)/cyclic adenosine monophosphate response element binding protein(CREB)pathway.Methods The mouse podocytes MPC5 were taken and divided into five groups:normal control group(5.5 mmol·L-1 glucose),high glucose group(30 mmol·L-1 glucose),icariin group(30 mmol·L-1glucose+5 μmol·L-1icariin),GDC-0349 group(30 mmol·L-1glucose+50 μmol·L-1 GDC-0349),icariin+GDC-0349 group(30 mmol·L-1 glucose+5 μmol·L-1 icariin+50 μmol·L-1 GDC-0349).Cultured for 48 hours,the tetramethylazozolium salt method was used to detect the viability of MPC5 cells;acridine orange staining was used to observe the autophagy of MPC5 cells;apoptosis of MPC5 cells was detected by flow cytometry;Western blotting was used to detect the expression of autophagy[microtubule associated protein one light chain 3(LC3)II,LC3Ⅰ,autophagy-related protein(Beclin-1)],apoptosis[Bcl-2 related X protein(Bax),B cell lymphoma-2(Bcl-2)]and mTOR/Akt/CREB pathway-related proteins of MPC5 cells.Results Compared with the normal control group,the cell viability,expression levels of Bcl-2,phosphorylated mTOR(p-mTOR)/mTOR,phosphorylated Akt(p-Akt)/Akt,phosphorylated CREB(p-CREB)/CREB protein of MPC5 cells in the high glucose group were significantly decreased(P＜0.05),the autophagy ability was enhanced,the autophagosome showed orange fluorescence,and the apoptosis rate,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bax protein expression levels were significantly increased(P＜0.05).Compared with the high glucose group,the cell viability,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bcl-2,p-mTOR/mTOR,p-Akt/Akt,p-CREB/CREB protein expression levels of MPC5 cells in icariin group were significantly increased,the autophagy ability was further enhanced,the number of autophagosomes was increased,the autophagosomes showed brick red fluorescence(P＜0.05),the apoptosis rate and Bax protein expression level were significantly decreased(P＜0.05),and the cell viability,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bcl-2,p-mTOR/mTOR,p-Akt/Akt and p-CREB/CREB proteins expression levels of MPC5 cells in GDC-0349 group were significantly decreased,the autophagy ability was weakened,the number of autophagosomes was reduced,the autophagosomes showed orange fluorescence(P＜0.05),and the apoptosis rate and Bax protein expression level were significantly increased(P＜0.05);icariin+GDC-0349 could reverse the effect of icariin on high glucose induced MPC5 cells(P＜0.05).Conclusion Icariin promotes elevated glucose-induced podocyte autophagy and inhibits apoptosis by activating the mTOR/Akt/CREB pathway.

Impact of evodiamine on the proliferation，migration and invasion of trophoblastic cells induced by high glucose and its mechanism / 中国药房

OBJECTIVE To investigate the impact of evodiamine on the proliferation， migration and invasion abilities of trophoblastic cells induced by high glucose and its potential mechanism based on advanced glycation end products （AGE）/receptor for advanced glycation end products （RAGE） signaling pathway. METHODS Human trophoblastic cells HTR-8/SVneo were divided into control group， high glucose group， evodiamine low-dose group （2 μmol/L）， evodiamine high-dose group （4 μmol/L）， pc-NC group （transfected with pc-NC plasmid+4 μmol/L evodiamine）， and pc-RAGE group （transfected with pc-RAGE plasmid+ 4 μmol/L evodiamine）. Cells in all groups except for the control group were cultured in a high sugar （25 mmol/L glucose） environment， and cells in all groups except for the control group and the model group were transfected with the corresponding plasmids and/or received the corresponding drug interventions. The survival rate， apoptotic rate， scratch healing rate， and invasion number， as well as the protein and mRNA expressions of AGE， RAGE， nuclear factor- κB p65 （NF- κB p65）， matrix metalloproteinase-2 （MMP-2）， and MMP-9 were examined in each group. RESULTS Compared with the control group， the cell survival rate， scratch healing rate， invasions number， and the mRNA and protein expressions of MMP-2 and MMP-9 in the high glucose group significantly decreased （P＜0.05）， while the apoptotic rate， the mRNA and protein expressions of AGE and RAGE， the mRNA expression of NF-κB p65， and the phosphorylation level of NF-κB p65 protein significantly increased （P＜0.05）； compared with the high glucose group， the above indexes of cells in evodiamine low-dose and high-dose groups were significantly improved， and the effect of the high-dose group was significantly better than that of the low-dose group （P＜0.05）； overexpression of RAGE attenuated the ameliorative effect of evodiamine onthe above indexes in high glucose-induced trophoblast cells （except for AGE mRNA and protein） （P＜0.05）. CONCLUSIONS Evodiamine can promote the proliferation， migration and invasion of high glucose-induced trophoblast cells and ameliorate their functional impairment， and the above effects are associated with the inhibition of the AGE/RAGE signaling pathway.

The protective effect of terpinen-4-ol on inflammatory injury of VSMCs induced by high glucose through improving autophagic flux disorder / 中国药理学通报

Aim To investigate the effect of terpinen-4-ol (T40) on inflammatory injury of vascular smooth muscle cells (VSMCs) induced by high glucose based on the improvement of autophagic flow disorder and involved molecular signals. Methods The scratch test was used to analyze the migration ability of VSMCs, the levels of IL-1β and IL-6 in cell culture supernatant were measured by ELISA, the expression levels of inflammation-related proteins NF-κb p65, p-NF-κb p65, IL-1β, IL-18 and autophagy-related proteins p62, LC3-HYLC3-I, Beclinl, p-Beclinl were de-tected by Western blot. Results T40 inhibited migration of VSMCs induced by high glucose, reduced the secretion and release of pro-inflammatory factors IL-1β and IL-6, inhibited the expression of p-NF-κb p65/ NF-κb p65, IL-1β, IL-18, downregulated the expression of p62, LC3-TJ/LC3- I and p-Beclinl at same time. After interfering the autophagic flux of VSMCs with autophagy inhibitor chloroquine (CQ) , T40 pre-treatment significantly inhibited the protein expression levels of the above inflammatory factors and autophagy-related signals which mediated by CQ. Conclusion T40 inhibits the inflammatory injury of VSMCs induced by high glucose through improving the autophagic flow disorder.

Effects of DNMT3A on mouse cardiac fibroblast proliferation and migration under high glucose environment / 中国药理学通报

Aim To investigate the effect of DNA methyltransferase 3A (DNMT3A) on the proliferation and migration of cardiac fibroblasts (CFs) in C57 mice under high glucose environment. Methods The hearts of C57 mice were taken from 1 to 3 days. After cutting and digesting, CFs were extracted by differential adherance centrifugattion and observed under microscope. After cell attachment, the cells were cultured under low glucose (5.5 mmol • L

Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells / 华西口腔医学杂志

OBJECTIVES@#This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients.@*METHODS@#HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay.@*RESULTS@#1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05).@*CONCLUSIONS@#1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

PPAR-γ inhibits the proliferation of human large cell lung cancer NCI-H460 cells through the NLRP3 inflammasome in a high-glucose microenvironment / 中国医科大学学报

Objective To investigate the effect of PPAR-γon the proliferation of human large cell lung cancer(NCI-H460)cells in a high-glucose microenvironment and to explore the associated molecular mechanism.Methods NCI-H460 cells were treated with normal-glucose(blank group),hypertonic(control group),and high-glucose(30 mmol/L;high-glucose group)media.The effects of a high-glucose microenvironment on the proliferation of NCI-H460 cells were analyzed using Cell Counting Kit 8(CCK-8)and colony-for-mation assays.The specific PPAR-γactivator,rosiglitazone,was used to treat NCI-H460 cells in a high-glucose microenvironment,and the expression of NLRP3-inflammasome-related proteins was detected by Western blotting.The effect of PPAR-γon the proliferation of NCI-H460 cells in a high-glucose microenvironment was analyzed by CCK-8 and clony-formation assays.The NLRP3 inflammasome agonist,NSS,combined with rosiglitazone,were used to treat NCI-H460 cells in a high-glucose microenvironment.CCK-8 and clony-formation assays were used to analyze whether the NLRP3 inflammasome was involved in the inhibitory effect of PPAR-γon the proliferation of NCI-H460 cells in a high-glucose microenvironment.Results A high-glucose microenvironment significantly induced the proliferation of NCI-H460 cells,increased the activity of the NLRP3 inflammasome,and reduced the expression levels of PPAR-γprotein.Rosiglitazone effectively inhibited NLRP3 inflammasome activity and NCI-H460 cell proliferation,and this effect was reversed by NLRP3 inflammasome agonist.Conclusion PPAR-γinhibits the proliferation of NCI-H460 cells in a high-glucose microenvironment by down-regulating the activity of the NLRP3 inflammasome.

miR-148a-3p improves mitochondrial injury and apoptosis induced by high-glucose through inhibiting CRT expression / 西安交通大学学报(医学版)

【Objective】 To evaluate the effects of miR-148a-3p on calreticulin (CRT) expression and mitochondrial function in cardiomyocytes incubated with high glucose. 【Methods】 miR-148a-3p minic and inhibitor were used to intervene the H9c2 cardiomyocytes of rats. The expression of CRT protein was detected. Then the cells were divided into control group, high-glucose group (HG), HG +miR-148a-3p minic group, HG + miR-148a-3p minic + TG (CRT agonist) group, HG + miR-148a-3p inhibitor group, and HG + miR-148a-3p inhibitor + CRT- (CRT-siRNA) group. The content of adenosine triphosphate (ATP) and the level of reactive oxygen species (ROS), the activity of mitochondrial respiratory chain complex enzyme and apoptotic rate were detected. 【Results】 miR-148a-3p minic significantly inhibited the expression of CRT protein in cardiomyocytes, while miR-148a inhibitor increased the expression of CRT. miR-148a-3p minic inhibited the decrease of ATP production, the increase of ROS production and cell apoptosis, and the inactivity of mitochondrial respiratory chain complex enzyme in cardiomyocytes induced by high glucose, while TG weakened the above effects of miR-148a-3p minic. miR-148a inhibitor aggravated the mitochondrial injury and apoptosis of cardiomyocytes induced by high glucose, but the effects of miR-148a-3p inhibitor were partially blocked by CRT-siRNA. 【Conclusion】 miR-148a-3p negatively regulates the expression of CRT in cardiomyocytes and protects the mitochondrial injury and apoptosis induced by high-glucose through inhibiting CRT.

Terpinen-4-ol inhibits proliferation of VSMCs exposed to high glucose via regulating KLF4/NF-κB signaling pathway / 中国中药杂志

This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G_1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G_1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.

Effects of miR-34a on polarization and inflammatory factor content of mouse macrophage cell line RAW264.7 under high glucose conditions / 基础医学与临床

Objective To detect the role of miR-34a on macrophage inflammation and polarization under high glucose conditions.Methods Mouse macrophages were collected and cultured during high glucose for 3－28 days.RT-qPCR was used to detect the expression of miR-34a,iNOS,MCP-1 and Arg-1 mRNA.Then miR-34a was over-expressed or silenced,ELISA was used to detect the expression of IL-6,IL-1β,TNF-α and qRT-PCR was used to detect the expression of iNOS and MCP-1 mRNA.Western blot was used to detect the expres-sion of Notch1.Results Expression of miR-34a increased under high glucose conditions in RAW264.7 cells continuously.Over-expression of miR-34a promoted the expression of MCP-1 and iNOS observed by RT-qPCR and increased the expression of IL-6,IL-1β and TNF-α detected by ELISA.Further studies showed that siRNA-Notch1 down-regulated the expression of miR-34a,MCP-1,iNOS,IL-6,IL-1β and TNF-α.Conclusions Chronic high glucose condition stimulates the expression of miR-34a which promotes M1 macrophage polarization and releasing of pro-inflammatory factors.

Impact of LncRNA TUG1 on high glucose-induced cardiomyocyte apoptosis by regulating the miR-181b-5p/PDCD4 axis / 天津医药

Objective To investigate the impact of long non-coding RNA(LncRNA)taurine up-regulated gene 1(TUG1)on high glucose-induced cardiomyocyte apoptosis by regulating miR-181b-5p/programmed cell death protein 4(PDCD4)axis.Methods Diabetic cardiomyopathy(DCM)cell model was established in vitro with high glucose(HG,25 mmol/L glucose).AC16 cells were divided into the NG(5.5 mmol/L glucose)group,the HG group,the HG+sh-NC group,the HG+sh-TUG1 group,the HG+miR-NC group,the HG+miR-181b-5p group,the HG+sh-TUG1+anti-miR-NC group,the HG+sh-TUG1+anti-miR-181b-5p group,the HG+miR-181b-5p+pcDNA group and HG+miR-181b-5p+pc-PDCD4 group.The Cell Counting Kit-8(CCK-8)method was applied to detect cell viability.Lactate dehydrogenase(LDH)assay was applied to detect LDH release.Quantitative real-time polymerase chain reaction(qRT-PCR)was applied to detect expression levels of TUG1,miR-181b-5p and PDCD4 mRNA.Flow cytometry was applied to detect apoptosis.Western blot assay was applied to detect levels of B-cell lymphoma 2-associated X(Bax),activated caspase 3(cleaved caspase 3)and PDCD4 proteins.Caspase-Glo3 assay was applied to assess caspase 3 activity.Dual-luciferase reporter assay was applied to verify the targeting relationship between TUG1 or PDCD4 and miR-181b-5p.Results Compared with the NG group,the cell activity decreased in the HG group,and LDH release,apoptosis rate,Bax,cleaved caspase 3 expression and caspase 3 activity increased(P＜0.05),which could be antagonized by TUG1 knockdown or miR-181b-5p overexpression(P＜0.05).Inhibition of miR-181b-5p was able to alleviate the impact of TUG1 silencing on cardiomyocyte viability and apoptosis under high glucose treatment(P＜0.05).The overexpression of PDCD4 attenuated the promotion effect of miR-181b-5p up-regulation on the viability of cardiomyocytes treated with high glucose and the inhibitory effect on apoptosis.TUG1 was able to increase the expression of PDCD4 through adsorption of miR-181b-5p(P＜0.05).Conclusion TUG1 promotes high glucose-induced cardiomyocyte apoptosis by down-regulating miR-181b-5p and up-regulating PDCD4.

Effect of micro ribonucleic acid-1-3p on high glucose-induced neovasculariza-tion of retinal microvascular endothelial cells through regulation of Annexin A2 expression / 眼科新进展

Objective To investigate the possible action mechanism of the micro ribonucleic acid-1-3p(miR-1-3p)/Annexin A2(AnxA2)molecular axis in high glucose(HG)-induced neovascularization of human retinal microvascular en-dothelial cells(HRMECs).Methods A cell injury model was established by culturing HRMECs in vitro and treating them with HG.The HRMECs were divided into the Con group(DMEM medium containing fetal bovine serum in volume fraction of 10％),HG group(cultured in 25 mmol·L-1 D-glucose),HG+miR-NC group(transfected with miR-NC),HG+miR-1-3p group(transfected with miR-1-3p mimics),HG+sh-NC group(transfected with sh-NC),HG+sh-AnxA2 group(transfect-ed with sh-AnxA2),HG+miR-1-3p+pcDNA group(transfected with miR-1-3p mimics+pcDNA),and HG+miR-1-3p+pcDNA-AnxA2 group(transfected with miR-1-3p mimics+pcDNA-AnxA2).After 48 h of transfection,cells were collected and cultured in 25 mmol·L-1 D-glucose medium for 24 h.Cell viability and number of migrating cells were detected using MTT and Transwell chamber experiments,respectively.The number of lumen formations was detected by the lumen forma-tion experiment.The dual luciferase reporter assay was adopted to detect the targeting relationship between miR-1-3p and AnxA2.Western blot was used to detect the protein levels of vascular endothelial growth factor(VEGF)and matrix metal-loproteinase-2(MMP-2).Results Compared with the Con group,the expression level of miR-1-3p in the HG group de-creased,while the levels of AnxA2 messenger ribonucleic acid(mRNA)and protein increased,with statistically significant differences(all P＜0.05).Compared with the Con group,the HG group showed an increase in cell viability,number of mi-grating cells,lumen formation and protein levels of VEGF and MMP-2,with statistically significant differences(all P＜0.05).Compared with the HG+miR-NC group,the HG+miR-1-3p group showed a decrease in cell viability,number of migrating cells,lumen formation and protein levels of VEGF and MMP-2,with statistically significant differences(all P＜0.05).Compared with the HG+sh-NC group,the HG+sh-AnxA2 group showed a decrease in cell viability,number of mi-grating cells,lumen formation and protein levels of VEGF and MMP-2,with statistically significant differences(all P＜0.05).Compared with the HG+miR-1-3p+pcDNA group,the HG+miR-1-3p+pcDNA-AnxA2 group showed an increase in cell viability,number of migrating cells,lumen formation and protein levels of VEGF and MMP-2,with statistically signifi-cant differences(all P＜0.05).Conclusion Overexpression of miR-1-3p can inhibit proliferation,migration and neovas-cularization of HRMECs by targetedly regulating AnxA2 expression.

Impact of downregulated miR-99a with high glucose on hepatic sinusoidal dysfunction and intervention of metformin / 中华内分泌代谢杂志

Objective:To observe the effect of high glucose downregulated microRNA(miR)-99a on hepatic sinus dysfunction and metformin intervention, and to explore the pathogenesis of diabetes-induced fatty liver and possible mechanism of metformin.Methods:The cultured human liver sinusoidal endothelial cells were randomly divided into normal control group, high glucose model group, miR-99a overexpression group, miR-99a overexpression negative control group, insulin-like growth factor 1 receptor(IGF-1R) inhibitor group, mammalian target of rapamycin(mTOR) inhibitor group, and metformin treatment group. The mRNA expressions of miR-99a were detected with realtime quantitative PCR(RT-qPCR), and the expression levels and distribution of IGF-1R, phosphorylated(p-)mTOR and vitronectin(VN) were detected by Western blotting and immunofluorescence. The ultrastructure of human liver sinusoidal endothelial cells was observed using scanning electron microscope.Results:Compared with normal control group, the mRNA expression of miR-99a was downregulated( P=0.008), while the protein expressions of IGF-1R, mTOR, and VN were significantly increased, and the diameter and number of fenestrae decreased significantly in high glucose model group. Compared with high glucose model group, after the treatment with metformin, the mRNA expression of miR-99a was upregulated, while the protein expressions of IGF-1R, mTOR, and VN were significantly decreased( P=0.001, P=0.016, P=0.005, respectively), the number of fenestras increased and the diameter became larger in miR-99a overexpression group, IGF-1R inhibitor group, mTOR inhibitor group, and metformin treatment group. After overexpression of miR-99a, the protein expressions of IGF-1R, p-mTOR, and VN were significantly reduced( P=0.007, P=0.013, P=0.003, respectively); After administration of IGF-1R inhibitors, the expressions of p-mTOR and VN significantly decreased( P=0.006, P=0.009, respectively), following treatment with the mTOR inhibitor, the expression of VN was significantly reduced( P=0.008), while the expression of IGF-1R remained unchanged( P=0.553). Conclusions:Downregulating of miR-99a with high glucose induced hepatic sinus dysfunction, which may be related to the regulation of IGF-1R/mTOR pathway. Metformin increased the expression of miR-99a, thereby inhibiting high glucose-induced hepatic sinusoidal dysfunction.