MicroRNA-377 knockdown suppresses high glucose-induced proliferation and inflammation in human mesangial cells

Objective To investigate the effect and potential mechanism of microRNA (miRNA)-377 on high glucose-induced proliferation and inflammation in human mesangial cells. Methods Cells were randomly divided into six groups: control group (5.5 mmol/L glucose), high glucose group (30.0 mmol/L glucose), negtive miRNA inhibitor transfection+high glucose group, negtive miRNA mimic transfection+high glucose group, miRNA-377 inhibitor transfection+high glucose group (miR-377i+high glucose group), miRNA-377 mimic transfection+high glucose group (miR-377m+high glucose group). miRNA-377 expression was detected by real-time PCR. Cell proliferation and cell cycle were detected by BrdU assay and flow cytometry, respectively. The release of tumor necrosis factor-α (TNF-α), interleukin (IL)-18, IL-6 and macrophages chemotaxis protein-1 (MCP-1) were evaluated by ELISA. The activations of NF-κB pathway, including the expressions of phosporylated (p)-IκBα, p-P65 and nuclear P65, were measured by Western blotting. Results Compared with those in control group, in high glucose group cell viability, miRNA-377 expression and cell proliferation rate increased (all P＜0.05), proportions of S phase cell and G2/M phase cell in cell cycle increased (all P＜0.05), the levels of TNF-α, IL-18, IL-6 and MCP-1 were higher (all P＜0.05), as well as the expressions of p-IκBα/IκBα, p-P65/P65 and nuclear P65 were increased (all P＜0.05). Compared with high glucose group, cell proliferation rate was restrained (P＜0.05), proportions of S phase cell and G2/M phase cell in cell cycle was descreased (all P＜0.05), the levels of TNF-α, IL-18, IL-6 and MCP-1 were lower (all P＜0.05), as well as the expressions of p-IκBα/IκBα, p-P65/P65 and nuclear P65 were reduced (all P＜0.05) in miR-377i+high glucose group. However, miR-377m+high glucose group presented opposite results (all P＜0.05). Conclusions miRNA-377 knockdown can partially suppress high glucose-induced human mesangial cell proliferation and cell cycle transition, and restrain inflammatory molecules release. Its mechanism may be related to the inhibition of NF-κB pathway.     

Angiotensin II up-regulates the release of inflammation factors via Toll-like receptor 4 signal in rat mesangial cells under high glucose

Objective To observe the regulation of Toll-like receptor 4 (TLR4) signal and the release of inflammation factors after angiotensin II (AngⅡ) stimulation in rat mesangial cells under high glucose condition, revealing the innate immune-related mechanism of injury by AngⅡ on mesangial cells under high glucose. Methods After synchronization, cells incubated with AngⅡ(10-7 mmo/L) and/or high glucose (25 mmol/L) were used as the stimulation group, cells without stimulation were as normal control (5.6 mmol/L glucose). To determine the role of TLR4 and the adaptor myeloid differentiation factor 88 (MyD88), equal number of HBZY-1 cells were added with 10-5 mmol/L irbesartan and/or TLR4 blocker (10 mg/L) for 1 h and then incubated with AngⅡ (10-7 mmo/L) and/or high glucose (25 mmol/L) for 12 h or 24 h respectively. Real-time PCR was used to analyze TLR4 mRNA and MyD88 mRNA expression after 12 h. Immunofluorescence was used to observe TLR4 protein expression after 24 h; Western blotting was used to observe TLR4, MyD88 and nuclear factor κB (NF-κB) protein; ELISA was used to detect the concentration of MCP-1, IL-6 in cell supernatant respectively. Results Compared with normal control group, TLR4 mRNA and MyD88 mRNA were highly expressed in high glucose or AngⅡ-induced HBZY-1 cells (P＜0.01), TLR4, MyD88 and NF-κB protein as well as MCP-1, IL-6 were also up-regulated significantly (P＜0.01). Compared with high glucose or AngⅡ group, MyD88 and NF-κB protein as well as MCP-1, IL-6 were further up-regulated markedly in AngⅡ and high glucose costimulated group (P＜0.01)．In HBZY-1 cells that were preincubated with irbesartan and/or TLR4 blocker, TLR4 and MyD88 protein expression were obviously inhibited, IL-6 and MCP-1 production were also decreased remarkably compared with high glucose and/or AngⅡ group (P＜0.01). Conclusions High glucose and AngⅡ stimulate the release of proinflammatory factors in rat glomerular mesangial cells via TLR4-MyD88 pathway. This process is inhibited by irbesartan or TLR4 blocker via modulation of the signal. AngⅡ has the positive-regulation potential on the release of inflammation factors via TLR4 signal in rat mesangial cells under high glucose condition.     

Impact of TLR4-siRNA transfection on the expression of TLR4/MyD88 Signaling in rat tubular epithelial cells under high glucose condition

Objective To observe the expression of toll like receptor 4(TLR4) Signaling and the release of inflammation factors in rat tubular epithelial cell(NRK-52E) under high glucose condition after TLR4-siRNA transfection. Methods Three TLR4-siRNA sequences were designed and synthesized. The transfection efficiency was observed by fluorescence microscope after transfection, and the expression of TLR4 mRNA was detected by real time PCR. The most effective siRNA was selected to be used for forward experiments. After transfection for 24 h, cells were stimulated with 25 mmol/L glucose and/or 10-7 mmol/L Angiotension Ⅱ(AngⅡ) for 12 h, 24 h; cells without stimulation were as normal control. Real-time PCR was used to analyze TLR4 and myeloid differentiation factor 88 (MyD88) mRNA expression; Western blot was used to observe TLR4/MyD88 and NF-κB protein expression. ELISA assay was used to detect the concentration of monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6) in cell supernatant after cells were stimulated for 24 h. Results TLR4/MyD88 mRNA and TLR4/MyD88/NF-κB protein were highly expressed under high glucose or AngⅡ co-incubated NRK-52E(P＜0.01), the MCP-1 and IL-6 levels were also increased markedly compared with normal control group (P＜0.01). TLR4/MyD88 mRNA and TLR4/MyD88/NF-kB protein expressions were obviously inhibited in cells that were transfected with TLR4-siRNA compared with high glucose group(P＜0.01), MCP-1 and IL-6 production decreased remarkably compared with high glucose or AngⅡ co-stimulated group(P＜0.01). Conclusions High glucose can lead to the activation of TLR4/MyD88/NF-kB signaling and the secretion of inflammation factors in NRK-52E, AngⅡ further augments these effects. The effect can be blocked efficiently by specific siRNA gene silence. TLR4 signaling plays a pivotal role in the innate-immune inflammatory reaction in NRK-52E.     

高糖和血管紧张素Ⅱ对人肾小管上皮细胞Toll样受体4信号表达及炎性和纤维化因子的影响

Objective To investigate the effects of angiotensinⅡ(AngⅡ) or high glucose on the toll-like receptor 4 (TLR4) expression, inflammatory cytokines and fibrotic factors in human tubular epithelial cells (HK-2), revealing the innate immune-related pathogenesis of diabetic nephropathy (DN) which may have clinical implications. Methods Three TLR4 siRNA sequences were designed and synthetized. After transfection, the most effective siRNA was selected to use for further expriments. The experiment consisted of 2 parts. Part 1: Cells were divided into three groups: normal-glucose group (NG, 5.5mmol/L glucose), mannose group (M, 5.5 mmol/L glucose+19.5 mmol/L mannose), High-glucose group (HG, 25 mmol/L glucose), preliminary validated the effects of high glucose and high osmotic pressure. Part 2: Cells were divided into seven groups: NG group, HG group, AngⅡgroup, AngⅡ+ negative group, HG+ negative group, AngⅡ+ siRNA group and HG+ siRNA group. Real time PCR was used to analyze the mRNA expression of TLR4, myeloid differentiation factor 88 (MyD88), heat shock protein 47 (HSP47). Western blotting was used to observe the protein expression of TLR4, MyD88, HSP47, NF-κB, type Ⅳ collagen (ColIV). ELISA was used to detect the expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6). Results Compared with NG group, TLR4, MyD88, HSP47 mRNA and TLR4, MyD88, NF-κB, ColⅣ, HSP47 protein were highly expressed under high glucose or AngⅡconditions (P＜0.01), and the expression levels of MCP-1 and IL-6 also increased significantly (P＜0.01). Compared with HG or AngⅡ group, the above indicators were obviously inhibited in the TLR4 siRNA groups (P＜0.01). Comparison between blank vector transfected groups and HG group as well as AngⅡ group indicated no statistic significance (P＞0.05). Conclusions Both AngⅡ and high glucose stimulate TLR4 expression, which result in the up-regulation of inflammatory and fibrotic factors in HK-2. Specific target of TLR4 gene silencing can block the TLR4 pathway that is activated by high glucose and AngⅡ, and thus reduce the inflammatory and fibtogenic factors' release. TLR4 signal is the common innate immune response pathway which induces the release of inflammatory and fibrotic factors in HK-2 under high glucose or high angiotension conditions.     

Regulation of melatonin on Toll-like receptor 4 signaling in diabetic db/db mice kidneys

Objective To investigate the regulation of melatonin (MT) on Toll-like receptor 4 (TLR4) signaling in diabetic db/db mice kidneys. Methods The 48 10-week-old male db/db mice were randomly divided into db/db group, db/db+MT 50 μg/kg group, db/db+MT 100 μg/kg group and db/db+MT 200 μg/kg group, each consisting of 12 mice. These mice received i.p. injections of MT These mice received i.p. injections of MT [dissoved in phosphate buffer solution (PBS)/ dimethylsulfoxide (DMSO) solution, given every day]. Alternatively, 12 db/m mice served as the control group. db/m and db/db group were injected i.p. with the same volume of PBS/DMSO solution. The animals were sacrificed after 12 weeks of dosage administration. Blood glucose (BG), body weight (BW), kidney weight (KW) and 24 h urinary albumin excretion rate (UAER) were determined; Kidney pathological lesions were evaluated by renal pathological staining. Immunohistochemistry of renal TLR4, NF-κB p65, and ED-1 was performed to determine the immunoreactivity. Western blotting was used to detect the expression of renal TLR4, myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF-3) and NF-κB p65, while the mRNA expressions of renal tumor necrosis factor -α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were much higher in db/db mice group (P＜0.01), while KW in db/db+MT (100, 200 μg/kg) groups and UAER level in db/db+MT (50, 100, 200 μg/kg) groups were distinctly decreased compared with those in db/db group (P＜0.01). In week 12 db/db mice, the glomerular mesangial expansion index and tubulointerstitial injury index were increased compared with those in db/m mice (P＜0.01). The above kidney histopathologic lesions were distinctly ameliorated by 50, 100, 200 μg/kg MT (P＜0.05). Immunohistochemistry intensity of renal TLR4, NF-κB p65 and ED-1 displayed obvious differences between db/m mice and db/db mice (P＜0.01), and that were remarkably decreased in db/db+MT (50, 100, 200 μg/kg) mice compared with db/db mice (P＜0.05). Western blotting showed that the protein expression of renal TLR4, MyD88, TRIF, IRF-3 and NF-κB p65 were stronger in db/db group compared with those in db/m group (P＜0.05) and weaker in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.05). Futhermore, the mRNA expressions of renal MCP-1 and TNF-α were higher in db/db group compared with those in db/m group (P＜0.01) and lower in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.01). Conclusion Melatonin may partly down-regulate TLR4 signaling pathway to inhibit Inflammatory reaction and alleviate kidney injury in diabetic db/db mice.     

Transient high glucose induces persistent inflammatory status in rat glomerular mesangial cell via histone methylation modification

Objective To investigate whether the effect of transient high glucose on inflammatory factors expression could be continuous in rat glomerular mesangial cell, and its relation with histone methylation modification. Methods Rat glomerular mesangial cells (HBZY-1) were divided into three groups: the high glucose group (25.0 mmol/L glucose), the hypertonic group (MA, 5.5 mmol/L glucose+19.5 mmol/L mannitol) and the normal-glucose control group (5.5 mmol/L glucose), which were cultured for 24 h respectively. All 3 groups were then changed with normal-glucose medium to culture for 24 h, 48 h and 72 h. Their protein, mRNA and supernatant were harvested. The protein expressions of mono-methylation of H3 lysine 4 (H3K4me1) was measured by Western blotting, and the mRNA expressions of NF-κB subunit p65 and set7/9 were determined by real time-quantitative PCR. The expression of monocyte chemoattractant protein 1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) were detected by enzyme-linked immunosorbent assay. Results (1) Compared with those in normal control group, the expressions of H3K4me1 protein and set7/9 mRNA were first up-regulated in high glucose group, then gradually down-regulated in the following 48 h normal-glucose medium (as compared with those at 0 h, all P＜0.05). At 72 h there was no statistic difference between high glucose group and normal control group (all P＞0.05). (2) Compared with those in normal control group, the up-regulated p65 mRNA, VCAM-1 and MCP-1 sustained at least for 72 h in high glucose group. Conclusions Transient high glucose can induce persistent inflammatory factors expression in rat glomerular mesangial cells, which may via histone modification.     

Effect and mechanism of chitosan inhibiting vascular smooth muscle cell hyperplasia of uremia patients

Objective To investigate the effect and mechanism of chitosan on vascular smooth muscle cell proliferation of uremia patients with arteriovenous fistula. Methods Primarily culturing the VSMCs of uremia patients with arteriovenous fistula and patients without uremia by explants adherent method, and taking the second generation. VSMCs from patients without uremia cultured with 20% FBS medium were non-uremia group, VSMCs of uremia patients cultured with 20% FBS medium were uremia group, VSMCs of uremia patients with 100 μg/ml chitosan were uremia+chitosan group. The expression of α-SMA was detected by immunohistochemistry. The changes of migration and invasion of VSMCs were detected by scratches and transwell migration assays. The mRNA expressions of TLR4 and PCNA were measured by real-time PCR. VSMCs of uremia patients with arteriovenous fistula were intervened with different doses of chitosan (0, 100 and 500 μg/ml), and the protein expressions of TLR4, MyD88 and NF-κB were detected by Western blotting. Results Compared with those in non-uremia group, in uremia group and uremia+chitosan group α-SMA was up-regulated, migration and invasion of VSMCs were enhanced, and mRNA expressions of TLR4 and PCNA were increased (all P＜0.05). Compared with those in uremia group, the level of α-SMA was significantly decreased, the ability of migration and invasion of VSMCs were decreased, and the mRNA expressions of TLR4 and PCNA were decreased (all P＜0.05). TLR4, MyD88 and NF-κB protein expressions were reduced in concentration-dependent manner by 100 and 500 μg/ml chitosan. Conclusions (1) In vitro, chitosan decreases the ability of migration and invasion of VSMCs of uremia patients with arteriovenous fistula. (2) Chitosan inhibits the proliferation of VSMCs, which may be relevant in the decreased expressions of TLR4, MyD88 and NF-κB.     

Effect of high glucose on the Notch signal pathway in mesangial cells and intervention of the cordyceps sinensis

Objective To investigate the expression of Notch signaling molecules, transforming growth factor-β (TGF-β) and fibronectin (FN) in mesangial cell induced by high glucose, and the underlying mechanism of cordyceps sinensis. Methods Rat glomerular mesangial cells were divided into following groups: normal control group (5.5 mmol/L glucose), hypertonic control group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), DAPT inhibitor group (25.0 mmol/L glucose+1 μmol/L DAPT), cordyceps sinensis intervention group (25.0 mmol/L glucose+10 mg/L cordyceps sinensis). Cell proliferation was detected by MTT. The protein and mRNA expression of Notch signaling molecules (Notch1, Jagged1 and Hes1), TGF-β and FN was detected by Western blotting and real time PCR. Results Compared with normal control group, high glucose induced mesangial cell proliferation, as well as the mRNA and protein expression of Notch1, Jagged1, Hes1, TGF-β1 and FN was up-regulated in high glucose group (all P＜0.05). Compared with that in high-glucose group, DAPT and cordyceps sinensis inhibited high glucose-induced mesangial cell proliferation and down-regulated the mRNA and protein expression of Notch pathway, TGF-β1 and FN (all P＜0.05). Conclusion By inhibiting the abnormal activation of Notch signaling pathway and TGF-β signaling pathway, cordyceps sinensis may alleviate high glucose-induced mesangial cell proliferation and reduce extracellular matrix accumulation, thus protecting kidney.     

Effect of tet methylcytosine dioxygenase 2 on the regulation of transforming growth factor-β1 expression in mesangial cells induced by high glucose

Objective To investigate the role of tet methylcytosine dioxygenase 2 (TET2) in the regulation of transforming growth factor-β1 (TGF-β1) expression in human glomerular mesangial cells induced by high glucose. Methods Cultured human glomerular mesangial cells were divided into normal control group (5.5 mmol/L glucose) and high glucose group (30.0 mmol/L glucose) which was cultured for 12 h to 72 h. The gene expression of TET2 in mesangial cells were inhibited by small molecule chemical called SC1, and which were divided into high glucose group (30.0 mmol/L glucose+DMEM), DMSO group (30.0 mmol/L glucose+0.1%DMSO) and SC1 group (30.0 mmol/L glucose+3 μmol/L SC1). The mRNA and protein expression of TGF-β1, TET1 to 3 and α-smooth muscle actin (α-SMA) was detected by quantitative real-time PCR and Western blotting. Methylation of CpG islands in the regulation region of TGF-β1 was detected by bisulfite sequencing PCR (BSP). The activity of mesangial cell proliferation was assessed by colorimetry of thiazolyl blue (MTT). Results Compared with normal control group, the mRNA and protein expression of TET2 in mesangial cells induced by high glucose was increased significantly in a time-dependent manner (all P＜0.05), but the expression of TET1 and TET3 was not affected. Meanwhile methylation rate of 4 CG sites from 24 h to 72 h were decreased in the first exon of TGF-β1 (P＜0.01), but not in the promoter. Compared with high glucose group, when the expression of TET2 was inhibited by SC1, the methylation rate of TGF-β1 was recovered evidently (P＜0.05), the mRNA and protein expression of TGF-β1 and α-SMA was suppressed, and the proliferation of mesangial cells was decreased (all P＜0.05). Conclusions Demethylation of the CpG island mediated by TET2 may play an important role in the expression of TGF-β1 and mesangial cell phenotype transformation induced by high glucose.     

Effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells

Objective To evaluate the effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells and to explore the possible mechanisms of KIM-1 involved in renal interstitial fibrosis of DN. Methods The rat renal tubular epithelial cells (NRK52E) were cultured in vitro and divided into five groups: Normal control group (D-glucose 5.6 mmol/L), Hypertonic group (D-glucose 5.6 mmol/L＋D-mannitol 24.4 mmol/L), High glucose group (D-glucose 30 mmol/L), Control siRNA group,KIM-1 siRNA group. ELISA assay was used to assess the levels of MCP-1 and FN in the cells supernatant; Western blotting was used to detect the protein expression of KIM-1; RT-PCR was used to detect mRNA expression of KIM-1, MCP-1 and FN. Results Compared with the control group, the protein and mRNA expression of KIM-1 in the high glucose group were increased at 12 h (P＜0.05), and reached the peak at 48 h (P＜0.05); the protein and mRNA expression of MCP-1 and FN in high glucose group were increased at 24 h significantly (P＜0.05), and peaked at 48 h (P＜0.05). Compared with the high glucose group, the protein and mRNA expressions of MCP-1 and FN in KIM-1 siRNA group were decreased (P＜0.05). Conclusions Down-regulating the expression of KIM-1 can significantly inhibit the expression of MCP-1 and FN, which suggests that KIM-1 may be involved in renal interstitial fibrosis of DN by regulating expression of MCP-1 and FN.     

TAK1信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用

Objective To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose. Methods Purity of mouse BMDM was detected by flow cytometry. The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol. Cells were divided into normal control group (RPMI 1640), osmolality control group (25 mmol/L mannitol), high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L D-glucose+300 nmol/L 5Z-7-oxozeaenol). Immunocytochemistry and flow cytometry were used to detect macrophage subtype. The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR. Expressions of p-TAK1, TAK1 binding protein (TAB1), p-JNK, p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting. Results The purity of BMDM was about 99.36%. Compared with normal control group, high glucose group had increased percentage of M1 macrophages, increased expression of MCP-1 and TNF-α mRNA (all P＜0.05). Moreover, p-TAK1, TAB1, p-JNK, p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P＜0.05). After treatment with inhibitor 5Z-7-oxozeaenol, the effects induced by high glucose were inhibited (P＜0.05). Conclusions High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM, which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.     

Fluorofenidone reduces renal fibrosis in diabetic kidney disease mice

Objective To investigate the effects of fluorofenidone (AKF-PD) on diabetic kidney disease in db/db mice and its possible mechanisms. Methods (1) Fifty-six mice aged 8 weeks (half male and half female), including 42 db/db mice and 14 wild-type mice were studied. Forty-two db/db mice randomly were divided into model group (mock-treated diabetic db/db mice), AKF-PD (250 mg?kg-1?d-1) treatment group and losartan (20 mg?kg-1?d-1) treatment group. Wild-type mice and model mice were treated with vehicle (0.5% sodium carboxymethylcellulose), while the treatment groups received either AKF-PD or losartan. After 18 weeks, the blood glucose and urinary albumin were measured, the pathological changes of kidney were observed by PAS staining. The protein expressions of type Ⅳ collagen and fibronectin (FN) in kidney tissue were detected by immunohistochemistry. (2) Mouse glomerular mesangial cells (MES-13 cells) were divided into six groups: normal glucose group (5.5 mmol/L glucose), hypertonic group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), AKF-PD group (25.0 mmol/L glucose+400 mg/L AKF-PD) and losartan group (25.0 mmol/L glucose+2 μmol/L losartan). After 72 h treatment, the expressions of type Ⅰ collagen, type Ⅳ collagen and transforming growth factor-β1 (TGF-β1) mRNA were detected by real-time PCR, and the content of TGF-β1 protein in the culture supernatant was detected by ELISA. Results (1) Compared with the wild type mice, model mice had increased weight, blood glucose and glomerulosclerosis index (all P＜0.01), accompanied with heavy albuminuria, glomerular hypertrophy, mesangial area expansion and deposition of collagen type Ⅳ and FN (all P＜0.01). Compared with model mice, in AKF-PD and losartan groups 24 h urinary albumin and glomerulosclerosis index decreased (all P＜0.01), glomerular hypertrophy and mesangial area expansion alleviated, and the protein expressions of collagen type Ⅳ and FN were inhibited (all P＜0.01). (2) Compared with the normal glucose group, the mRNA expressions of type Ⅰ collagen and type Ⅳ collagen increased in high glucose group, meanwhile the mRNA and protein expressions of TGF-β1 increased (all P＜0.01). In AKF-PD and losartan groups the expressions of type Ⅰ collagen, type Ⅳ collagen and TGF-β1 were inhibited as compared with high glucose group (all P＜0.05). Conclusion Fluorofenidone may play an anti-fibrotic effect in db/db mice by reducing the expression of TGF-β1 and inhibiting collagen synthesis in glomerular mesangial cells.     

Effects of benazepril on the expressions of intergrin-linked kinase and α-smooth muscle actin in glomerular mesangial cells exposed to high glucose

Objective To investigate the effect of benazepril on intergrin-linked kinase (ILK) and α-smooth muscle actin (α-SMA) expression in glomerular mesangial cells induced by high-glucose. Methods The mesangial cells from SD rat (HBZY-1) were cultured conventionally and randomly divided into four groups: normal glucose (D-glucose 5.5 mmol/L, group NG), mannitol-treated group (mannitol 20 mmol/L, group MG), high glucose (D-glucose 30 mmol/L, group HG), Benazepril-treated high glucose group (D-glucose 30 mmol/L＋Benazepril 10 μmol/L, group ACEI). Cells from NG, MG, HG, ACEI gronps were harvested after 3, 6, 12, 24, 48 and 72 hours of treatment respectively. The mRNA expressions of ILK and α-SMA were detected by RT-PCR. The protein levels of ILK and α-SMA were detected by Western blotting and immunofluorescence. Results The expressions of ILK mRNA and protein in HG group were significantly increased compared with those in NG group (all P＜0.05). The increased expressions of ILK and α-SMA in HG group were time-dependent and the expression reached the peak at 48 h (ILK, P＜0.05) or 72 h (α-SMA, P＜0.01). The expressions of ILK and α-SMA in ACEI group were lower than those in HG group (all P＜0.01), but failed to rescue to the same level as those in NG. There was no significant differences of ILK expressions between MG group and NG group at the same time point (P＞0.05). The expressions of α-SMA mRNA and protein in MG were higher than that in NG (P＜0.05), which suggest that high osmotic pressure could cause the increasing of α-SMA. Conclusions Benazepril can decrease the expressions of ILK and α-SMA to inhibit the process of fibrosis in DN and mediate the phenotypic transformation of glomerular mesangial cells. The phenotypic transformation of glomerular mesangial cells in glucose may also depend on high osmotic pressure in DN.     

MicroRNA-148b influences high glucose-induced endoplasmic reticulum stress in rat mesangial cell by targeting AMPKα1

Objective To observe the expression of microRNA-148b (miR-148b) induced by high glucose in rat mesangial cells, and to explore its effect on its target gene AMP-activated protein kinase α1 (AMPKα1) and extracellular matrix excretion. Methods Rat mesangial cells were divided into 3 groups: normal glucose (NG, 5.5 mmol/L glucose) group, hypertonic (MA, 5.5 mmol/L glucose+ 19.5 mmol/L mannitol) group and high-glucose (HG, 25.0 mmol/L glucose) group. MiR-148b expression was detected by real time PCR. Then miR-148b inhibitor was transfected to rat mesangial cells. Their protein expressions of AMPKα1, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), fibronectin (FN) and collagen Ⅳ were detected by Western blotting. The expression of AMPKα1 mRNA was detected by real time PCR. The expression of collagen Ⅳ was also detected by immunofluorescence. Results Compared with NG group, HG group showed up-regulated miR-148b expression, down-regulated AMPKα1 mRNA and protein expressions, and up-regulated CHOP, GRP78, collagen Ⅳ and FN expressions (all P＜0.05). HG-induced mesangial cells with miR-148b inhibitor had up-regulated AMPKα1 mRNA and protein expressions, and down-regulated CHOP, GRP78, collagen Ⅳ, FN expressions as compared with HG-induced cells without miR-148b inhibitor (all P＜0.05). Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells, then activate endoplasmic reticulum stress to induce extracellular matrix excretion. MiR-148b inhibitor up-regulates AMPKα1 expression, inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.     

细胞因子信号传导抑制蛋白1抑制高糖诱导的肾小球系膜细胞单核细胞趋化蛋白1的表达

目的 探讨细胞因子信号传导抑制蛋白1(SOCS-1)对高糖状态下肾小球系膜细胞单核细胞趋化蛋白1（MCP-1）表达的影响。 方法 体外培养人肾小球系膜细胞,应用脂质体2000分别转染pCR3.1-SOCS-1表达质粒和pCR3.1 空质粒载体,G418筛选阳性克隆。分别采用低糖（5.5 mmol/L）、高糖（30 mmol/L）、低糖+甘露醇（24.5 mmol/L甘露醇）和JAK-STAT信号通路抑制剂AG490 （10 μmol/L）进行刺激。Western印迹检测系膜细胞SOCS-1、信号转导和转录活化因子1、3（STAT1、STAT3）及其磷酸化蛋白（p-STAT1、p-STAT3）的表达。ELISA法和放免法测定细胞上清液中MCP-1、FN和Ⅳ型胶原的含量。RT-PCR法检测SOCS-1和MCP-1 mRNA的表达。 结果 高糖刺激系膜细胞SOCS-1蛋白和mRNA表达呈时间依赖性变化, 4 h表达达到峰值,然后逐渐减低,24 h达基线水平。与低糖组相比,高糖组系膜细胞STAT1和STAT3磷酸化水平显著上调（P < 0.01）; MCP-1 mRNA水平表达显著上调[（0.39±0.05）比（0.16±0.02）,P < 0.01];上清液中MCP-1[（459±67）比（241±19） ng/L]、FN[（5.84±0.61）比（3.41±0.31） mg/L]和Ⅳ型胶原[（16.45±2.30）比（9.56±1.52） μg/L] 含量均显著增加（均P < 0.01）。与空载体对照组相比,SOCS-1过表达组系膜细胞STAT1和STAT3的磷酸化水平显著下降（P < 0.05）;MCP-1 mRNA表达下调[（0.34±0.04）比（0.42±0.05）,P < 0.05]; 上清液中MCP-1[(387±47）比（463±56） ng/L]、 FN[（4.61±0.57）比（5.76±0.74） mg/L]和Ⅳ型胶原[(13.4±2.32)比（17.1±2.57） μg/L] 含量显著减少（均P < 0.05）。与高糖组相比,AG490组系膜细胞MCP-1 mRNA（0.31±0.04）表达显著下调;上清液中MCP-1[（361±53） ng/L]、FN[（5.46±0.71）mg/L]和Ⅳ型胶原[（15.2±1.97） μg/L]含量均减少。 结论 SOCS-1过表达抑制高糖状态下肾小球系膜细胞MCP-1及细胞外基质的分泌可能部分是通过影响STAT1和STAT3的激活而实现。     

罗格列酮对高糖环境中大鼠系膜细胞活性氧及单核细胞化学吸引蛋白质1的抑制作用

目的 观察罗格列酮对高糖培养基中大鼠系膜细胞活性氧（ROS）及单核细胞化学吸引蛋白质1（MCP-1）mRNA、蛋白表达的抑制作用,并探讨相关机制。 方法 将培养的大鼠系膜细胞分为以下6组：对照组（C组,普通MEM培养基,含5.6 mmol/L葡萄糖）、甘露醇组（M组,24.2 mmol/L甘露醇＋C组）、高糖组（H组,30 mmol/L高糖MEM培养基）、小剂量罗格列酮干预组（R1组,H组＋10 μmol/L罗格列酮）、大剂量罗格列酮干预组（R2组,H组＋20 μmol/L罗格列酮）、N-乙酰半胱氨酸（NAC）干预组（N组,H组＋5 mmol/L NAC）。采用激光共聚焦法检测ROS水平,分别采用RT-PCR和ELISA法检测MCP-1 mRNA及蛋白含量。 结果 C组和M组间ROS、MCP-1的表达差异无统计学意义,H组ROS水平较C组增高4.1倍（P < 0.01）,分别采用罗格列酮（20 μmol/L）和NAC干预后,ROS水平显著降低（P < 0.01）;罗格列酮（20 μmol/L）和NAC均可显著抑制高糖环境中MCP-1 mRNA的高表达（P < 0.01）。H组中MCP-1蛋白水平[（940.9±20.3） ng/L]显著高于C组[（403.0±8.1） ng/L]（P < 0.01）,而R2组和N组的MCP-1蛋白水平[（562.5±15.3） ng/L,（539.8±8.3） ng/L]显著低于H组（P < 0.01）。 结论 罗格列酮可通过减少ROS而降低高糖所诱导的MCP-1表达,而这可能是罗格列酮发挥直接肾脏保护作用的机制之一。     

Exogenous administration of PACAP alleviates traumatic brain injury in rats through a mechanism involving the TLR4/MyD88/NF-κB pathway

Pituitary adenylate cyclase-activating polypeptide (PACAP) is effective in reducing axonal damage associated with traumatic brain injury (TBI), and has immunomodulatory properties. Toll-like receptor 4 (TLR4) is an important mediator of the innate immune response. It significantly contributes to neuroinflammation induced by brain injury. However, it remains unknown whether exogenous PACAP can modulate TBI through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. In this study, we investigated the potential neuroprotective mechanisms of PACAP pretreatment in a weight-drop model of TBI. PACAP38 was microinjected intracerebroventricularly before TBI. Brain samples were extracted from the pericontusional area in the cortex and hippocampus. We found that TBI induced significant upregulation of TLR4, with peak expression occurring 24?h post-trauma, and that pretreatment with PACAP significantly improved motor and cognitive dysfunction, attenuated neuronal apoptosis, and decreased brain edema. Pretreatment with PACAP inhibited upregulation of TLR4 and its downstream signaling molecules MyD88, p-IκB, and NF-κB, and suppressed increases in the levels of the downstream inflammatory agents interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), in the brain tissue around the injured cortex and in the hippocampus. Administration of PACAP both in vitro and in vivo attenuated the ability of the TLR4 agonist lipopolysaccharide (LPS) to increase TLR4 protein levels. Therefore, PACAP exerts a neuroprotective effect in this rat model of TBI, by inhibiting a secondary inflammatory response mediated by the TLR4/MyD88/NF-κB signaling pathway in microglia and neurons, thereby reducing neuronal death and improving the outcome following TBI.     

尿激酶型纤溶酶原激活物对高糖诱导大鼠系膜细胞增殖及表型转化的影响及其机制

目的 通过体外培养大鼠系膜细胞（MC）,观察尿激酶型纤溶酶原激活物（uPA）对高糖环境下MC增殖及表型转化的影响及其可能的信号转导机制。 方法 体外培养大鼠MC,分为4组：对照组、高糖组、高糖+渥曼青霉素组、高糖+uPA组。MTT法检测各组MC增殖情况。流式细胞仪分析各组MC细胞周期改变。Western印迹法检测各组MC表达CDK2与p27Kip1变化,并测定MC中信号蛋白Akt的活性。激光共聚焦显微镜检测各组MC中α-SMA表达方式及表达量变化。 结果 培养24 h后高糖组MC增殖程度较对照组显著增加（P < 0.01）,渥曼青霉素与uPA组可明显抑制细胞增殖（P < 0.01）。高糖刺激Akt活性,渥曼青霉素与uPA组Akt活性均较高糖组显著降低（均P < 0.01）。高糖组MC培养24 h后, p27Kip1蛋白表达较对照组显著减少（P < 0.01）;高糖+渥曼青霉素组、高糖+uPA组p27Kip1蛋白表达均较高糖组显著增加（均P < 0.01）;各组CDK2蛋白表达无明显变化。高糖组MC培养24 h后,胞质中α-SMA表达较对照组显著增加（P < 0.01）,并在核周出现聚集;高糖+渥曼青霉素组、高糖+uPA组α-SMA表达量均较高糖组显著减少（均P < 0.01）,其分布与对照组无显著差异。 结论 uPA可能通过抑制Akt信号分子活性,上调p27Kip1表达,拮抗高糖所致MC增殖与表型转化。     

血管紧张素Ⅱ通过TLR4-MyD88途径诱导大鼠肾小管上皮细胞炎性因子释放

目的 观察血管紧张素Ⅱ（AngⅡ）刺激肾小管上皮细胞（NRK-52E）后肿瘤坏死因子α（TNF-α）和热休克蛋白47（HSP47）的表达,分析Toll样受体4（TLR4）信号通路的变化与上述因子的关联,探讨AngⅡ促进NRK-52E细胞炎性反应、纤维化的天然免疫机制。 方法 细胞同步化后,将其分为4组：对照组、AngⅡ（10-7 mmol/L）组、坎地沙坦（10-5 mmol/L）+AngⅡ组、TLR4阻断剂（20 mg/L）+AngⅡ组,培养6 h后RT-PCR法检测TLR4及转接信号髓分化因子88（MyD88）mRNA表达水平;12 h后免疫荧光法检测细胞表面TLR4蛋白表达;24 h后以ELISA法检测细胞上清液TNF-α及HSP47的浓度。 结果 与对照组相比,AngⅡ显著上调NRK-52E细胞 TLR4、MyD88 mRNA和TLR4蛋白表达（均P < 0.01）,并诱导细胞TNF-α和HSP47的释放（均P < 0.01）。与AngⅡ组相比,TLR4阻断剂和坎地沙坦干预均显著抑制 AngⅡ对细胞TLR4、MyD88的刺激效应（均P < 0.01）;坎地沙坦抑制AngⅡ诱导的细胞 TNF-α、HSP47的释放（均P < 0.01）,TLR4阻断剂对细胞 TNF-α、HSP47的下调呈剂量依赖性。 结论 AngⅡ对NRK-52E细胞天然免疫信号TLR4、MyD88具有激活效应,该信号激活可能是AngⅡ促进肾小管细胞炎性相关因子释放的重要机制之一。坎地沙坦抑制肾小管细胞炎性因子的体外效应也与其调节该信号通路有关。