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Objective:To explore the expression of serum connective tissue growth factor (CTGF), glyoxalase Ⅰ (GLO-I) and pyruvate kinase M2 (PKM2) in endometrial cancer and their relationship with clinicopathological characteristics.Methods:A total of 96 endometrial cancer patients in Yuechi County People's Hospital from February 2015 to February 2017 were selected as the research group, 48 patients with endometrial hyperplasia during the same period were selected as the benign control group, and 48 patients with healthy physical examination during the same period were selected as the healthy control group. The serum levels of CTGF, GLO-Ⅰ, and PKM2 in the three groups were analyzed. The correlation between serum levels of CTGF, GLO-Ⅰ and PKM2 in the research group was analyzed, and the relationship between each serum index and clinicopathological characteristics was analyzed.Results:The levels of serum CTGF, GLO-Ⅰ and PKM2 in the research group were higher than those in the benign control group and healthy control group: (184.31 ± 37.14) μg/L vs. (110.45 ± 20.59), (17.28 ± 0.42) μg/L; (95.17 ± 16.56) pmol/L vs. (56.29 ± 10.14), (9.08 ± 0.66) pmol/L; (20.25 ± 6.13) μg/L vs. (13.11 ± 4.58), (9.05 ± 2.74) μg/L; and the levels of serum CTGF, GLO-Ⅰ and PKM2 in the benign control group were higher than those in the healthy control group, there were statistical differences ( P<0.05). The results of Pearson correlation analysis showed that the level of CTGF had positive correlation with GLO-Ⅰ and PKM2 ( r = 0.713, 0.741, P<0.05), and the level of GLO-Ⅰ had positive correlation with PKM2 ( r = 0.823, P<0.05). The results of Spearman correlation analysis showed that the levels of CTGF, GLO-Ⅰ, PKM2 had positive correlation with FIGO stage ( r = 0.609, 0.704, 0.721; P<0.05), myometrial invasion depth ( r = 0.753, 0.695, 0.719; P<0.05), lymph node metastasis ( r = 0.776, 0.744, 0.640; P<0.05); had negative correlation with the degree of differentiation ( r = - 0.711, - 0.720, - 0.668; P<0.05). Conclusions:Serum CTGF, GLO-I, PKM2 expression levels are abnormally elevated in patients with endometrial cancer, which are significantly related to multiple clinicopathological characteristics.
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Objective:To investigate the regulatory effect of miR-26a in radiation-induced heart disease (RIHD) mice.Methods:C57/BL6 mice were used to establish RIHD models. The cardiac function, fibrosis, the expression levels of collagen 1 (COL1) and connective tissue growth factor (CTGF), and miR-26a were detected in RIHD mice. Whether CTGF was the target gene of miR-26a was verified by dual luciferase kit. Moreover, cardiac fibroblasts were transfected with miR-26a up and miR-26a down lentivirus vectors to construct the miR-26a overexpression and underexpression cell models. The expression of CTGF, proliferation, and apoptosis of cardiac fibroblasts were detected.Results:In the RIHD mice, heart function was decreased, myocardial fibrosis was remodeled, the expression levels of COL1 and CTGF were up-regulated, and the expression level of miR-26a was down-regulated. Dual luciferase reporter assay confirmed that CTGF was the target gene regulated by miR-26a. Overexpression of miR-26a could inhibit the expression of CTGF, suppress the proliferation of cardiac fibroblasts, promote cell apoptosis and secrete collagen. Underexpression of miR-26a yielded the opposite results.Conclusion:MiR-26a affects the function of cardiac fibroblasts by targeting CTGF and probably mediates the process of radiation-induced myocardial fibrosis, which may become a new regulatory target of RIHD.
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Objective:To investigate the effect and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomal microRNA-1297 (miR-1297) on hippocampal neuron damage in depressed rats.Methods:BMSCs and BMSCs-derived exosomes were prepared and identified. Rats were first injected with corticosterone to establish the model of depression, and then injected with BMSCs-derived exosomes. Superoxide dismutase (SOD), malondialdehyde (MDA), lactate dehydrogenase (LDH), TNF-α and IL-1β in rat serum samples, hippocampal tissues and neurons were detected. Expression of miR-1297 in hippocampal tissues and neurons was detected by RT-qPCR. A rat hippocampal neuron injury model was established to investigate the role of BMSC-derived exosomes and miR-1297 in neuronal apoptosis and proliferation. The targeting relationship between miR-1297 and connective tissue growth factor (CTGF) was analyzed using dual luciferase reporter genes.Results:In the hippocampus of depressed rats, the expression of miR-1297 was low, while the expression of CTGF was elevated. Exosomes derived from BMSCs can inhibit the expression of CTGF by up-regulating the level of miR-1297, thereby inhibiting neuronal cell apoptosis in the hippocampus of depressed rats, while increasing the level of SOD, and reducing inflammatory damage, and ultimately improving the behavioral function of depressed rats.Conclusions:Depressed rats showed decreased expression of miR-1297 and increased expression of CTGF. BMSC-derived exosomes inhibited CTGF expression through up-regulating miR-1297, thereby improving hippocampal neuron damage in rats with depression.
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Objective:To investigate the efficacy of atenolol combined with perindopril in the treatment of chronic heart failure in older adult patients and its effects on ventricular function, serum connective tissue growth factor (CTGF), and nexin.Methods:120 older adult patients with chronic heart failure who received treatment in the Department of Cardiology, the First People's Hospital of Fuyang District from January 2016 to January 2018 were included in this study. They were randomly assigned to receive either basic treatment + atenolol treatment (control group, n = 60) or atenolol combined with perindopril treatment (observation group, n = 60). Clinical efficacy and clinical symptom, serum CTGF, nexin level and ventricular function pre- and post-treatment, as well as adverse reactions were compared between the control and observation groups. Results:After treatment, effective rate in the observation group was significantly higher than that in the control group (91.6% vs. 78.3%, χ2 = 4.183, P = 0.041). After treatment, CTGF and nexin levels in the control and observation groups were decreased compared with before treatment (both P < 0.05). After treatment, CTGF and nexin levels in the observation group were (4.42 ± 0.46) μg/L and (0.82 ± 0.03) μg/L, respectively, which were significantly lower than those in the control group [(4.82 ± 0.51) μg/L, (0.98 ± 0.04) μg/L, t = 18.153, 4.511, 19.335, 24.787, all P < 0.05]. After treatment, left ventricular end diastolic diameter and left ventricular end systolic diameter in the control and observation groups were deceased compared with before treatment (both P < 0.05). After treatment, left ventricular end diastolic diameter and left ventricular end systolic diameter in the observation group were (48.73 ± 4.41) mm and (41.13 ± 4.15) mm, respectively, which were significantly lower than those in the control group [(56.01 ± 4.67) mm, (47.45 ± 4.17) mm, t = 5.700, 8.799, 8.317, 8.351, all P < 0.05]. After treatment, left ventricular ejection fraction in the control and observation groups was significantly increased compared with before treatment. After treatment, left ventricular ejection fraction in the observation group was significantly higher than that in the control group [(44.86 ± 4.59) % vs. (39.05 ± 4.69) %, P < 0.05]. Conclusion:Atenolol combined with perindopril in the treatment of older adult patients with chronic heart failure can reduce clinical symptoms, improve ventricular function, decrease serum CTGF and nexin levels and is highly safe. Therefore, this method is worthy of clinical application.
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Objective:To investigate the effects of thrombolytic therapy time on serum inflammatory factors, cathepsin S, connective tissue growth factor (CTGF), left ventricular ejection fraction (LVEF) and left ventricular end diastolic diameter (LVEDD) in patients with acute ST-elevation myocardial infarction.Methods:The clinical data of 119 patients with acute ST-elevation myocardial infarction who received thrombolytic therapy in the People's Hospital of Taierzhuang District of Zaozhuang from January 2019 to May 2020 were retrospectively analyzed. These patients were assigned to three groups according to different time points at which thrombolytic therapy was performed: group A (the time from onset to thrombolytic therapy ≤ 3 hours, n = 27), group B (3 hours < the time from onset to thrombolytic therapy ≤ 6 hours, n = 39), group C (6 hours < the time from onset to thrombolytic therapy ≤ 12 hours, n = 53). Recanalization rate, recanalization time, ST segment resolution rate at 2 and 12 hours, serum levels of inflammatory factors [including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and high-sensitivity C-reactive protein (hs-CRP)], cathepsin S, CTGF, LVEF, and LVEDD, and incidence of cardiovascular adverse events. Results:Recanalization time in group C was (148.73 ± 15.37) minutes, which was significantly longer than that in groups A and B [(89.34 ± 8.95) minutes, (98.76 ± 9.33) minutes]. Recanalization rate and ST segment resolution rate at 2 and 12 hours in group C were 45.28%, (40.17 ± 4.77) %, (73.92 ± 8.24) %, respectively, which were significantly lower than those in the groups A and B [96.30%, 79.49%, (47.42 ± 5.12)%; (83.68 ± 9.33)%, (43.56 ± 4.87)%, (78.73 ± 8.44)%] ( t/ χ2 = 248.088, 4.244, 20.204, 11.146, 18.508, 19.861, 6.271, 4.789, 17.995, 10.932, 3.339, 4.111, 4.100, 3.828, 3.100, 2.244, all P < 0.05). At 2 and 12 hours after thrombolytic therapy, IL-6, TNF-α and hs-CRP levels in group C were (23.29 ± 2.12) ng/L, (27.03 ± 2.75) ng/L, (6.49 ± 2.37) mg/L, (22.73 ± 2.05) ng/L, (26.24 ± 2.37) ng/L and (6.01 ± 2.53) mg/L, respectively, which were significantly higher than those in groups A and B ( t = 54.578, 54.578, 10.638, 8.584, 8.735, 5.199, 7.909, 7.171, 3.597, 1.382, 1.584, 1.008, 7.237, 5.190, 4.364, 8.829, 11.114, 2.585, 3.172, 6.815, 2.196, all P < 0.05). At 2 and 12 hours after thrombolytic therapy, cathepsin S and CTGF levels in group C were (29.97 ± 3.98) μg/L, (30.03 ± 4.79) μg/L, (28.05 ± 2.13) μg/L, (28.29 ± 4.31) μg/L, respectively, which were significantly higher than those in groups A and B [(31.74 ± 3.56) μg/L, (29.87 ± 4.91) μg/L; (20.81 ± 2.35) μg/L, (16.94 ± 3.46) μg/L; (30.95 ± 3.79) μg/L, (29.93 ± 4.95) μg/L; (26.37 ± 2.44) μg/L, (21.46 ± 4.79) μg/L, t = 93.870, 68.555, 15.039, 12.562, 6.345, 7.679, 3.096, 1.966, 13.882, 3.514, 11.863, 7.164, 9.239, 4.199, all P < 0.05). At 2 and 12 hours after thrombolytic therapy, LVEF and LVEDD in group C were (42.81 ± 4.77)%, (52.64 ± 4.71) mm, (43.13 ± 5.11)%, (51.57 ± 4.01) mm, respectively, which were significantly lower than those in groups A and B [(42.61 ± 4.58)%, (52.31 ± 4.47) mm, (46.33 ± 4.35)%, (47.75 ± 3.41) mm, (42.73 ± 4.79)%, (52.79 ± 4.76) mm, (44.79 ± 4.44)%, (49.93 ± 3.73) mm, t = 4.285, 9.193, 3.060, 4.214, 1.970, 2.953, 0.333, 1.259, 2.779, 1.626, 4.229, 1.996, 1.404, 2.416, all P < 0.05). The total incidence of cardiovascular adverse events was 7.41%, 12.82% and 33.96% in groups A, B and C, respectively ( χ2 = 4.383, all P < 0.05). Conclusion:The earlier the thrombolytic therapy time after acute ST-elevation myocardial infarction, the higher the recanalization rate and ST segment resolution rate, the milder the inflammatory reaction, atherosclerosis, the better the cardiac remodeling, the better the recovery of cardiac function, and the lower the incidence of cardiovascular adverse events.
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@#Drug transportation is impeded by various barriers in the hypoxic solid tumor, resulting in compromised anticancer efficacy. Herein, a solid lipid monostearin (MS)-coated CaO2/MnO2 nanocarrier was designed to optimize doxorubicin (DOX) transportation comprehensively for chemotherapy enhancement. The MS shell of nanoparticles could be destroyed selectively by highly-expressed lipase within cancer cells, exposing water-sensitive cores to release DOX and produce O2. After the cancer cell death, the core-exposed nanoparticles could be further liberated and continue to react with water in the tumor extracellular matrix (ECM) and thoroughly release O2 and DOX, which exhibited cytotoxicity to neighboring cells. Small DOX molecules could readily diffuse through ECM, in which the collagen deposition was decreased by O2-mediated hypoxia-inducible factor-1 inhibition, leading to synergistically improved drug penetration. Concurrently, DOX-efflux-associated P-glycoprotein was also inhibited by O2, prolonging drug retention in cancer cells. Overall, the DOX transporting processes from nanoparticles to deep tumor cells including drug release, penetration, and retention were optimized comprehensively, which significantly boosted antitumor benefits.
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Objectives: Assessment ofplasma level of connective tissue growth factor in congestive heart failure children,assessment of its diagnostic and prognostic role and correlate its level with clinical and echocardiographic assessment of congestive heart failure.Methods: Connective tissue growth factorlevel in the plasma was measured in 40 children; 20 of them have congestive heart failure, and 20 are healthy then,correlated with clinical parameters. Results: The diagnostic and prognostic value of itwas evaluated. Wecompareditslevels in both patientsand healthy children. We found that connective tissue growth factor level was significantly increased in diseased children. Fractional shortening and ejection fraction correlated negatively with the plasma levelof connective tissue growth factor. Heart rate, respiratory rate and calibrated integrated backscatter correlated positively with connective tissue growth factor. Connective tissue growth factorwas significantly correlated with the class of heart failure according to Ross classification.Conclusions: Plasma connective tissue growth factor has a promising diagnostic and prognostic value as a biomarker for congestive heart failure in children with high sensitivity and specificity.
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BACKGROUND: Chronic nerve compression leads to muscle atrophy and fibrosis. Previous studies mainly focus on its pathogenesis. However, little is known about the dorsal root ganglia(DRG) responses to chronic nerve compression injury. OBJECTIVE: To investigate the effect of chronic sciatic nerve compression on fibrosis of the DRG. METHODS: Animal models of chronic sciatic nerve compression were made in rats according to the method described by Mackinnon. L4-6 ipsilateral and contralateral DRG were harvested 3 weeks post injury. Real-time RT-PCR, immunofluorescence and western blot were performed to determine the expression levels of transforming growth factor-β, connective tissue growth factor, and collagen type I in ipsilateral and contralateral DRG. RESULTS AND CONCLUSION: Three weeks after injury, the m RNA and protein expression of transforming growth factor-β, connective tissue growth factor and collagen type I were increased significantly in the ipsilateral DRG as compared with the contralateral DRG(P < 0.05). Transforming growth factor-β and connective tissue growth factor mainly expressed in DRG neurons and axons, while collagen type I formed a net structure that surrounded DRG neurons and axons. These findings indicate that chronic sciatic nerve compression can induce fibrotic changes in the DRG that appears to be associated with an increase in transforming growth factor-β and connective tissue growth factor expression in DRG neurons.
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OBJECTIVE: To observe the effect of herbal cake-partitioned moxibustion on renal function and expression of connective tissue growth factor (CTGF), integrin-linked kinase (ILK) and bone morphogenetic protein-7 (BMP-7) in chronic renal failure (CRF) rabbits, so as to reveal its mechanisms underlying improvement of CRF. METHODS: Twenty-four male New Zealand rabbits were randomly divided into control, model, medication and herbal cake-partitioned moxibustion (moxibustion) groups (n=6 rabbits in each group). The CRF model was established by gavage of suspension of Adenine (150 mg·kg-1·d-1) for 21 days. Herbal cake-partitioned moxibustion was applied to "Mingmen"(GV4) and bilateral "Shenshu"(BL23), "Pishu"(BL20) and for 5 moxa-cones every time. Rabbits of the medication group was treated by gavage of Losartan Potassium (2.33 mg·kg-1·d-1). All the treatments were conducted once daily,12 times a course for consecutive 3 courses with a two-day rest after each course of treatment. Serum creatinine (Scr), blood urea nitrogen (BUN) and 24 h urine protein contents were detected by using an automatic biochemical analyzer. The expression levels of CTGF, ILK and BMP-7 proteins and mRNA in the kidney tissue were determined by Western blot and quantitative real time-PCR, separately. RESULTS: Following modeling, Scr and BUN and 24 h urine protein contents were significantly increased in the model group in comparison with the control group (P0.05). CONCLUSION: Herbal cake-partitioned moxibustion can improve renal function in CRF rabbits, which may be related to its effects in suppressing the expression of ILK and CTGF, and in up-regulating the expression of BMP-7 in the kidney tissue.
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OBJECTIVE： To study improvement effects of Panax notoginsenoside（PNS） on cisplatin-induced renal injury model rats and its effects on related factors. METHODS： Totally 72 SD rats were randomly divided into blank group， model group， positive drug group and PNS low-dose， medium-dose， high-dose groups， with 12 rats in each group. Except for blank group， other groups were given cisplatin via tail vein （3 mg/kg×4 times） to establish renal injury model. Since the first day after the first injection of cisplatin， positive group was given anfostine solution intraperitoneally （1.0 mg/kg）； PNS groups were given PNS solution intraperitoneally （15.63， 31.35， 62.70 mg/kg）； blank group and model groups were given constant volume of normal saline 0.2 mL， for consecutive 60 d. The 24 h urine of rats was collected； the contents of β-N-acetylaminoglycosidase（NAG） and 24 h urine protein （Upro/24 h） were detected； the serum contents of Scr and BUN were detected. mRNA and protein expression of CTGF， TGF-β1， Col-1， TIMP-1 and PAI-1 in renal tissue were determined by RT-PCR and immunohistochemistry respectively. RESULTS： Compared with blank group， the contents of NAG and Upro/24 h in urine， serum contents of Scr and BUN， mRNA and protein expression levels of CTGF， TGF-β1， Col-1， TIMP-1 and PAI-1 in renal tissue were increased significantly （P＜0.05）. Compared with model group， the contents of above urine and serum biochemical indicators were decreased significantly in PNS groups； mRNA expression of CTGF and TGF-β1 and protein expression of CTGF， TGF-β1， Col-1 and TIMP-1 in renal tissue of rats in PNS groups， mRNA expression of Col-1 in PNS high-dose group， and mRNA expression of TIMP-1 and protein expression of PAI-1 in PNS medium-dose and high-dose groups were decreased significantly （P＜0.05）. Compared with positive group， the contents of NAG and Upro/24 h in urine were decreased significantly in PNS medium-dose and high-dose groups （P＜0.05）. CONCLUSIONS： PNS can effectively improve the renal function of cisplatin-induced renal injury model rats， and relieve cisplatin-induced renal fibrosis by decreasing the expression of renal fibrosis related factors as CTGF， TGF-β1， Col-1， TIMP-1 and PAI-1 in renal tissue.
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@#AIM: To investigate the effect of connective tissue growth factor(CTGF)on the proliferation and migration and transformation in Tenon's capsule fibroblasts(Tfb)of primary open angle glaucoma(POAG)patients.<p>METHODS: Tfb were cultured from Tenon's tissues in POAG patients <i>in vitro</i>. The free-serum DMEM-F12 containing 1.0, 10.0, 100.0ng/mL of CTGF was added into medium for 24h and 48h in different experimental group respectively, and only equal volume of free-serum DMEM-F12 was added in the negative control group. After 24h, the cell proliferation was analyzed through MTT assay, and migration was evaluated by crutch method. After 48h, Semi-quantitative RT-PCR was used to observed the mRNA expression of α-smooth muscl actin(α-SMA), and expression of α-SMA protein was examined by immunochemistry.<p>RESULTS: The proliferation values <i>A</i> of the cells in 1.0, 10.0, 100.0ng/mL of CTGF group respectively were 0.436±0.009, 0.643±0.009, 0.679±0.006, and 0.423 ±0.156 in the negative control group. The migrated cell number was 34.600±3.507, 70.400±2.074, 80.000±2.915 in different concentrations of CTGF group respectively, and 31.000±3.536 in the negative control group. And also in different experimental groups respectively, the absorbance ratio of α-SMA/β-actin was 0.873±0.161, 1.213±0.312, 1.352±0.376, and 0.851±0.158 in the negative control group, the expressing levels <i>A</i> of α-SMA protein in Tfb were 0.110±0.026, 0.141±0.017, 0.175±0.027, and 0.108±0.020 in the negative control group. The statistics of the above experimental data showed that, comparing with the negative control group, the 10.0 and 100.0ng/mL CTGF groups was statistically significant different(<i>P</i><0.05), but there was no statistical different between the 1.0ng/mL CTGF group and the negative control group(<i>P</i>>0.05). <p>CONCLUSION: The proliferation, migration, and phenotypic transformation of Tfb can be promoted in CTGF group in POAG patients. These findings suggest that CTGF may play a role in the development of filtering bleb scarring.
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Objective To investigate the effect of microRNA-133b(miR-133b)on cardiac fibrosis and its mechanism.Methods Human cardiac fibroblasts(CFs)were harvested.The proliferation of CFs was detected by CCK8 during the overexpression and knock-down of miR-133b.The expressions of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),collagen Ⅰ,and collagen Ⅲ were detected with qRT-PCR and Western blot analysis after miR-133b overexpression or downexpression.Target genes of miR-133b were predicted by bioinformatics software.Dual-luciferase activity assay were used to verify a target gene of miR-133b.Results qRT-PCR showed that the expression level of miR-133b in the miR-133b mimic group was significantly higher than that in the negative control group(=26.219,=0.000).The expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that in the negative control group(=6.738,=0.003).After 21,45,69,93,and 117 hours of transfection,the proliferation ability of CFs significantly decreased in the miR-133b mimic group but significantly increased in the miR-133b group(all <0.05,compared with the negative control group).After overexpression of miR-133b,the mRNA and protein levels of CTGF(=9.213,=0.001;=8.195,=0.001),α-SMA(=6.511, =0.003;=4.434,=0.011),collagenⅠ(=3.172,=0.034;=4.053,=0.015)and collagen Ⅲ(=6.404,=0.003;=5.319,=0.006)were significantly down-regulated.After the expression of miR-133b was knocked down,the mRNA and protein levels of CTGF(=9.439,=0.001;=14.100,=0.000),α-SMA(=4.519,=0.011;=4.377,=0.012),collagen Ⅰ(=5.966,=0.004;=5.514,=0.005)and collagen Ⅲ(=4.622,=0.010;=4.996,=0.008)were significantly increased.The relative luciferase activity of the cells co-transfected with miR-133b mimic and WT 3'UTR expression vector was significantly lower than that of the cells co-transfected with mimic control and WT 3'UTR expression vectors(=5.654,=0.005);however,there was no significant difference in relative luciferase activity between cells co-transfected with miR-133b mimic and MUT 3'UTR expression vectors and cells co-transfected with mimic control and MUT 3'UTR expression vectors(=0.380,=0.724).Conclusion miR-133b may affect the activation and proliferation of CFs by targeting CTGF and thus improve cardiac fibrosis.
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Humans , Actins , Metabolism , Cell Proliferation , Cells, Cultured , Collagen , Metabolism , Connective Tissue Growth Factor , Metabolism , Fibroblasts , Cell Biology , Fibrosis , MicroRNAs , Genetics , Myocardium , PathologyABSTRACT
Aim To observe the effects of UCN on TGF-β1 and CTGF in rats with DCM, and to explore the protective effects of UCN on DCM and its possible signaling pathway. Methods The rats were divided into five groups: Control group, DCM group, UCN group, UCN + Astressin group, and UCN + Triciribine group. After 12 weeks of feeding and 4 weeks of treatment, blood glucose, urinary sugar, urine volume and the levels of TGF-β1, CTGF in serum were determined, and the expression of TGF-β1, CTGF, Akt, GSK-3β, p-Akt and p-GSK-βof cardiomyocytes were also determined. Results The myocardial HE staining in DM rats was consistent with DCM. The levels of TGF-β1 and CTGF in serum and myocardium of rats with DCM increased significantly, while the levels of p-Akt and p-GSK-3J3 in myocardium of rats with DCM decreased significantly ( P < 0. 05 ). The levels of TGF-β1 and CTGF in UCN group decreased, both as-tressin and triciribine could inhibit the role of UCN, and the expression of p-Akt and p-GSK-3(3 in myocardial cells in UCN group increased significantly, then astressin could inhibit the role of UCN ( P < 0. 01). Conclusions The protective effects of UCN on DCM might be related to the activation of Akt/GSK-30 signaling pathway after binding with CRH-R receptor, and then decreasing the expression of TGF-β1 and CTGF.
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Objective: To investigate the mechanism of p38 mitogen activated protein kinase (MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor β 1 (TGF-β 1)/connective tissue growth factor (CTGF). Methods: The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun N-terminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR (qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA (siRNA), p38 siRNA, siRNA+3 ng/mL TGF-β 1, and p38 siRNA+3 ng/mL TGF-β 1 in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38 (p-p38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRT-PCR; the protein expression of CTGF in each group was detected by Western blot. Results: p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ ( P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly ( P<0.05), while those in groups C and D increased significantly ( P<0.05); and those indicators significantly increased in group C than in group D ( P<0.05). Conclusion: TGF-β 1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.
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Objective: To investigate the effect of transforming growth factor β 1 (TGF-β 1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression. Methods: The ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-β 1, 50 ng/mL CTGF, 3 ng/mL TGF-β 1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-β 1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes. Results: The morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance ( A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different ( P0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E ( P0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E ( P0.05). Conclusion: TGF-β 1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-β 1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.
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Objective • To investigate the effects of silencing connective tissue growth factor (Ctgf) gene on the growth, cell cycle and the expression of TGF-β1, Smad3 and Smad7 of rat hepatic stellate cell line HSCT6. Methods • The recombinant lentivirus vector pCDH/Ctgf-shRNA of Ctgf gene was constructed by RNA interference. The recombinant vector was packaged to obtain highly infectious pCDH/Ctgf-shRNA lentivirus particles for HSCT6 infection. The expression of green fluorescent protein (GFP) in the transfected HSCT6 cells was observed under fluorescence microscope. The effects of Ctgf-shRNA lentivirus on the growth of HSCT6 cells were tested by CCK-8. The effects of Ctgf-shRNA lentivirus on the cell cycle of HSCT6 cells were analyzed by flow cytometry (FCM). The effects of Ctgf-shRNA lentivirus on the expression of mRNA of Ctgf, Tgf-β1, Smad3 and Smad7, and their proteins in HSCT6 cells were detected by real-time PCR and Western blotting, respectively. Results • The lentiviral vector pCDH/Ctgf-shRNA has been constructed successfully. The HSCT6 cells transfected by Ctgf-shRNA lentivirus significantly expressed GFP under fluorescence microscope. The results of CCK-8 confirmed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was slower than that of controls and the differences were statistically significant after being cultured for 72 h (P<0.05). The results of FCM revealed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was blocked in the S phase of cell cycle. The results of real-time PCR and Western blotting showed that the Ctgf-shRNA lentivirus effectively silenced Ctgf gene, down-regulated the expression of genes and encoding proteins of TGF-β1, and Smad3 of HSCT6 and up-regulated the expression of genes and encoding proteins of Smad7 of HSCT6 cells. The differences between transfected cells and controls were statistically significant (P<0.05). Conclusion • Silencing Ctgf gene can effectively inhibit the growth of HSCT6 cells, down-regulate the expression of TGF-β1 and Smad3 and up-regulate the expression of Smad7. The inhibition of the growth of HSCT6 cells may be closely related to interference of the TGF-β1/Smads (Smad3 and Smad7) signaling pathway.
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Objective To explore the potential effects of neutrophil extracellular traps (NETs) on rheumatoid arthritis synovial fibroblasts (RA-FLSs).Methods The synovial tissues of RA patients were isolated and cultured in vitro.Peripheral blood neutrophils were extracted from healthy volunteers and used to stimulate NETs' formation,following with NETs' extraction.MTS proliferation assay was used to evaluate the effect of NETs on the proliferation of RA-FLSs.QRT-polymerase chain reaction (q-PCR) was used to determine the expression of connective tissue growth factor (CTGF) mRNA in cells treated with NETs-stimulated RA-FLSs for 60 h.The results were processed using paired sample t-test and one-way analysis of variance (ANOVA).Results The isolated and purified neutrophils could form NETs by in vitro stimulation.The concentration of extracted NETs-DNA was 58.5 ng/μl (1×106 cells).Compared with the control group (0 μl NETs),NETs could promote the proliferation of RA-FLSs.With the increase of NETs' concentration,the proliferation of RA-FLSs was also enhanced (F=99.519,P＜0.05).Compared with the control group (0 μl NETs),10 μl NETs could significandy promote the proliferation of RA-FLSs (t=-12.226,P＜0.01).Pretreatment of NETs with DNase Ⅰ inhibited its effect on promoting the proliferation of RA-FLSs (t=-2.376,P=0.049),NETs stimulated the upre-gulation of CTGF mRNA expression in RA-FLSs [(30.7±0.5),t=12.13,P＜0.01].Conclusion NETs can promote the proliferation of RA-FLSs and stimulate the up-regulation of CTGF mRNA in RA-FLSs in vitro.
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BACKGROUND: Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. METHODS: Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and MPGΔNLS) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8 mm biopsy punch wound at 1 day to 8 wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. RESULTS: CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p < 0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF siRNA/MPGΔNLS complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of 128 ± 22 nm (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean 90 ± 20 nm, n = 563). CONCLUSION: Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.
Subject(s)
Biopsy , Cell-Penetrating Peptides , Cicatrix , Collagen , Connective Tissue Growth Factor , Connective Tissue , Down-Regulation , Ear , Microscopy, Electron, Transmission , Preventive Medicine , Regeneration , RNA, Small Interfering , Wounds and InjuriesABSTRACT
Objective To investigate whether pyrrolidine dithiocarbamate (PDTC) can attenuate the acute radiation-induced heart damage (RIHD) by inhibiting the activation of NF-κB and its downstream signaling pathways in rat models. Methods Twenty-one male adult Sprague-Dawley (SD) rats were randomly divided into the blank control, irradiation and PDTC plus irradiation groups (n=7 for each group). In the irradiation and PDTC+ irradiation groups,the rats received 6 MV X-ray at a single fraction of 20.0 Gy. In the PDTC+ irradiation group, intraperitonal injection of PDTC was administered at a dose of 120 mg/kg body weight,30 minutes prior to radiation, once daily for 1-14 days. On the 14thday,pathological changes of myocardial tissue were observed. Masson's trichrome staining was performed to calculate the collagen volume fraction (CVF) of myocardial cells. The expression levels of NF-κB family members including p50, p65,HIF-1α,connective tissue growth factor (CTGF) and collagen type 1(COL-1) proteins and mRNA were quantitatively measured by Western blot and quantitative real-time PCR (qPCR). Statistical analysis was conducted by using t-test. Results HE staining demonstrated that compared with the irradiation group, the severity of myocardial edema was alleviated,the infiltration of inflammatory cells was mitigated and the quantity of fibroblasts was reduced in the PDTC+irradiation group. Masson's trichrome staining revealed that PDCT intervention could decrease the deposition of collagen fiber in the interstitial tissues. Semi-quantitative analysis demonstrated that the CVF value in the PDTC+irradiation group was (9.99± 0.32)%, significantly lower compared with (22.05±0.21)% in the irradiation group (P<0.05). Western blot and qRT-PCR demonstrated that the expression levels of p50,p65,and HIF-1αproteins and mRNA in the PDTC+ irradiation group were significantly down-regulated compared with those in the irradiation group (all P<0.05). Compared with the irradiation group,the expression levels of CTGF protein and mRNA tended to decline (all P>0.05),and the expression levels of COL-1 protein and mRNA were equally inclined to decrease (P<0.05 and P>0.05). Conclusion PDTC can alleviate the acute RIHD by suppressing the activation of NF-κB and its downstream HIF-1α transcription.
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Objective To construct the connective tissue growth factor (CTGF) recombinant interference vector (shRNA) and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells.Methods The human CTGF shRNA was constructed and the high-titer CTGF shRNA lentivirus particles was acquired via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells.The optimal multiplicity and onset time of lentivirus infection were identified by tracing down the red florescent protein in interference vector.The cells were classified into three groups:blank control group,infection control group and CTGF knockdown group.The differences in cells migrating ability was observed through Transwell allay.The mRNA and protein expression of CTGF,fibronectin,a-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were quantified through real-time PCR testing and Western blot system.Data between the three groups were examined via one-way analysis of variance.Results The result showed that an optimal multiplicity of 20 and onset time of 72 hours were the requirements to optimize lentivirus infection.Transwell allay result showed a contrast in the number of migrated cells in the CTGF knockdown group and that in the blank control group and infection control group (F=20.64,P=0.002).Real-time PCR testing showed a contrast in related gene expression (CTGF,fibronectin,α-SMA and Col Ⅰ) in the CTGF knocked-down group and that in the blank control group and infection control group (F=128.83,124.44,144.76,1 374.44;P=0.000,0.000,0.000,0.000).Western blot system showed the statistical significance of the contrasted number of related protein expression (CTGF,fibronectin,α-SMA and Col Ⅰ) in the knockdown group and that in the blank control group (F=22.55,41.60,25.73,161.68;P=0.002,0.000,0.001,0.000).Conclusion The success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression.