Protein Expression Levels Of Collagen Research Articles

Therapeutic effect and mechanism of Yougui Wan in rats with intervertebral disk degeneration

ObjectiveTo explore the potential mechanism of Yougui Wan on deformed lumbar intervertebral disk structure in rats.MethodsThirty male Sprague–Dawley rats were randomly divided into 3 groups, with 10 rats in each group. The animals in the blank control group were healthy rats without specific treatment, and those in the model group and traditional Chinese medicine (TCM) group were used to establish the intervertebral disk degeneration (IDD) model by puncturing the annulus. Four weeks after modeling, rats in the TCM group were administered Yougui Wan by gavage for 2 consecutive weeks. Serum interleukin-6 (IL-10), macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNF-α) levels were measured by ELISA, and the protein expression levels of collagen II and Notch1 in intervertebral disk tissues were examined by Western blotting. Apoptosis was detected by the TUNEL method.ResultsCompared with those in the blank group, IL-10, MIF and TNF-α levels in the model group and TCM group were increased (P < 0.05), the protein expression levels of collagen II were decreased, and the protein expression levels of Notch1 were increased. Compared with those in the model group, the levels of IL-10 in the TCM group were increased (P < 0.05), the levels of MIF and TNF-α were decreased (P < 0.05), the protein expression levels of collagen II were increased, and the protein expression levels of Notch1 were decreased.ConclusionYougui Wan can inhibit the inflammatory response in IDD rats, reduce the degradation of extracellular matrix, reduce apoptosis in nucleus pulposus cells, and alleviate intervertebral disk degeneration. The mechanism may be related to the regulation of the Notch signaling pathway.

Treprostinil inhibits functional activation of scleroderma (SSc) vascular smooth muscle cells (vSMCs) by inhibiting Yap and activating PPARG signaling.

Background:&#x0D; Progressive vascular wall thickness and fibrosis are the hallmarks of SSc vasculopathy. Overexpression of TGFB1 in SSc and activation of vSMCs are important steps in the pathogenesis of SSc vascular disease.&#x0D; Objectives:&#x0D; In this study, we examined the expression levels of COL1, PCNA, PFKP, IP, EP2, IP, and PTGIS in SSc skin, the effects of Treprostinil (prostacyclin analog) on cell proliferation, TGFB1-induced collagen expression in SSc-vSMCs, PPARG expression and Yap nuclear translocation.&#x0D; Methods: &#x0D; SSc and control skin biopsies were fixed and 10uM serial sections were cut for histological examination. vSMCs were isolated from involved skin and matched healthy subjects. The expression and distribution of collagen, PCNA, PFKP, IP, EP2, PTGIS, PPARG, and Yap were measured by immunohistochemical staining or immunofluorescent staining. Cell proliferation was measured by MTT assay. The mRNA expression levels were detected by qPCR.&#x0D; Results:&#x0D; The protein expression levels of collagen, PCNA, PFKP, and EP2 were increased, while the expression levels of PTGIS and IP were decreased in vSMCs of SSc-skin, compared to the control. These results suggested that defective PGI2-IP signaling in SSc-vSMCs may contribute to vessel wall thickness and vascular fibrosis in SSc. Treprostinil inhibited vSMCs proliferation, COL1A1, and PFKP mRNA expression in SSc-vSMCs in a dose-dependent fashion. Treprostinil also inhibited TGFB1-induced COL1A1 mRNA expression in SSc-vSMCs via engagement of EP2. The PPARG expression was significantly increased in treprostinil-treated SSc-vSMCs. Treprostinil decreased the nuclear location of YAP which is induced by 10%FBS and TGFB1.&#x0D; Conclusion:&#x0D; Defective PGI2-IP signaling in SSc-vSMCs is associated with enhanced expression of collagen, PCNA, and PFKP in SSc vessel walls. The antiproliferative and antifibrotic activity of treprostinil is mediated through the inhibition of YAP nuclear translocation and enhanced PPARG expression. YAP and PPARG might be promising therapeutic targets for the treatment of SSc-related vasculopathy.

Phloretin ameliorates heart function after myocardial infarction via NLRP3/Caspase-1/IL-1β signaling

Objectives/AimsInflammation is crucial in structural and electrical remodeling after myocardial infarction (MI), affecting cardiac pump function and conduction pathways. Phloretin possesses an anti-inflammation role by inhibiting the NLRP3/Caspase-1/IL-1β pathway. However, the effects of Phloretin on cardiac contractile and electrical conduction function after MI remained unclear. Therefore, we aimed to investigate the potential role of Phloretin in a rat model of MI. MethodsRats were assigned into four groups: Sham, Sham+Phloretin, MI and MI+Phloretin, with ad libitum food and water. In the MI and MI+Phloretin groups, the left anterior descending coronary artery was occluded for 4 weeks, while the Sham and Sham+Phloretin groups received sham operation. The Sham+Phloretin group and the MI+Phloretin group received oral administration of Phloretin. In vitro, H9c2 cells were subjected to hypoxic conditions to simulate an MI model, with Phloretin for 24 h. Cardiac electrophysiological properties were assessed following MI, including the effective refractory period (ERP), action potential duration (APD)90 and ventricular fibrillation (VF) incidence. Echocardiography evaluated left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV) to assess cardiac function. Serum type B natriuretic peptide (BNP) level was applied to evaluate the degree of Heart failure (HF). The fibrosis area and severity were assessed by Masson staining and protein expression levels of collagen 3, collagen 1, TGF-β and α-SMA. Western blot analysis estimated the protein expression levels of NLRP3, Pro Caspase-1, Caspase-1, ASC, IL-18, IL-1β, pp38, p38, and Connexin43(Cx43) to elucidate the influence of inflammation on electrical remodeling after MI. ResultsOur findings demonstrate that Phloretin inhibits the NLRP3/Caspase-1/IL-1β pathway, leading to the upregulation of Cx43 by limiting p38 phosphorylation, which further decreases susceptibility to ventricular arrhythmias (VAs). Additionally, Phloretin attenuated fibrosis by inhibiting inflammation to prevent HF. In vitro experiments also provided strong evidence supporting the inhibitory effects of Phloretin on the NLRP3/Caspase-1/IL-1β pathway. ConclusionOur results suggest that Phloretin could suppress the NLRP3/Caspase-1/IL-1β pathway to reverse structural and electrical remodeling after MI to prevent the occurrence of VAs and HF.

Therapeutic hypothermia alleviates myocardial ischaemia-reperfusion injury by inhibiting inflammation and fibrosis via the mediation of the SIRT3/NLRP3 signalling pathway.

Therapeutic hypothermia (TH) may attenuate myocardial ischaemia–reperfusion injury, thereby improving outcomes in acute myocardial infarction. However, the specific mechanism by which TH alleviates MIRI has not been elucidated so far. In this study, 120 healthy male Sprague‐Dawley rats were randomly divided into five groups. Haemodynamic parameters, myocardial infarction area, histological changes and the levels of cardiac enzymes, caspase‐1 and inflammatory cytokines were determined. In addition, the extent of myocardial fibrosis, the degree of cardiomyocyte apoptosis and the expression levels of SIRT3, GSDMD‐N, fibrosis‐related proteins and inflammation‐related proteins were estimated.TH reduced myocardial infarct area and cardiac enzyme levels, improved cardiomyopathic damage and haemodynamic indexes, and attenuated myocardial fibrosis, the protein expression levels of collagen I and III, myocardial apoptosis, the levels of inflammatory cytokines and inflammation‐related proteins. Notably, the immunofluorescence and protein expression levels of SIRT3 were upregulated in the 34H+DMSO group compared to the I/R group, but this protective effect was abolished by the SIRT3 inhibitor 3‐TYP. After administration of Mcc950, the reversal effects of 3‐TYP were significantly abolished, and TH could protect against MIRI in a rat isolated heart model by inhibiting inflammation and fibrosis. The SIRT3/NLRP3 signalling pathway is one of the most important signalling pathways in this regard.

Knockdown of lncRNA-NEAT1 expression inhibits hypoxia-induced scar fibroblast proliferation through regulation of the miR-488-3p/COL3A1 axis.

Long non-coding (lnc)RNA nuclear-enriched transcripts 1 (NEAT1) has been demonstrated to be involved in the inhibition of hypoxia-induced scar fibroblast proliferation, but the specific mechanism remains undetermined. The present study found that with the decrease of oxygen concentration, lncRNA NEAT1 was upregulated in hypoxia-induced scar fibroblasts, which promoted the mRNA and protein expression levels of collagen (COL)-I, COL-III and α-smooth muscle actin, thereby suppressing hypoxia-induced scar fibroblasts proliferation. In addition, the microRNA (miR)-488-3p/COL3A1 axis was involved in lncRNA NEAT1's regulation of the proliferation of hypoxia-induced scar fibroblasts. In conclusion, the knockdown of lncRNA-NEAT1 expression can inhibit hypoxia-induced scar fibroblasts proliferation through regulation of the miR-488-3p/COL3A1 axis, which will provide a novel therapeutic target for the treatment of hypertrophic scars.

Mechanism and Experimental Verification of Luteolin for the Treatment of Osteoporosis Based on Network Pharmacology.

PurposeTo explore the molecular mechanism of luteolin in the treatment of osteoporosis (OP) by network pharmacological prediction and experimentation.MethodsThe target proteins of luteolin were obtained with the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). OP-related proteins were extracted from the Comparative Toxicogenomics Database (CTD) and GeneCards and DisGeNET databases. We imported the common protein targets of luteolin and OP into the STRING database to obtain the relationships between the targets. The common target proteins of luteolin and OP were assessed by KEGG and GO enrichment analyses with the DAVID database. Animal experiments were conducted to verify the effect of luteolin on bone mineral density in ovariectomised (OVX) rats. Finally, the effects of luteolin on key signalling pathways were verified by cell experiments in vitro.ResultsForty-four targets of luteolin involved in the treatment of OP, including key target proteins such as TP53, AKT1, HSP90AA1, JUN, RELA, CASP3, and MAPK1, were screened. KEGG enrichment analysis found that luteolin inhibits OP by regulating the PI3K-Akt, TNF, oestrogen and p53 signalling pathways. The results of animal experiments showed that bone mass in the low-dose luteolin group (Luteolin-L group, 10 mg/kg), high-dose luteolin group (Luteolin-H group, 50 mg/kg) and positive drug group was significantly higher than that in the OVX group (P<0.05). Western blot (WB) analysis showed that the protein expression levels of Collagen I, Osteopontin and RUNX2 in bone marrow mesenchymal stem cells (BMSCs) cultured with 0.5, 1 and 5 μM luteolin for 48 h were significantly higher than those in the dimethyl sulfoxide (DMSO) group (P<0.05). In vitro cell experiments showed that the p-PI3K/PI3K and p-Akt/Akt expression ratios in BMSCs cultured with 0.5, 1 and 5 μM luteolin for 48 h were also significantly higher than those in the DMSO group (P<0.05).Conclusions Luteolin has multitarget and multichannel effects in the treatment of OP. Luteolin could reduce bone loss in OVX rats, which may be due to its ability to promote the osteogenic differentiation of BMSCs by regulating the activity of the PI3K-Akt signalling pathway.

MiR-320a in serum exosomes promotes myocardial fibroblast proliferation via regulating the PIK3CA/Akt/mTOR signaling pathway in HEH2 cells.

MicroRNAs (miRNAs/miRs) serve an important role in the pathogenesis of chronic heart failure (CHF). A number of reports have illustrated the regulatory effect of serum exosomal miRNA on myocardial fibrosis. The present study aimed to investigate the expression of miR-320a in serum exosomes, as well as the effect of miR-320a on myocardial fibroblast proliferation. Serum exosome samples from 10 patients with CHF and 5 healthy volunteers were obtained and characterized. mRNA and protein expression levels were measured via reverse transcription-quantitative PCR and western blotting, respectively. The content of soluble growth stimulation expressed gene 2 (sST2) was determined via ELISA. HEH2 cell viability and apoptosis were detected by performing MTT assays and flow cytometry, respectively. The results demonstrated that serum miR-320a expression levels and sST2 content were significantly increased in patients with CHF compared with healthy controls, and the expression of serum miR-320a was significantly correlated with clinical CHF indexes. miR-320a expression levels were significantly increased in exosomes isolated from patients with CHF compared with those isolated from healthy controls. Phosphoinositide-3-kinase catalytic α polypeptide gene (PIK3CA) expression levels and sST2 content were increased in HEH2 cells following transfection with miR-320a mimics compared with NC-mimic, whereas miR-320a inhibitor displayed contrasting effects by reduced the cell viability and apoptosis in myocardial fibroblasts compared with the NC-inhibitor group. The protein expression levels of collagen I, collagen III, α-smooth muscle actin, phosphorylated (p)-mTOR (ser 2448)/mTOR, p-Akt (ser 473)/Akt, p-Akt (thr 308)/Akt and PIK3CA were significantly increased in miR-320a mimic-transfected HEH2 cells compared with the NC-mimics groups. By contrast, miR-320a inhibitor notably downregulated the expression levels of these proteins compared with the NC-inhibitor group. Collectively, the results of the present study demonstrated that miR-320a promoted myocardial fibroblast proliferation via regulating the PIK3CA/Akt/mTOR signaling pathway in HEH2 cells, suggesting that serum exosomal miR-320a may serve as a potential biomarker for the diagnosis of CHF.

MicroRNA‑133b alleviates doxorubicin‑induced cardiomyocyte apoptosis and cardiac fibrosis by targeting PTBP1 and TAGLN2.

Doxorubicin is one of the most important chemotherapeutic drugs for the treatment of malignant tumors, but the cardiotoxicity of doxorubicin severely limits its clinical application. Increasing numbers of microRNAs (miRNAs/miRs) have been found to be dysregulated in doxorubicin-treated cardiomyocytes or animal hearts. The current study aimed to investigate the role of miR-133b in doxorubicin-induced cardiomyocyte injury. Doxorubicin was used to treat HL-1 cardiomyocytes to mimic cardiomyocyte injury in vitro. A mouse model of cardiac injury was generated by chronic intraperitoneal injections of doxorubicin. Masson's trichrome staining was performed on cardiac tissues to reveal cardiac fibrosis. Bioinformatics analysis and luciferase reporter assays were applied to explore the downstream targets of miR-133b. Flow cytometry and western blotting were conducted to detect cardiomyocyte apoptosis. Protein expression levels of collagen I, III and IV, and fibronectin were detected to reveal extracellular matrix deposition. The results revealed that doxorubicin decreased miR-133b expression in the treated HL-1 cardiomyocytes and mouse hearts. Overexpression of miR-133b restrained cardiomyocyte apoptosis, inhibited collagen accumulation and alleviated cardiac fibrosis in vivo. Mechanistically, polypyrimidine tract binding protein 1 (PTBP1) and transgelin 2 (TAGLN2) were confirmed to bind to miR-133b after prediction and screening. Moreover, miR-133b negatively regulated the protein expression levels of PTBP1 and TAGLN2. Finally, overexpression of PTBP1 or TAGLN2 reversed the effects of miR-133b on apoptosis and collagen accumulation. Thus, the current results indicated that miR-133b alleviated doxorubicin-induced cardiomyocyte apoptosis and cardiac fibrosis by targeting PTBP1 and TAGLN2, implying that miR-133b may be a potential biomarker for doxorubicin-induced cardiac injury.

Chrysin inhibited epithelial-mesenchymal transition of type Ⅱ alveolar epithelial cell by regulating NF-κB/Twist 1 signaling pathway

This study aimed to assess whether chrysin(ChR) can inhibit epithelial-mesenchymal transition(EMT) of type Ⅱ alveolar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway. Sixty rats were randomly divided into the control group, the bleomycin(BLC) group, BLC+ChR(50 mg·kg~(-1)) group and BLC+ChR(100 mg·kg~(-1)) group, with 15 rats in each group. The pulmonary fibrosis model was induced by intratracheal injection of BLC(7 500 U·kg~(-1)). Rats were orally administered with different doses of ChR after BLC injection for 28 days. The cells were divided into control group, TGF-β1 group(5 ng·mL~(-1)), and TGF-β1+ChR(1, 10, 100 μmol·L~(-1)) groups. The type Ⅱ alveolar epithelial cells were treated with TGF-β1 for 24 h, and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1, 10 and 100 μmol·L~(-1)). The morphological changes and collagen deposition in lung tissues were analyzed by HE staining, Masson staining and immunohistochemistry. The mRNA and protein expression levels of collagen Ⅰ, E-cadherin, zonula occludens-1(ZO-1), vimentin, alpha smooth muscle actin(α-SMA), inhibitor of nuclear factor kappa B alpha(IκBα), nuclear factor-kappa B p65(NF-κB p65), phospho-NF-κB p65(p-p65) and Twist 1 in lung tissues and cells were detected by qPCR and Western blot, respectively. The animal experiment results showed that as compared with the BLC group, after administration of ChR for 28 days, bleomycin-induced pulmonary fibrosis in rats was significantly relieved, collagen Ⅰ expression in lung tissues was significantly reduced(P&lt;0.05 or P&lt;0.01), and EMT of alveolar epithelial cells was obviously inhibited [the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], concomitantly with significantly reduced IκBα and p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P&lt;0.05 or P&lt;0.01). The cell experiment results showed that different doses of ChR(1, 10 and 100 μmol·L~(-1)) significantly reduced TGF-β1-induced collagen Ⅰ expression(P&lt;0.05 or P&lt;0.01), significantly inhibited EMT of type Ⅱ alveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin and α-SMA were decreased(P&lt;0.05 or P&lt;0.01)], and inhibited IκBα and p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P&lt;0.05 or P&lt;0.01). The results suggest that ChR can reverse EMT of type Ⅱ alveolar epithelial cell and alleviate pulmonary fibrosis in rats, and its mechanism may be associated with reducing IκBα phosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer, thus down-regulating Twist 1 expression.

Plumbagin attenuates traumatic tracheal stenosis in rats and inhibits lung fibroblast proliferation and differentiation via TGF-β1/Smad and Akt/mTOR pathways

ABSTRACT Traumatic tracheal stenosis (TS) is a serious respiratory disease characterized by hyperplasia of airway granulation. Plumbagin (PLB) is a natural naphthoquinone component with anti-fibrotic properties. This research aimed to explore the roles of PLB in alleviating TS and the underlying mechanisms. For in vitro studies, lung fibroblasts (IMR-90 cells), with/without PLB treatment or TGF-β1 induction, were used. The viability and proliferation of IMR-90 cells were examined by CCK-8 and EdU incorporation assays. The differentiation of IMR-90 cells was assessed by detecting the mRNA and protein expression levels of collagen (COL)-1 and alpha-smooth muscle actin (α-SMA). Besides, immunofluorescence assay was conducted to evaluate the localization of α-SMA in TGF-β1-induced IMR-90 cells. Moreover, the combination of PLB with/without TβRI (SB-431,542), PI3K/Akt (Ly294002) or mTOR (rapamycin) inhibitor was pretreated on IMR-90 cells after TGF-β1 induction. For in vivo studies, a rat model of TS was established. The pathological features and severity of TS were determined by hematoxylin and eosin staining. The protein levels of TGF-β1/Smad and Akt/mTOR pathways were detected for both in vitro and in vivo models. PLB effectively inhibited the proliferation and differentiation of TGF-β1-induced IMR-90 cells, and suppressed TGF-β1/Smad and Akt/mTOR signaling pathways both in vivo and in vitro. Furthermore, PLB reduced the degree of TS in rats. Taken together, our results indicate that PLB regulates lung fibroblast activity and attenuates TS in rats by inhibiting TGF-β1/Smad and Akt/mTOR signaling pathways. In conclusion, this study implies that PLB may serve as a promising therapeutic compound for TS.

Ginsenoside Rb1 attenuates age-associated vascular impairment by modulating the Gas6 pathway

Context Ginsenoside Rb1 (Rb1) exerts many beneficial effects and protects against cardiovascular disease. Objective To investigate whether Rb1 could attenuate age-related vascular impairment and identify the mechanism. Materials and methods Female C57BL/6J mice aged 2 and 18 months, randomly assigned to Young, Young + 20 mg/kg Rb1, Old + vehicle, Old + 10 mg/kg Rb1 and Old + 20 mg/kg Rb1 groups, were daily intraperitoneal injected with vehicle or Rb1 for 3 months. The thoracic aorta segments were used to inspect the endothelium-dependent vasorelaxation. Left thoracic aorta tissues were collected for histological or molecular expression analyses, including ageing-related proteins, markers relevant to calcification and fibrosis, and expression of Gas6/Axl. Results We found that in Old + vehicle group, the expression of senescence proteins and cellular adhesion molecules were significantly increased, with worse endothelium-dependent thoracic aorta relaxation (58.35% ± 2.50%) than in Young group (88.84% ± 1.20%). However, Rb1 treatment significantly decreased the expression levels of these proteins and preserved endothelium-dependent relaxation in aged mice. Moreover, Rb1 treatment also reduced calcium deposition, collagen deposition, and the protein expression levels of collagen I and collagen III in aged mice. Furthermore, we found that the downregulation of Gas6 protein expression by 41.72% and mRNA expression by 52.73% in aged mice compared with young mice was abrogated by Rb1 treatment. But there was no significant difference on Axl expression among the groups. Conclusions Our study confirms that Rb1 could ameliorate vascular injury, suggesting that Rb1 might be a potential anti-ageing related vascular impairment agent.

Mitochondrial dysfunction drives persistent vascular fibrosis in rats after short-term exposure of PM2.5

Nowadays, the great majority of toxicological studies have focused on immediate cardiovascular effects of simultaneous exposure to long-term or short-term PM2.5; yet, whether the persistent vascular fibrosis will be induced after short-term PM2.5 exposure and its related underlying mechanisms remain unclear. In this study, we adopted SD rats treated with PM2.5 for 1 month and followed by 12 months and 18 months recovery. Results from Doppler ultrasonography and histopathological analysis found that PM2.5-evoked vascular fibrosis was comprised of structural injury, including thickening of aortic media and carotid intima media thickness (CIMT), narrow left common carotid artery (LCCA), collagen deposition, impaired elasticity and functional alterations in aortal stiffness during long-term recovery. The protein expression levels of collagen I, collagen III, proliferating cell nuclear antigen (PNCA), TGF-β and osteopontin (OPN) remained elevated trends in PM2.5-treated groups for the related period than in control groups. Additionally, PM2.5 upregulated the protein expression levels of superoxide dismutase 2 (SOD2), mitochondrial fission related proteins (Drp1 and Fis1), while downregulated the protein expression levels of mitochondrial fusion related proteins (Mfn2 and OPA1). Moreover, PM2.5 significantly activated the mitophagy-related protein expression, including LC3, p62, PINK, Parkin. In summary, our results demonstrated that short-term PM2.5 exposure could trigger mitophagy, further lead to mitochondrial dysfunction which regulated persistent vascular fibrosis during long-term recovery.

Bidirectional ephrinB2‑EphB4 signaling regulates the osteogenic differentiation of canine periodontal ligament stem cells.

The aim of the present study was to evaluate the effect of ephrinB2 gene-transfected canine periodontal ligament stem cells (cPDLSCs) on the regulation of osteogenic differentiation. cPDLSCs were transfected with a transgenic null-control green fluorescent protein (GFP) vector (termed Vector-cPDLSCs) or with NFNB2 GFP-Blasticidin (termed EfnB2-cPDLSCs). Subsequently, the osteogenic differentiation of Vector-cPDLSCs and EfnB2-cPDLSCs was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), alkaline phosphatase (ALP) assay and Alizarin Red S staining. The migratory abilities of cPDLSCs, Vector-cPDLSCs and EfnB2-cPDLSCs were also assessed. Following osteogenic induction of Vector-cPDLSCs and EfnB2-cPDLSCs, the protein expression levels of collagen I, Runt-related transcription factor 2, osteocalcin, ephrin type-B receptor 4 (EphB4), phospho-EphB4, ephrinB2 and phosphoephrinB2 were analyzed by western blot assays. Following gene transfection, the RT-qPCR and western blotting results revealed that the mRNA and protein expression levels of ephrinB2, respectively, were significantly increased in EfnB2-cPDLSCs compared with that in Vector-cPDLSCs (P<0.05). ALP and Alizarin Red S staining assays revealed increased ALP activity and mineralization nodules, respectively, in EfnB2-cPDLSCs. Cell proliferation and migration assays revealed that EfnB2-cPDLSCs exhibited enhanced proliferation and migration compared with Vector-cPDLSCs (P<0.05). In conclusion, the findings of the current study indicated that ephrinB2 gene-modified cPDLSCs exhibited enhanced osteogenic differentiation, with the ephrinB2 reverse signaling and EphB4 forward signaling pathways serving a key role in this process. Furthermore, ephrinB2 gene modification was observed to promote the migration and proliferation of cPDLSCs.

Triptolide inhibits NLRP3 inflammasome activation and ameliorates podocyte epithelial-mesenchymal transition induced by high glucose

The aim of this paper was to explore the effects of triptolide( TP),the effective component of Tripterygium wilfordii on improving podocyte epithelial-mesenchymal transition( EMT) induced by high glucose( HG),based on the regulative mechanisms of Nod-like receptor protein 3( NLRP 3) inflammasome in the kidney of diabetic kidney disease( DKD). The immortalized podocytes of mice in vitro were divided into the normal( N) group,the HG( HG) group,the low dose of TP( L-TP) group,the high dose of TP( HTP) group and the mannitol( MNT) group,and treated by the different measures,respectively. More specifically,the podocytes in each group were separately treated by D-glucose( DG,5 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) or HG( 30 mmol·L~(-1)) + TP( 5 μg·L~(-1))or HG( 30 mmol·L~(-1)) + TP( 10 μg·L~(-1)) or DG( 5 mmol·L~(-1)) + MNT( 24. 5 mmol·L~(-1)). After the treatment of HG or TP at 24,48 and 72 h,firstly,the activation of podocyte proliferation was investigated. Secondly,the protein expression levels of the epithelial markers in podocytes such as nephrin and ZO-1,the mesenchymal markers such as collagen Ⅰ and fibronectin( FN) were detected,respectively. Finally,the protein expression levels of NLRP3 and apoptosis-associated speck-like protein( ASC) as the key signaling molecules of NLRP3 inflammasome activation,as well as the downstream effector proteins including caspase-1,interleutin( IL)-1β and IL-18 were examined,severally. The results indicated that,for the cultured podocytes in vitro,HG could cause the low protein expression levels of nephrin and ZO-1,induce the high protein expression levels of collagen Ⅰ and FN and trigger podocyte EMT. Also HG could cause the high protein expression levels of NLRP3,ASC,caspase-1,IL-1β and IL-18 and induce NLRP3 inflammasome activation. On the other hand,the co-treatment of TP( L-TP or H-TP) and HG for podocytes could recover the protein expression levels of nephrin and ZO-1,inhibit the protein expression levels of collagen Ⅰ and FN and ameliorate podocyte EMT. Also the co-treatment of TP( L-TP or H-TP) and HG could down-regulate the protein expression levels of NLRP3 and ASC,inhibit NLRP3 inflammasome activation and reduce the protein expression levels of the downstream effector molecules including caspase-1,IL-1β and IL-18. On the whole,HG could activate NLRP3 inflammasome and induce podocyte EMT in vitro. TP at the appropriate dose range could inhibit NLRP3 inflammasome activation and ameliorate podocyte EMT,which may be one of the critical molecular mechanisms of TP protecting againstpodocyte inflammatory injury in DKD.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts

BackgroundConjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery.MethodsCygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-β1, and HIF1α were determined by RT-PCR and western blotting respectively.ResultsAfter Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-β1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05).ConclusionOur study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-β1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

Soluble cMet levels in urine are a significant prognostic biomarker for diabetic nephropathy

Hepatocyte growth factor and its receptor cMet activate biological pathways necessary for repair and regeneration following kidney injury. Here, we evaluated the clinical role of urinary cMet as a prognostic biomarker in diabetic nephropathy (DN). A total of 218 patients with DN were enrolled in this study. We examined the association of urine cMet levels and long-term outcomes in patients with DN. The levels of urinary cMet were higher in patients with decreased renal function than in patients with relatively preserved renal function (5.25 ± 9.62 ng/ml versus 1.86 ± 4.77 ng/ml, P = 0.001). A fully adjusted model revealed that a urinary cMet cutoff of 2.9 ng/mL was associated with a hazard ratio for end-stage renal disease of 2.33 (95% confidence interval 1.19–4.57, P = 0.014). The addition of urinary cMet to serum creatinine and proteinuria provided the highest net reclassification improvement. We found that in primary cultured human glomerular endothelial cells, TGFβ treatment induced fibrosis, and the protein expression levels of collagen I, collagen IV, fibronectin, and αSMA were decreased after administration of an agonistic cMet antibody. In conclusion, elevated levels of urinary cMet at the time of initial diagnosis could predict renal outcomes in patients with DN.

MiR‑203 inhibits the expression of collagen‑related genes and the proliferation of hepatic stellate cells through a SMAD3‑dependent mechanism.

Activation of hepatic stellate cells (HSCs) is a pivotal event during hepatic fibrogenesis. Activated HSCs are the main source of collagen and other extracellular matrix (ECM) components, and emerging antifibrotic therapies are aimed at preventing ECM synthesis and deposition. MicroRNAs (miRNAs) have been demonstrated to exert regulatory effects on HSC activation and ECM synthesis. In the present study, the HSC-T6 rat hepatic stellate cell line was transiently transfected with a miRNA (miR)-203 mimic, which is an artificial miRNA that enhances the function of miR-203, with a miR-203 inhibitor or with a scramble miRNA negative control. mRNA and protein expression levels of collagen (COL) 1A1, COL3A1, α-smooth muscle actin (α-SMA) and mothers against decapentaplegic homolog 3 (SMAD3) were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The interaction between miR-203 and the 3′-untranslated region (UTR) of SMAD3 mRNA was examined using a dual-luciferase reporter assay. The proliferative capabilities of activated HSCs were measured using an MTT assay. The present results demonstrated that the mRNA and protein expression levels of COL1A1, COL3A1, α-SMA and SMAD3 were significantly upregulated following transfection of HSC-T6 cells with the miR-203 inhibitor. Conversely, COL1A1, COL3A1, α-SMA, and SMAD3 mRNA and protein expression appeared to be downregulated in rat HSCs transfected with miR-203 mimics. Notably, the inhibition of miR-203 expression was revealed to promote HSC proliferation, whereas increased miR-203 expression suppressed the proliferative capabilities of HSC-T6 cells. Furthermore, SMAD3 was revealed to be a direct target of miR-203. The present study suggested that miR-203 may function to prevent the synthesis and deposition of ECM components, including COL1A1, COL3A1 and α-SMA, and to inhibit the proliferation of HSCs through a SMAD3-dependent mechanism. Therefore, it may be hypothesized that miR-203 has potential as a novel target for the development of alternative therapeutic strategies for the treatment of patients with hepatic fibrosis in clinical practice.

Shensong Yangxin (SSYX) ameliorates disordered excitation transmission by suppressing cardiac collagen hyperplasia in rabbits with chronic myocardial infarction.

The traditional Chinese medicine Shensong Yangxin (SSYX) can improve the clinical symptoms of arrhythmia in an integrated manner. This study aimed to investigate the electrophysiological effect of SSYX on the hearts of myocardial-infarcted rabbits and further explore the mechanism by which SSYX alleviates myocardial fibrosis. Myocardial infarction (MI) was established in rabbits by ligation of the left circumflex coronary. The rabbits were treated with SSYX (0.5 g/kg/d) or saline for 8 weeks by oral administration. Microelectrode array (MEA) technology was used in vivo for extracellular electrophysiological recordings of the infarct border zone. Masson's trichrome staining was used to observe myocardial fibrosis. Western blotting was performed to evaluate the protein expression levels of collagen I (COL I) and collagen III (COL III). Quantitative real-time polymerase chain reaction (real-time PCR) was performed to evaluate the TGF-β1 and MMP-2 mRNA expression levels. The results showed that the total activation time (TAT) and the dispersion of TAT were significantly increased and the excitation propagation markedly disordered after MI. SSYX could significantly decrease TAT and the dispersion of TAT, and significantly ameliorate the chaotic spread pattern of excitation. Furthermore, SSYX treatment could significantly decrease COL I and COL III protein levels and down-regulate TGF-β1 and MMP-2 mRNA expression levels in MI rabbits. It was concluded that SSYX may ameliorate cardiac electrophysiological abnormalities in infarcted hearts by decreasing the protein levels of COL I and COL III, down-regulating the mRNA expression levels of TGF-β1 and MMP2, and thereby reducing adverse cardiac remodeling.

A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model

IntroductionIn recent years, nucleus pulposus cell (NPC) transplantation has been used to treat intervertebral disc degeneration (IDD); however, the degenerative nature of NPCs influences its effectiveness. Nucleus pulposus-derived stem cells (NPSCs), which are self-renewing, have high expansion potential and can adapt to the intervertebral disc (IVD) microenvironment and may have a better regenerative capacity, which is favourable for treating IDD. The aim of this study was to compare the effectiveness of transplantation with NPSCs and NPCs on the regeneration of the IVD in rabbit models. MethodsNPSCs and NPCs were isolated from human degenerate nucleus pulposus tissue by differential adhesion method, and stem cell surface markers were detected by flow cytometry. Degenerative discs in rabbits were randomly distributed into three groups: NPSCs, NPCs and vehicle control group; the normal discs served as the normal control group. Cells of the P3 generation were prepared for transplantation. About 20 μl of cell suspension (NPSCs or NPCs) or DMEM was injected into corresponding discs, while the normal discs were left untreated. After 8 weeks, disc height was evaluated using X-ray, water content was evaluated by MRI, and gene and protein expression levels of collagen II and aggrecan in the nucleus were determined by real-time PCR and ELISA. ResultsNPCs and NPSCs from the P3 generation were polygonal and spindle-shaped, respectively. Both NPSCs and NPCs strongly expressed surface markers CD73, CD90, and CD105 and weakly expressed CD34 and CD45. The relative rates of expression of CD73, CD90, and CD105 were higher in NPSCs than in NPCs. After 8 weeks, X-ray results showed no significant difference in disc height index among the groups (p > 0.05). MRI revealed that the intensity of the nucleus pulposus signal was increased in NPSCs (p < 0.05). The results from PCR and ELISA demonstrated that NPSCs promoted gene and protein expression of aggrecan instead of collagen II (p < 0.05). ConclusionCompared to NPCs, NPSCs harvested by differential adhesion method displayed a higher positive rate of stem cell surface markers and showed superior regenerative effectiveness for treating IDD in rabbit models. Therefore, NPSCs are potential candidates for cell therapy for the regeneration of the IVD.

Inhibitory effects of fasudil on renal interstitial fibrosis induced by unilateral ureteral obstruction.

Renal fibrosis is the major cause of chronic kidney disease, and the Rho/Rho-associated coiled-coil kinase (ROCK) signaling cascade is involved in the renal fibrotic processes. Several studies have reported that ROCK inhibitors attenuate renal fibrosis. However, the mechanism of this process remains to be fully elucidated. The present study assessed the inhibitory effect of fasudil, a ROCK inhibitor using immunohistochemistry, reverse transcription-quantitative polymerase chain reaction and western blot analyses, in vivo and in vitro, to elucidate the mechanisms underlying renal interstitial fibrosis. In mice induced with unilateral ureteral obstruction (UUO), collagen accumulation, the expression of fibrosis-associated genes and the content of hydroxyproline in the kidney increased 3, 7, and 14 days following UUO. Fasudil attenuated the histological changes, and the production of collagen and extracellular matrix in the UUO kidney. The expression of α-smooth muscle actin (α-SMA) and the transforming growth factor-β (TGFβ)-Smad signaling pathway, and macrophage infiltration were suppressed by fasudil in the kidneys of the UUO mice. The present study also evaluated the role of intrinsic renal cells and infiltrated macrophages using NRK-52E, NRK-49F and RAW264.7 cells. The mRNA and protein expression levels of collagen I and α-SMA increased in the NRK-52E and NRK-49F cells stimulated by TGF-β1. Hydroxyfasudil, a bioactive metabolite of fasudil, attenuated the increase in the mRNA and protein expression levles of α-SMA in the two cell types. However, the reduction in the mRNA expression of collagen I was observed in the NRK-49F cells only. Hydroxyfasudil decreased the mRNA expression of monocyte chemoattractant protein-1 (MCP-1) induced by TGF-β1 in the NRK-52E cells, but not in the NRK-49F cells. In the RAW264.7 cells, the mRNA expression levels of MCP-1, interleukin (IL)-1β, IL-6 and tumor necrosis factor α were increased significantly following lipopolysaccharide stimulation, and were not suppressed by hydroxyfasudil. These data suggested that the inhibition of ROCK activity by fasudil suppressed the transformation of renal intrinsic cells into the myofibroblast cells, and attenuated the infiltration of macrophages, without inhibiting the expression or the activation of cytokine/chemokines, in the progression of renal interstitial fibrosis.