GRK2 promotes activation of lung fibroblast cells and contributes to pathogenesis of pulmonary fibrosis through increasing Smad3 expression

Pulmonary fibrosis is a chronic, progressive, and irreversible interstitial lung disease. Transforming growth factor beta1 (TGF-β1) plays a major role in lung fibroblast cell differentiation to myofibroblast cells and production of extracellular matrix, which are hallmarks of pulmonary fibrosis. G protein-coupled receptor kinase-2 (GRK2) has been shown to play controversial roles in TGF-β1-induced signal transduction in different cell types; however, the roles of GRK2 in TGF-β1-induced activation of lung fibroblast cells and development of pulmonary fibrosis have not been revealed. In this study, we found that GRK2 levels were induced in lungs and isolated fibroblast cells in a murine model of pulmonary fibrosis, as well as TGF-β1-treated lung fibroblasts. GRK2 levels were not changed in lungs in the injury phase of pulmonary fibrosis. Post-treatment with GRK2 inhibitor reduced ECM accumulation in lungs in bleomycin-challenged mice, suggesting that GRK2 activation contributes to the progressive phase of pulmonary fibrosis. Inhibition or downregulation of GRK2 attenuates fibronectin, collagen, and α-smooth muscle actin expression in TGF-β1-induced lung fibroblast cells or myofibroblast cells isolated from pulmonary fibrosis patients. Further, we showed that GRK2 regulates Smad3 expression, indicating that inhibition of GRK2 attenuates ECM accumulation through downregulation of Smad3 expression. This study reveals that GRK2 is a therapeutic target in treating pulmonary fibrosis and inhibition of GRK2 dampens pulmonary fibrosis by suppression of Smad3 expression, eventually attenuating TGF-β1 signal pathway and ECM accumulation.

Exploring the Effect of Dapagliflozin on Alcoholic Kidney Injury and Renal Interstitial Fibrosis in Rats Based on TIMP-1/MMP-24 Pathway

Objective. To establish a rat model of alcoholic kidney injury and detect the expression of TIMP-1/MMP-24 in the kidneys of rats with alcoholic kidney injury at the molecular pathological level, so as to explore the mechanism of alcohol abuse leading to kidney injury and renal interstitial fibrosis as well as the alleviation of alcohol-induced kidney injury and inhibition of renal interstitial fibrosis by dapagliflozin. Methods. 48 male rats were randomly divided into 4 groups: control group, alcohol group, alcohol + dapagliflozin group, and alcohol + losartan group, each with 12 rats. Different drugs were administered by gavage for modeling and treatment. Six days later, the rats were sacrificed, blood was collected from the heart to separate the serum, and the blood creatinine (Scr) and urea nitrogen (BUN) contents were detected biochemically. After blood collection, the kidney tissue was taken and fixed in10% neutral formalin. The expression of renal tissue inflammatory factors (CRP, IL-6, and TNF-α) and renal fibrosis indexes (LN, HA, and TGF-β1) were detected; MMP-24 and TIMP-1 in the kidney tissue of rats in different treatment groups were detected, and Smad3 expression was also detected. Results. After treatment, the general condition of the alcohol + dapagliflozin group and the alcohol + losartan group improved to different degrees. The weight first decreased and then gradually increased over time. There was no statistical difference in the weight change between the two groups; Compared with the control group, the Scr level, BUN content, renal index, inflammatory factors, and renal fibrosis indexes in the alcohol group were significantly increased ( P < 0.05 ); after 6 weeks of treatment, in the alcohol + dapagliflozin group and alcohol + losartan group, Scr level, BUN content, kidney index, inflammatory factors, and renal fibrosis indexes were significantly decreased ( P < 0.05 ); the expression of MMP-24 in the kidney tissue of the control group was upregulated, and the expression of TIMP-1 and Smad3 was downregulated; MMP-24 expression was downregulated, and TIMP-1 and Smad3 expression was significantly upregulated ( P < 0.05 ) in the rats of the alcohol group. After dapagliflozin and losartan treatment, MMP-24 expression gradually increased and TIMP-1 and Smad3 expression gradually decreased ( P < 0.05 ). Conclusion. Long-term large-scale alcohol intake can cause kidney tissue damage and fibrotic lesions. The expression of fibrotic cytokines such as TIMP-1 and Smad3 will increase, and the expression of MMP-24 will be decreased. However, dapagliflozin and losartan have certain therapeutic effects on the abovementioned lesions. The mechanism may be downregulating TIMP-1 and Smad3 and upregulating the expression of MMP-24 and other cytokines in the kidney.

The Effect of Autophagy Induced by Mothers Against Decapentaplegic Homolog 3 (Smad3) Regulated by MicroRNA (miRNA)-23b on Renal Injury in Septic Rats

This study intends to investigate the mechanism by how microRNA (miRNA)-23b alleviates kidney damage in septic rats. Herein, septic rat model, control group and sham-operated model were set up to assess kidney tissue damage. Tissues were extracted from the rats and isolated into cells. Then cells were transfected with plasmids expressing miR-23b followed by analysis of the expression of miR-23b, Smad3, TLR4, HMGB1 and autophagy-related proteins (LC3, beclin-1) by western blot and RT-qPCR. The level of TNF-α, IL-6 and BUN and SCr were elevated in the model group and decreased after upregulation of miR-23b with increased LC3-II, Smad3 and Beclin-1 expression. miR-23b mimic group showed highest miR-23b expression followed by miR-23b NC group and miR-23b inhibitor group. The levels of TLR4, and HMGB1 and positive rate of NF-κBp65 in miR-23b mimic group were significantly lower than those in miR-23b inhibitor group (p < 0.05). Importantly, miR-23b directly targeted Smad3 and inhibited its expression. In conclusion, overexpressed miR-23b induces autophagy by promoting Smad3 expression, alleviates kidney damage in septic rats, and reduces inflammation and inactivates NF-κB signaling pathway.

Human cytomegalovirus blocks canonical TGFβ signaling during lytic infection to limit induction of type I interferons

Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFβ interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.

Chloride intracellular channel 4 (CLIC4) expression is transcriptionally regulated by crosstalk of the TGF-beta, Wnt and Hedgehog signalling pathways

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts. CLIC4 expression and activity are regulated by TGF-beta signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show Wnt3a/beta-catenin and Smoothened/GLI signalling cooperate with SMAD3 to regulate CLIC4 expression in normal dermal fibroblasts, and that inhibition of SMAD3 expression or activity abolishes Wnt and Hh-dependent CLIC4 induction. We further show that expression of the profibrotic marker alpha-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Our data highlight novel mechanisms that regulate CLIC4 expression that present targetable pathways to prevent fibroblast activation in SSc and other fibrotic conditions.

Dahuang Zhechong Pills Suppress Silicosis Fibrosis Progression via p38 MAPK/TGF-β1/Smad Pathway In Vitro

Background. Dahuang Zhechong pills (DHZCP) is a classic Chinese medicinal prescription in “Treatise on Cold Pathogenic and Miscellaneous Diseases (Shanghan Zabing Lun),” and it has the function of tonifying blood, nourishing Yin, and removing blood stasis. Previous studies have shown that DHZCP could alleviate SiO2 induced pulmonary fibrosis in mice. This study aims to further explore the preventive and therapeutic effects of DHZCP against silicosis fibrosis and the underlying mechanisms in vitro. Methods. We used the experimental model of SiO2-induced MH-S cells to evaluate the therapeutic potential of DHZCP. MH-S cells induced by SiO2 were intervened with the drug-containing serum of DHZCP, and the effects of drug-containing serum of DHZCP on the MH-S cells were detected by CCK8, ELISA, flow cytometry, western blot, and immunofluorescence. Results. DHZCP improved cell viability by reducing apoptosis. It also decreased the levels of TNF-α, IL-1β, IL-6 in the supernatant of MH-S cells induced by SiO2, inhibited the expression of p38 MAPK, blocked the activation of NF-κB, and controlled the upstream inflammatory response by multiple targeting. Concomitantly, we observed upregulation of Smad7 and a marked decline in TGF-β1, α-SMA, Smad2, Smad3 expression in MH-S cells treated with DHZCP. Conclusion. To sum up, we conclude that DHZCP protects against SiO2-induced silicosis by reducing the persistent irritation of inflammation, regulating the p38 MAPK/TGF-β1/Smad pathway.

Protective Effect of MicroRNA-23b on Kidney Injury in Septic Rats Through Regulation of Mothers Against Decapentaplegic Homolog 3 (Smad3)

Our study aims to investigate the mechanism whereby microRNA (miRNA)-23b alleviates kidney damage in septic rats. Herein, we set up septic rat model, control group and sham-operated model to evaluate the kidney tissue damage. The glomerular mesangial cells isolated from rats were transfected with plasmids expressing miR-23b followed by analysis of the expression of miR-23b, Smad3, TLR4, HMGB1 and expression of autophagy-related proteins (LC3, beclin-1) by western blot and RT-qPCR. The level of TNF-α, IL-6 and BUN and SCr were significantly elevated in the model group and decreased after overexpression of miR-23b with elevated LC3-II, Smad3 and Beclin-1 expression. miR-23b mimic group presented highest expression of miR-23b, followed by miR-23b NC group, and miR-23b inhibitor group. The levels of TLR4, and HMGB1 and positive rate of NF-κBp65 in miR-23b mimic group were significantly lower than those in miR-23b inhibitor group (p < 0.05). Importantly, miR-23b has a targeted relationship with Smad3. Overexpression of miR- 23b induces autophagy by promoting the Smad3 expression, alleviates kidney damage in septic rats, and reduces inflammation and inactivates NF-κB signaling pathway.

The Dual Effect and Mechanism of (-)-epicatechin on Hypoxic-induced Proliferation and Apoptosis of Cardiac Fibroblasts

Objective: Cardiovascular diseases impose a considerable economic burden on health services and remain a threat to human being. (-)-Epicatechin [(-)-EPI], a traditional Chinese medicine is applied to treatment against the cardiovascular diseases. Herein, we aimed at investigating the underlying mechanism whereby (-)-EPI affects myocardial fibrosis (MF).Methods: The efficacy of (-)-EPI was determined in mouse models of acute myocardial infarction (AMI) and hypoxia-treated fibroblasts. Western blot and RT-qPCR analyses were conducted to determine TGF-β1, SMAD2 and SMAD3 expression, as collagen content was detected. CCK-8 assay and flow cytometry were carried out to detect fibroblast proliferation and apoptosis. HE and Masson staining reflected the histological change of myocardial tissuesResults: Compared to sham-operated mice, AMI group exhibited MF and hypoxia-treated cardiac fibroblasts proliferation was restrained and apoptosis was increased. Treatment with (-)-EPI significantly attenuated the MF condition and restored fibroblast proliferation and apoptosis, whereas these effects were abrogated by the TGF-β1 agonist HY-100347A. (-)-EPI administration caused a decline in TGF-β1, SMAD2 and SMAD3 expression. Mechanistically, (-)-EPI targeting TGF-β signaling inhibits collagen deposition and attenuates MF.Conclusion: Collectively, (-)-EPI could improve MF following AMI through down-regulation of TGF-β1 signaling, providing a novel insight into treatment against the cardiovascular diseases.