scholarly journals MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Liu Hua ◽  
Lei Chen ◽  
Limin Wei ◽  
Hongli Jiang
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Mediators ◽  
Mesangial Cells ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Pyrrolidine Dithiocarbamate ◽  
Tgf Β1

Abstract Background and Aims Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4). Conclusion DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.

scholarly journals Overexpression of IκBα in cardiomyocytes alleviates hydrogen peroxide-induced apoptosis and autophagy through inhibiting NF-κB activation

2020 ◽  
Author(s):  
Min Han ◽  
Xiao-Cui Chen ◽  
Ming-Hui Sun ◽  
Min-Tao Gai ◽  
Yi-Ning Yang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Western Blot ◽  
Cell Viability ◽  
Signaling Pathway ◽  
Nuclear Translocation ◽  
Neonatal Rat ◽  
Cell Counting ◽  
Pyrrolidine Dithiocarbamate ◽  
Induced Apoptosis

Abstract Background: Inflammation and oxidative stress play a predominant role in the initiation and progression of ischemia/reperfusion (I/R) injury, of which nuclear factor kappa B (NF-κB) is a crucial mediator. Overexpression of the inhibitor of κB alpha (IκBα) gene is hypothesized to have protective effects against apoptosis and autophagy in cardiomyocytes subjected to hydrogen peroxide (H2O2) through inhibiting the NF-κB pathway.Methods: The IκBαS32A, S36A gene was transfected via adeno-associated virus serotype 9 (AAV9) delivery into neonatal rat ventricular cardiomyocytes (NRVMs) prior to H2O2 treatment. NRVMs were divided into control, H2O2, GFP +H2O2, IκBα+H2O2, and pyrrolidine dithiocarbamate (PDTC)+H2O2 groups. Nuclear translocation of NF-κB p65 subunit was evaluated by immunofluorescence and Western blot. Cell viability was assessed by Cell Counting Kit-8 assay. Supernatant lactate dehydrogenase (LDH) and intracellular malondialdehyde (MDA) were measured to identify H2O2-stimulated cytotoxicity. Apoptosis was determined by Annexin V-PE/7-AAD staining, and the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Western blot was used to detect apoptosis- and autophagy-related proteins.Results: IκBα transfection significantly increased cell viability and ΔΨm, but decreased the supernatant LDH and cellular MDA levels in cardiomyocytes exposed to H2O2. Meanwhile, IκBα overexpression decreased H2O2-induced apoptosis by upregulating the Bcl-2/Bax ratio and reduced autophagy by downregulating the expression of Beclin-1 and the LC3-Ⅱ/LC3-Ⅰ ratio. These effects partly accounted for the ability of IκBα to inhibit the NF-κB signaling pathway, as evidenced by decreases in p65 phosphorylation and nuclear translocation. Indeed, the effects of inactivation of NF-κB signaling with the specific inhibitor, PDTC, resembled the cardioprotective effects of IκBα during H2O2 stimulation.Conclusion: IκBα overexpression can ameliorate H2O2-induced apoptosis, autophagy, oxidative injury, and ΔΨm loss through inhibition of the NF-κB signaling pathway. These findings suggest that IκBα transfection can successfully resist oxidative stress-induced damage through inhibiting NF-κB activation, which may provide a potential therapeutic target for prevention of myocardial I/R injury.

Study on Receptor-Regulated Smad Protein of Acute Promyelocytic Leukemia Cell Differentiation Process in TGF-β1 Signaling Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4078-4078
Author(s):  
Jiayao Lin ◽  
Fangyuan Chen ◽  
Hua Zhong ◽  
Hairong Wang ◽  
Renrong Ouyang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Transcriptional Activity ◽  
Control Groups ◽  
Nb4 Cells ◽  
The Third ◽  
Realtime Pcr ◽  
Smad3 Protein ◽  
Tgf Β1

Abstract It was reported that several members of TGF-β1 signaling pathway had the situation of reduced expression, absence or abnormal function in mononuclear cell of acute leukemia patients. Our preliminary experiments also found that the expression of TGF-β1 receptors and several downstream Smad proteins decreased considerably than those after induced differentiation in a APL cell line (NB4) and primarily cells of APL patients. In order to further investigate the important links in TGF-β1 pathway in induced differentiation of APL cells by ATRA, R-Smad, namely the change of Smad2 and Smad3 protein expression, we detected mRNA and the protein expression of Smad2,Smad3 by Realtime-PCR and Western blot after we brought ATRA to act on cell NB4 and primary cells of patient for 0, 3, 6, 12, 24, 48, 72 and 96 hours. Cells treated with dehydrated alcohol were harvested at the given time as control group. As to NB4, The expression of Smad2 and Smad3 mRNA tested showed a significant increase treated with ATRA measured by PCR. In contrast, the expression levels in the control groups remained almost unchanged with time(p<0.01). After ATRA induced, Smad2 and Smad3 levels emerged a gradual rising trend, reached maximum value up to 48h (126.11±1.06,7780.09±288.58)and then gradually declined. Western blots showed that the degree of Smad2 protein was increased at 3h, which peaked at 48h, before returning to baseline level at 72h. Simultaneously, elevation of Smad3 was observed. The expression level was increased within 6h, up to the peak at 72h which was later than Smad2, and then dropped down. In the aspect of the primary cells, Western results showed that three De novo patients had extremely low Smad2 and Smad3 protein expression, but after activated by ATRA for 48h, their levels were increased significantly. These experiment results showed that a R-Smad reduction or absence might exist in APL, and that APL cells differentiation might something to do with up-regulating of expression of R-Smad. Its concrete mechanism might be associated with PML-RARα fusion protein. We analyzed the influence of PML-RARα on the transcriptional activity of the (CACA) 9-lux reporter gene plasmid(including nine tandem repeated CAGA sequence that may combine Smad3 and Smad4) and found that PML-RARα may inhibit the transcription regulation of R-Smad. In IF experiment, we observed that there was a phenomenon that PML-RARa protein in NB4 cells combined with Smad2, Smad3 protein respectively. The phenomenon presented an obvious falling tendency after activated by ATRA for 24, 48 and 72 hours. HK Lin et al. found that cytoplasmic PML (cPML) in normal cells could mediate the formation of complex TGF-β1 receptor/R-Smad/SARA, and thus activate TGF-β1 signaling pathway. We guess that PML-RARa may combine with the R-Smad, therefore, block the transcription control for genes of TGF-β1 pathway. Figure 1. The expression of R-Smad mRNA and protein in ATRA treated NB4 cells. The cells were exposed to ATRA (experimental groups) or not (control groups). At given time (0h, 3h, 6h, 12h, 24h, 48h, 72h, 96h) cells were harvest and subjected to Realtime-PCR and western blot Figure 1. The expression of R-Smad mRNA and protein in ATRA treated NB4 cells. The cells were exposed to ATRA (experimental groups) or not (control groups). At given time (0h, 3h, 6h, 12h, 24h, 48h, 72h, 96h) cells were harvest and subjected to Realtime-PCR and western blot Figure 2. The influence of PML-RARα fusion protien on the transcriptional activity of the (CACA) 9-lux reporter gene plasmid in NB4 cells. In the first group, with plasmid (CACA) 9-lux (nine Smad3 repeat sequences included). In the second group, with (CACA) 9-lux and empty vector, and in the third group, with (CACA) 9-lux and plasmid PML-RARα. The transcriptional activity of the third group was lower than the other two groups. (p<0.05) Figure 2. The influence of PML-RARα fusion protien on the transcriptional activity of the (CACA) 9-lux reporter gene plasmid in NB4 cells. In the first group, with plasmid (CACA) 9-lux (nine Smad3 repeat sequences included). In the second group, with (CACA) 9-lux and empty vector, and in the third group, with (CACA) 9-lux and plasmid PML-RARα. The transcriptional activity of the third group was lower than the other two groups. (p<0.05)

scholarly journals Osteopontin Promotes Cell Migration and Invasion, and Inhibits Apoptosis and Autophagy in Colorectal Cancer by activating the p38 MAPK Signaling Pathway

2017 ◽  
Vol 41 (5) ◽  
pp. 1851-1864 ◽  
Author(s):  
Ren-hong Huang ◽  
Ying-jun Quan ◽  
Jin-hong Chen ◽  
Ting-feng Wang ◽  
Ming Xu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Western Blot ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Hct116 Cell ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Cell Counting ◽  
Migration And Invasion

Background: Osteopontin (OPN) is highly expressed in colorectal cancer (CRC) and is associated with disease progression in vivo. High levels of OPN have been demonstrated to predict low survival rates in CRC. Autophagy is a process of self-digestion, which is thought to play a significant role in carcinogenesis. However, the mechanisms of OPN's effects on CRC cell autophagy have not been elucidated. Therefore, we aimed to investigate possible mechanisms of OPN's effects on CRC autophagy. Methods: HCT116 cell proliferation, apoptosis, and migration and invasion ability were identified by cell counting k¡t-8 assay, flow cytometry, wound healing assay, and transwell chamber invasion assay, respectively. The ratios of proteins LC3-II/LC3-I, P62, and Atg7 were analyzed by Western-blot. Expressions of Beclin-1, Atg4b, Bnip3, and Vps34, both in transcriptional and translational levels, were analyzed and compared by RT-PCR and Western blot. Immunofluorescence and co-focusing experiments were used to investigate the formation of autophagosomes. Results: The results showed that OPN can promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis. It was also demonstrated that OPN could inhibit cell autophagy. Further experiments revealed that the inhibitory effect of OPN on autophagy could be reversed by blocking the p38 MAPK pathway in HCT116 cells. Conclusion: OPN is involved in HCT116 cell progression and is capable of inhibiting cell autophagy possibly by activating the p38 MAPK signaling pathway, implying that OPN could be a potential novel molecular therapeutic biomarker in patients with CRC.

scholarly journals Resistin is a survival factor for porcine ovarian follicular cells

Reproduction ◽  
2015 ◽  
Vol 150 (4) ◽  
pp. 343-355 ◽  
Author(s):  
Agnieszka Rak ◽  
Eliza Drwal ◽  
Anna Wróbel ◽  
Ewa Łucja Gregoraszczuk
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Dna Fragmentation ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Inhibitory Effect ◽  
Ovarian Cell ◽  
Proliferation And Apoptosis ◽  
Mrna And Protein Expression

Previously, we demonstrated the expression of resistin in the porcine ovary, the regulation of its expression and its direct effect on ovarian steroidogenesis. The objective of this study was to examine the effect of resistin on cell proliferation and apoptosis in a co-culture model of porcine granulosa and theca cells. First, we analysed the effect of resistin at 1 and 10 ng/ml alone or in combination with FSH- and IGF1 on ovarian cell proliferation with an alamarBlue assay and protein expression of cyclins A and B using western blot. Next, the mRNA and protein expression of selected pro-apoptotic and pro-survival regulators of cell apoptosis, caspase-9, -8 and -3 activity and DNA fragmentation using real time PCR, western blot, fluorescent assay and an ELISA kit, respectively, were analysed after resistin treatment. Furthermore, we determined the effect of resistin on the protein expression of ERK1/2, Stat and Akt kinase. Using specific inhibitors of these kinases, we also checked caspase-3 activity and protein expression. We found that resistin, at both doses, has no effect on cell proliferation. The results showed that resistin decreased pro-apoptotic genes, which was confirmed on protein expression of selected factors. We demonstrate an inhibitory effect of resistin on caspase activity and DNA fragmentation. Finally, resistin stimulated phosphorylation of the ERK1/2, Stat and Akt and kinases inhibitors reversed resistin action on caspase-3 activity and protein expression to control. All of these results showed that resistin has an inhibitory effect on porcine ovarian cell apoptosis by activation of the MAPK/ERK, JAK/Stat and Akt/PI3 kinase signalling pathways.

scholarly journals The effect of histone deacetylase inhibitor Trichostatin A on IL-17A-induced transformation of MRC5 into myofibroblasts and its mechanism

2021 ◽  
Author(s):  
qian Zhang ◽  
Zexin Guo ◽  
Jing Zhang ◽  
Wei Zhou ◽  
Yueqing Wu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Trichostatin A ◽  
Combined Treatment ◽  
Collagen I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Mrc5 Cell ◽  
Tgf Β1

Objective To elucidate potential IL-17A- and TSA-mediated regulation of fibroblasts transformation. Methods MTT assay, HDAC1 activity assay, cell immunofluorescence and Western blot were employed to detect the expression of related indicators. Results MRC5 cells expressed only a small amount of Vimentin. IL-17A treatment upregulated MRC5 cell proliferation, in a concentration-dependent manner. TSA treatment, however, suppressed MRC5 cell proliferation. IL-17A treatment also upregulated HDAC1 activity in MRC5 cells, in a concentration-dependent manner. Using immunofluorescence, we demonstrated that IL-17A-treated MRC5 cells had markedly elevated Vimentin, Collagen-I and a-SMA levels, compared to controls. However, a combined treatment of IL-17A and TSA resulted in markedly reduced levels of the Vimentin, Collagen-I and a-SMA, compared to IL-17A alone, yet the amount was higher than controls. Using western blot analysis, we also revealed that the IL-17A-treated MRC5 cells had markedly elevated levels of Vimentin, a-SMA, HDAC1, p-Smad2, and p-Smad3, and markedly reduced level of Smad7, compared to controls. In TSA intervention group, the expression effect of the above protein was opposite. Moreover, no discernible difference was observed in the levels of Smad2 and Smad3 among the treated and un-treated groups. Conclusion IL-17A stimulates proliferation of MRC5 cells and increases HDAC1 activity and protein expression. It also transforms MRC5 cells into myofibroblasts via activation of the TGF -β1/ Smads signaling network. TSA, on the other hand, strongly suppresses TGF -β1/ Smads pathway-mediated fibrosis by ceasing HDAC1 activity and protein expression.

scholarly journals Ergosterol Ameliorates Diabetic Nephropathy by Attenuating Mesangial Cell Proliferation and Extracellular Matrix Deposition via the TGF-β1/Smad2 Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 483 ◽  
Author(s):  
Zhonghua Dong ◽  
Yueyue Sun ◽  
Guangwei Wei ◽  
Siying Li ◽  
Zhongxi Zhao
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Diabetic Nephropathy ◽  
Signaling Pathway ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Diabetic Mice ◽  
Mesangial Cell Proliferation ◽  
Tgf Β1

(1) Background: Diabetic nephropathy, a microvascular complication of diabetes, is one of the principal causes of end-stage renal disease worldwide. The aim of this study was to explore the therapeutic effects of ergosterol on diabetic nephropathy. (2) Methods: Streptozotocin (STZ)-induced C57BL/6 diabetic mice were treated with ergosterol (10, 20, 40 mg/kg/day) for 8 weeks by oral gavage. The in vitro study employed rat mesangial cells exposed to 30 mM glucose for 48 h in the presence of 10 or 20 μM ergosterol. (3) Results: Ergosterol treatment improved body weights, ameliorated the majority of biochemical and renal functional parameters and histopathological changes, and reduced extracellular matrix (ECM) deposition in diabetic mice. In vitro, ergosterol suppressed proliferation, reduced the levels of ECM proteins, and increased the expression of matrix metalloproteinase-2 and -9 in high glucose-induced mesangial cells; Furthermore, ergosterol markedly improved transforming growth factor-β1 (TGF-β1) expression, enhanced phosphorylation levels of drosophila mothers against decapentaplegic 2 (Smad2), and regulated the downstream factors in vivo and in vitro. (4) Conclusions: Ergosterol alleviated mesangial cell proliferation and the subsequent ECM deposition by regulating the TGF-β1/Smad2 signaling pathway.

scholarly journals Prodigiosin inhibits cholangiocarcinoma cell proliferation and induces apoptosis via suppressing SNAREs-dependent autophagy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dijie Zheng ◽  
Shiyu Chen ◽  
Kun Cai ◽  
Linhan Lei ◽  
Chunchen Wu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cancer Biology ◽  
Bacterial Species ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Dependent Manner ◽  
Western Blot Assay

Abstract Background Prodigiosin (PG), a natural red pigment produced by numerous bacterial species, has been a eye-catching research point in recent years for its anticancer activity. However, the role of PG in the cancer biology of cholangiocarcinoma (CCA) remains vague. Methods The proliferation of CCA cells was detected by Cell Counting Kit-8(CCK-8), Colony formation assay and 5-ethynyl-2′-deoxyuridine (EdU) assay. Cell apoptosis was evaluated by flow cytometry assay and western blot assay. The effects of PG or SNAREs on cell autophagy were measured by autophagy flux assay and western blot assay. Xenograft mouse models were used to assess the role of PG in CCA cells in vivo. Results PG could inhibit the proliferation and viability of CCA cells in a concentration- and time-dependent manner via suppressing the late stage of autophagy. Mechanistically, PG inhibits the fusion of autophagosomes and lysosomes by blocking STX17 and SNAP29, components of soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs)complex. When STX17 and SNAP29 were overexpressed, the inhibitory effect of PG on CCA cells autophagy was relieved. In addition, PG showed obvious inhibitory effects on cancer cell viability but no toxic effects on organs in xenotransplantation models. Conclusion Taken together, our results demonstrated that PG inhibits CCA cell proliferation via suppressing SNAREs-dependent autophagy, implying that PG could be a potential chemotherapy drug for advanced CCA.

Effect of PI3K/AKT/mTOR Signaling Pathway on Regulating and Controlling the Anti-Invasion and Metastasis of Hepatoma Cells by Bufalin

2021 ◽  
Vol 16 ◽  
Author(s):  
Xia Sheng ◽  
Pengfei Zhu ◽  
Yi Zhao ◽  
Jinwei Zhang ◽  
Haijia Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Inhibitory Effect ◽  
Mtor Signaling ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Mtor Signaling Pathway ◽  
Invasion And Metastasis ◽  
Blotting Technique ◽  
Adhesion And Invasion

Background: Autophagy plays a "double-edged sword" in the process of tumorigenesis, development and metastasis. Objective: In this study, we explored the effect of PI3K/AKT/mTOR autophagy related signaling pathway on regulating and controlling the invasion and metastasis of liver cancer cells by Bufalin. Methods: The cell counting , migration , adhesion and invasion assay were used to evaluate the effect of Bufalin on the cell proliferation, invasion and metastasis. The protein expression of PI3K/AKT/mTOR signaling pathway were detected by Western Blotting technique. Results: After inhibiting autophagy of HCC-LM3 cells, the inhibitory effect of Bufalin on adhesion, migration and invasion of HCC-LM3 cells was significantly enhanced. The synergistic inhibition was strongest when different autophagy inhibitors were combined with 3MA and CQ. After inhibiting autophagy, Bufalin significantly inhibited the protein expression of P-AKT, Cyclin D1, MMP-2, MMP-9 and VEGF in HCC-LM3 cells. The protein expression of PTEN and E-Cadherin in HCC-LM3 cells was significantly increased. Conclusion: The present study shows that the anti-tumor effect of Bufalin mainly inhibit the proliferation, extracellular matrix degradation and angiogenesis of HCC by influencing autophagy. These findings confirm the capability of Bufalin in inhibiting metastasis of HCC and in parallel to current patents could be applied as a novel therapeutic strategy in the prevention of metastasis of HCC.

scholarly journals Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qilu Wei ◽  
Ning Kong ◽  
Xiaohui Liu ◽  
Run Tian ◽  
Ming Jiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fast Green ◽  
Expression Levels ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Safranin O ◽  
Tgf Β1

Abstract Background Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. Methods Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-β1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson’s trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. Results The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-β1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-β1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-β1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson’s trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. Conclusions PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

scholarly journals Knockdown of long noncoding RNA HOTAIR inhibits osteoarthritis chondrocyte injury by miR-107/CXCL12 axis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jipeng Lu ◽  
Zhongxiong Wu ◽  
Ying Xiong
Keyword(s):  
Cell Proliferation ◽  
Western Blot ◽  
Cell Apoptosis ◽  
Noncoding Rna ◽  
Underlying Mechanism ◽  
Joint Disease ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Cxcl12 Expression ◽  
Ecm Degradation

Abstract Background Osteoarthritis (OA) is a joint disease characterized via destruction of cartilage. Chondrocyte damage is associated with cartilage destruction during OA. Long noncoding RNAs (lncRNAs) are implicated in the regulation of chondrocyte damage in OA progression. This study aims to investigate the role and underlying mechanism of lncRNA homeobox antisense intergenic RNA (HOTAIR) in OA chondrocyte injury. Methods Twenty-three OA patients and healthy controls without OA were recruited. Chondrocytes were isolated from OA cartilage tissues. HOTAIR, microRNA-107 (miR-107) and C-X-C motif chemokine ligand 12 (CXCL12) levels were measured by quantitative real-time polymerase chain reaction and western blot. Cell proliferation, apoptosis and extracellular matrix (ECM) degradation were measured using cell counting kit-8, flow cytometry and western blot. The target interaction was explored by bioinformatics, luciferase reporter and RNA immunoprecipitation assays. Results HOTAIR expression was enhanced, and miR-107 level was reduced in OA cartilage samples. HOTAIR overexpression inhibited cell proliferation, but induced cell apoptosis and ECM degradation in chondrocytes. HOTAIR knockdown caused an opposite effect. MiR-107 was sponged and inhibited via HOTAIR, and knockdown of miR-107 mitigated the effect of HOTAIR silence on chondrocyte injury. CXCL12 was targeted by miR-107. CXCL12 overexpression attenuated the roles of miR-107 overexpression or HOTAIR knockdown in the proliferation, apoptosis and ECM degradation. CXCL12 expression was decreased by HOTAIR silence, and restored by knockdown of miR-107. Conclusion HOTAIR knockdown promoted chondrocyte proliferation, but inhibited cell apoptosis and ECM degradation in OA chondrocytes by regulating the miR-107/CXCL12 axis.

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