OBJECTIVE: To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. METHODS: Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor β ₁ (TGF-β ₁), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. RESULTS: The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L ( P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β ₁, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression. CONCLUSION VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.
Rats ; Animals ; Chondrocytes/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Collagen Type II/metabolism* ; Interleukin-6 ; Lipopolysaccharides/pharmacology* ; NF-E2-Related Factor 2/pharmacology* ; Inflammation/drug therapy* ; Osteoarthritis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Dipeptides ; para-Aminobenzoates

Objective To explore the effects and mechanisms of a traditional Chinese medicine (TCM) prescription, "Fang-gan Decoction" (FGD), in protecting against SARS-CoV-2 spike protein-induced lung and intestinal injuries in vitro and in vivo.Methods Female BALB/c mice and three cell lines pretreated with FGD were stimulated with recombinant SARS-CoV-2 spike protein (spike protein). Hematoxylin-eosin (HE) staining and pathologic scoring of tissues, cell permeability and viability, and angiotensin-converting enzyme 2 (ACE2) expression in the lung and colon were detected. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of inflammatory factors in serum and cell supernatant. The expression of NF-κB p65, p-NF-κB p65, p-IκBα, p-Smad2/3, TGF-β1, Caspase3, and Bcl-2 was evaluated by Western blotting.Results FGD protected against the damage to the lung and colon caused by the spike protein in vivo and in vitro according to the pathologic score and cell permeability and viability (P<0.05). FGD up-regulated ACE2 expression, which was reduced by the spike protein in the lung and colon, significantly improved the deregulation of inflammatory markers caused by the spike protein, and regulated the activity of TGF-β/Smads and NF-κB signaling.Conclusion Traditional Chinese medicine has a protective effect on lung and intestinal tissue injury stimulated by the spike protein through possible regulatory functions of the NF-κB and TGF-β1/Smad pathways with tissue type specificity.
Mice ; Animals ; Female ; Humans ; NF-kappa B/metabolism* ; Spike Glycoprotein, Coronavirus/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; Angiotensin-Converting Enzyme 2/pharmacology* ; COVID-19 ; SARS-CoV-2/metabolism* ; Lung ; Antineoplastic Agents ; Transforming Growth Factor beta/pharmacology* ; Epithelial Cells/metabolism* ; Colon

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease. Anti-fibrosis treatment is a significant therapy for heart disease, but there is still no thorough understanding of fibrotic mechanisms. This study was carried out to ascertain the functions of cytokine receptor-like factor 1 (CRLF1) in cardiac fibrosis and clarify its regulatory mechanisms. We found that CRLF1 was expressed predominantly in cardiac fibroblasts. Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction, but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-‍β1 (TGF‍-‍β1). Gain- and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts (NMCFs) with or without TGF-‍β1 stimulation. CRLF1 overexpression increased cell viability, collagen production, cell proliferation capacity, and myofibroblast transformation of NMCFs with or without TGF‍-‍β1 stimulation, while silencing of CRLF1 had the opposite effects. An inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and different inhibitors of TGF-‍β1 signaling cascades, comprising mothers against decapentaplegic homolog (SMAD)‍-dependent and SMAD-independent pathways, were applied to investigate the mechanisms involved. CRLF1 exerted its functions by activating the ERK1/2 signaling pathway. Furthermore, the SMAD-dependent pathway, not the SMAD-independent pathway, was responsible for CRLF1 up-regulation in NMCFs treated with TGF-‍β1. In summary, activation of the TGF-‍β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression. CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway. CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.
Animals ; Humans ; Mice ; Disease Models, Animal ; Fibroblasts/metabolism* ; Fibrosis ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 3/metabolism* ; Myocardial Infarction/metabolism* ; Receptors, Cytokine/metabolism* ; Signal Transduction ; Transforming Growth Factor beta1/pharmacology*

Although sympathetic blockade is clinically used to treat pain, the underlying mechanisms remain unclear. We developed a localized microsympathectomy (mSYMPX), by cutting the grey rami entering the spinal nerves near the rodent lumbar dorsal root ganglia (DRG). In a chemotherapy-induced peripheral neuropathy model, mSYMPX attenuated pain behaviors via DRG macrophages and the anti-inflammatory actions of transforming growth factor-β (TGF-β) and its receptor TGF-βR1. Here, we examined the role of TGF-β in sympathetic-mediated radiculopathy produced by local inflammation of the DRG (LID). Mice showed mechanical hypersensitivity and transcriptional and protein upregulation of TGF-β1 and TGF-βR1 three days after LID. Microsympathectomy prevented mechanical hypersensitivity and further upregulated Tgfb1 and Tgfbr1. Intrathecal delivery of TGF-β1 rapidly relieved the LID-induced mechanical hypersensitivity, and TGF-βR1 antagonists rapidly unmasked the mechanical hypersensitivity after LID+mSYMPX. In situ hybridization showed that Tgfb1 was largely expressed in DRG macrophages, and Tgfbr1 in neurons. We suggest that TGF-β signaling is a general underlying mechanism of local sympathetic blockade.
Mice ; Animals ; Receptor, Transforming Growth Factor-beta Type I/metabolism* ; Transforming Growth Factor beta/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; Hyperalgesia/metabolism* ; Radiculopathy/metabolism* ; Pain/metabolism* ; Analgesics/pharmacology* ; Ganglia, Spinal/metabolism*

OBJECTIVE: To observe whether metformin (MET) inhibits transforming growth factor-β₁ (TGF-β₁)/Smad3 signaling pathway by activating adenosine activated protein kinase (AMPK), so as to alleviate the pulmonary fibrosis caused by paraquat (PQ) poisoning in mice. METHODS: Male C57BL/6J mice were randomly divided into the Control group, PQ poisoning model group (PQ group), MET intervention group (PQ+MET group), AMPK agonist group (PQ+AICAR group), and AMPK inhibitor group (PQ+MET+CC group), according to a random number table method. A mouse model of PQ poisoning was established by one-time peritoneal injection of 1 mL PQ solution (20 mg/kg). The Control group was injected with the same volume of normal saline. After 2 hours of modeling, the PQ+MET group was given 2 mL of 200 mg/kg MET solution by gavage, the PQ+AICAR group was given 2 mL of 200 mg/kg AICAR solution by intraperitoneal injection, the PQ+MET+CC group was given 2 mL of 200 mg/kg MET solution by gavage and then 1 mL complex C (CC) solution (20 mg/kg) was intraperitoneally injected, the Control group and PQ group were given 2 mL of normal saline by gavage. The intervention was given once a day for 21 consecutive days. The 21-day survival rate of ten mice in each group was calculated, and the lung tissues of remaining mice were collected at 21 days after modeling. The pathological changes of lung tissues were observed under light microscope after hematoxylin-eosin (HE) staining and Masson staining, and the degree of pulmonary fibrosis was evaluated by Ashcroft score. The content of hydroxyproline in lung tissue and oxidative stress indicators such as malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. The protein expressions of E-cadherin, α-smooth muscle actin (α-SMA), phosphorylated AMPK (p-AMPK), TGF-β₁ and phosphorylated Smad3 (p-Smad3) in lung tissue were detected by Western blotting. RESULTS: Compared with the Control group, the 21 days survival rate was significantly reduced, lung fibrosis and Ashcroft score were significantly increased in PQ group. In addition, the content of hydroxyproline, MDA and the protein expressions of α-SMA, TGF-β₁ and p-Smad3 in lung tissue were significantly increased, while the activity of SOD and the protein expressions of E-cadherin and p-AMPK were significantly decreased in PQ group. Compared with the PQ group, the 21 days survival rates of mice were significantly improved in the PQ+MET group and PQ+AICAR group (70%, 60% vs. 20%, both P < 0.05). The degree of pulmonary fibrosis and the Ashcroft score were significantly reduced (1.50±0.55, 2.00±0.63 vs. 6.67±0.52, both P < 0.05). The content of hydroxyproline and MDA in lung tissue, as well as α-SMA, TGF-β₁ and p-Smad3 protein expressions were significantly reduced [hydroxyproline (mg/L): 2.03±0.11, 3.00±0.85 vs. 4.92±0.65, MDA (kU/g): 2.06±1.48, 2.10±1.80 vs. 4.06±1.33, α-SMA/GAPDH: 0.23±0.06, 0.16±0.06 vs. 1.00±0.09, TGF-β₁/GAPDH: 0.28±0.03, 0.53±0.05 vs. 0.92±0.06 p-Smad3/GAPDH: 0.52±0.04, 0.69±0.06 vs. 1.11±0.10, all P < 0.05], SOD activity and the protein expressions of E-cadherin and p-AMPK were significantly increased [SOD (μmol/g): 39.76±1.35, 33.03±1.28 vs. 20.08±1.79, E-cadherin/GAPDH: 0.91±0.08, 0.72±0.08 vs. 0.26±0.04, p-AMPK/GAPDH: 0.62±0.04, 0.60±0.01 vs. 0.20±0.04, all P < 0.05]. However, these protective effects of MET were inhibited by the addition of AMPK inhibitor CC solution. CONCLUSIONS MET can effectively alleviate the degree of pulmonary fibrosis in mice poisoned with PQ, and its mechanism may be related to the activation of AMPK and inhibition of TGF-β₁/Smad3 signaling pathway, which can be inhibited by AMPK inhibitor CC.
Mice ; Male ; Animals ; Pulmonary Fibrosis/drug therapy* ; Paraquat ; AMP-Activated Protein Kinases/pharmacology* ; Metformin/pharmacology* ; Hydroxyproline/pharmacology* ; Saline Solution ; Mice, Inbred C57BL ; Lung/metabolism* ; Transforming Growth Factor beta1/pharmacology* ; Cadherins ; Superoxide Dismutase

OBJECTIVE: Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis. METHODS: The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting. RESULTS: Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment. CONCLUSION Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.
Humans ; Transforming Growth Factor beta/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; RNA, Long Noncoding/pharmacology* ; Drugs, Chinese Herbal/pharmacology* ; MicroRNAs/genetics* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/metabolism* ; Cell Proliferation ; Transforming Growth Factors/pharmacology*

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-β1(TGF-β1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-β1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.
NF-kappa B/metabolism* ; Hepatic Stellate Cells ; Transforming Growth Factor beta1/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Intercellular Adhesion Molecule-1/metabolism* ; Isodon ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Toll-Like Receptor 4/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Lipopolysaccharides/pharmacology* ; Signal Transduction ; Colchicine/pharmacology* ; Caspases

Objective To investigate the effect of 1, 25-(OH)₂-VitD3 (VitD3) on renal tubuleinterstitial fibrosis in diabetic kidney disease. Methods NRK-52E renal tubular epithelial cells were divided into control group (5.5 mmol/L glucose medium treatment), high glucose group (25 mmol/L glucose medium treatment) and high glucose with added VitD3 group (25 mmol/L glucose medium combined with 10^-8 mmol/L VitD3). The mRNA and protein expression of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in NRK-52E cells were detected by real-time quantitative PCR and Western blot analysis respectively. The expression and localization of Snail1, SMAD3 and SMAD4 were detected by immunofluorescence cytochemical staining. The binding of Snail1 with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor (CAR) was detected by chromatin immunoprecipitation. The interaction among Snail1, SMAD3/SMAD4 and E-cadherin were detected by luciferase assay. Small interfering RNA (siRNA) was used to inhibit the expression of Snail1 and SMAD4, and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR. SD rats were randomly divided into control group, DKD group and VitD3-treated group. DKD model was established by injection of streptozotocin (STZ) in DKD group and VitD3-treated group. After DKD modeling, VitD3-treated group was given VitD3 (60 ng/kg) intragastric administration. Control group and DKD group were given normal saline intragastric administration. In the DKD group and VitD3-treated group, insulin (1-2 U/kg) was injected subcutaneously to control blood glucose for 8 weeks. The mRNA and protein levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively. Immunohistochemistry was used to detect the expression and localization of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissue. Results Compared with the control group, the mRNA and protein expressions of Snail1, SMAD3, SMAD4 and α-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues were up-regulated, while E-cadherin expression was down-regulated. After the intervention of VitD3, the expression levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in the DKD model improved to be close to those in the control group. Chromatin immunoprecipitation showed that Snail1 and SMAD3/SMAD4 bound to CAR promoter IV, while VitD3 prevented Snail1 and SMAD3/SMAD4 from binding to CAR promoter IV. Luciferase assay confirmed the interaction among Snail1, SMAD3/SMAD4 and E-cadherin. After the mRNA of Snail1 and SMAD4 was inhibited by siRNA, the expression of E-cadherin induced by high glucose was up-regulated. Conclusion VitD3 could inhibit the formation of Snail1-SMAD3/SMAD4 complex and alleviate the renal tubulointerstitial fibrosis in DKD.
Animals ; Rats ; Cadherins/genetics* ; Diabetes Mellitus/pathology* ; Diabetic Nephropathies/pathology* ; Epithelial-Mesenchymal Transition ; Fibrosis/pathology* ; Glucose/pharmacology* ; Kidney/pathology* ; Rats, Sprague-Dawley ; RNA, Messenger ; RNA, Small Interfering ; Transforming Growth Factor beta1/metabolism* ; Vitamin D/pharmacology*

OBJECTIVE: To elucidate the renoprotective effect of resveratrol (RSV) on sphingosine kinase 1 (SphK1) signaling pathway and expression of its downstream molecules including activator protein 1 (AP-1) and transformation growth factor-β1 (TGF-β1) in lipopolysaccharide (LPS)-induced glomerular mesangial cells (GMCs). METHODS: The rat GMCs line (HBZY-1) were cultured and randomly divided into 5 groups, including control, LPS (100 ng/mL), and 5, 10, 20 µmol/L RSV-treated groups. In addition, SphK1 inhibitor (SK-II) was used as positive control. GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h. GMCs proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The proteins expression of SphK1, p-c-Jun and TGF-β1 in GMCs were detected by Western blot, and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay (EMSA). The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016. RESULTS: LPS could obviously stimulate GMCs proliferation, elevate SphK1, p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1 (P<0.05 or P<0.01), whereas these effects were significantly blocked by RSV pretreatment. It was also suggested that the effect of RSV was similar to SK-II (P>0.05). Moreover, RSV exhibited good binding affinity towards SphK1, with docking scores of -8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1. CONCLUSION RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression, which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.
Animals ; Rats ; Lipopolysaccharides/pharmacology* ; Mesangial Cells ; Resveratrol/pharmacology* ; Transcription Factor AP-1 ; Transforming Growth Factor beta1 ; Intercellular Signaling Peptides and Proteins ; Cell Proliferation ; DNA ; Cells, Cultured

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (COL1A1) gene promoter in immortalized human hepatic stellate cells, so as to estimate the activation status of the cells and provide a new cell model for the screening and study of anti-hepatic fibrosis drugs. METHODS: The promoter sequence of human COL1A1 was amplified from the genomic DNA of human hepatocarcinoma cell line HepG2. Based on the pLVX-AcGFP1-N1 plasmid, the recombinant plasmid pLVX-COL1A1-enhanced green fluorescent protein (EGFP) was constructed, in which the enhanced green fluorescent protein gene expression was regulated by the COL1A1 promoter. The monoclonal cell line was acquired by stably transfecting pLVX-COL1A1-EGFP into the immortalized human hepatic stellate cell line LX-2 by the lentivirus packaging system and screening. The cell line was treated with transforming growth factor-β1 (TGF-β1) or co-treated with TGF-β1 and drugs with potential anti-hepatic fibrosis effects. The EGFP fluorescence intensity in cells was analyzed by the fluorescence microscope and ImageJ 1.49 software using a semi-quantitative method. The COL1A1 and EGFP mRNA were detected by reverse transcription real-time quantitative PCR (RT-qPCR), and corresponding proteins were detected by Western blot. RESULTS: The recombinant plasmid pLVX-COL1A1-EGFP with the expression of EGFP regulated by COL1A1 promoter was successfully constructed. Kozak sequence was added to enhance the expression of EGFP, which was identified by double digestion and sequencing. The LX-2 monoclonal cell line LX-2-CE stably transfected with pLVX-COL1A1-EGFP was obtained. After co-treatment with TGF-β1 and 5 μmol/L dihydrotanshinone Ⅰ with potential anti-hepatic fibrosis effect for 24 h, the total fluorescence intensity and the average fluorescence intensity of LX-2-CE were lower than those in TGF-β1 single treatment group (P < 0.05), the intracellular mRNA and protein levels of COL1A1 and EGFP were also lower than those in the TGF-β1 single treatment group (P < 0.05). CONCLUSION A reporter system for estimating activation of hepatic stellate cells based on COL1A1 promoter regulated EGFP expression is successfully constructed, which could visually report the changes in COL1A1 expression, one of the activation-related markers of hepatic stellate cells, in vitro. It provides a new cell model for the screening and study of anti-hepatic fibrosis drugs.
Humans ; Transforming Growth Factor beta1/pharmacology* ; Hepatic Stellate Cells/pathology* ; Liver Cirrhosis/genetics* ; Collagen Type I/pharmacology* ; RNA, Messenger/metabolism*