Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.
Animals ; Rats ; Cell Proliferation ; Collagen Type I/metabolism* ; Fibrosis ; Hepatic Stellate Cells ; HMGB1 Protein/genetics* ; Liver Cirrhosis/pathology* ; MicroRNAs/metabolism*

OBJECTIVE: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts <i>in vitroi> and promote the tendon-bone healing in rabbits. METHODS: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. RESULTS: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( <i>Pi><0.05), Fibronectin significantly increased at 3 days ( <i>Pi><0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( <i>Pi><0.05). CCK-8 detection showed that there was no significant difference ( <i>Pi>>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. CONCLUSION Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Pregnancy ; Female ; Humans ; Rabbits ; Animals ; Vascular Endothelial Growth Factor A/metabolism* ; Fibronectins/metabolism* ; Collagen Type I/genetics* ; Tenascin/metabolism* ; Collagen/metabolism* ; Anterior Cruciate Ligament/surgery* ; Mesenchymal Stem Cells ; Tendons/metabolism* ; Fibroblasts/metabolism*

OBJECTIVE: To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF). METHODS: Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting. RESULTS: The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs. CONCLUSIONS STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.
Rats ; Animals ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; RNA-Seq ; Transcriptome/genetics* ; Heart Failure/drug therapy* ; Collagen ; Collagen Type I/metabolism* ; Fibrosis ; Myocardium/pathology*

OBJECTIVE: To establish a visual reporting system for evaluating the activity of collagen Ⅰ α 1 chain (<i>COL1A1i>) 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 <i>COL1A1i> 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 <i>COL1A1i> 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 <i>COL1A1i> and <i>EGFPi> 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 <i>EGFPi> regulated by <i>COL1A1i> promoter was successfully constructed. Kozak sequence was added to enhance the expression of <i>EGFPi>, 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 (<i>Pi> < 0.05), the intracellular mRNA and protein levels of <i>COL1A1i> and <i>EGFPi> were also lower than those in the TGF-β1 single treatment group (<i>Pi> < 0.05). CONCLUSION A reporter system for estimating activation of hepatic stellate cells based on <i>COL1A1i> promoter regulated <i>EGFPi> expression is successfully constructed, which could visually report the changes in <i>COL1A1i> expression, one of the activation-related markers of hepatic stellate cells, <i>in vitroi>. 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*

Objective To explore the effect of overwork (OW) on extracellular matrix of arterial vessel wall in rats. Methods Random number grouping method was employed to assign 18 Sprague-Dawley rats into three groups(<i>ni>=6):the control group(no special treatment),group OW(forced swimming twice a day for 15 days),and sleep deficiency(SD)+OW group(in addition to forced swimming twice a day,the rats were put on the platforms in water to limit sleep for 15 days).On the 16th day,the abdominal aorta and common carotid artery were collected after blood sampling from heart under deep anesthesia.A part of the abdominal aorta sample was taken for Masson staining of collagen fiber,and Verhoeff-Van Gieson staining was carried out for the elastic fiber of common carotid artery.Image J was employed for the quantitative analysis of collagen fiber and elastic fiber content.The expression of collagen 1(Col-1) protein was quantified by immunohistochemistry and the ultrastructure of vascular matrix was examined by transmission electron microscopy.The other part of the abdominal aorta sample was used to determine the mRNA levels of matrix metalloproteinase(MMP)-1,MMP-2,MMP-9,tissue inhibitor of metalloproteinases-1(TIMP-1),and Col-1 by quantitative real-time polymerase chain reaction. Results Compared with that in control group,the content of collagen fiber in groups OW and SD+OW had no significant change(all <i>Pi>>0.05);the content of elastic fiber in groups OW and SD+OW decreased(all <i>Pi><0.001) and had no significant difference between each other(<i>Pi>>0.05).The vascular vessel wall of group OW showed slight fiber breakage,while that of group SD+OW presented wormhole-like or spongy fiber fragmentation.The mRNA levels of MMP-1 and MMP-2 in groups OW and SD+OW had no significant difference between each other(<i>Pi>>0.05) but were higher than that in control group(all <i>Pi><0.001).The mRNA levels of MMP-9 and TIMP-1 had no significant difference among the three groups(all <i>Pi>>0.05).Groups OW and SD+OW had lower mRNA level(all <i>Pi><0.001) and protein level(all <i>Pi><0.001) of Col-1 than control group,while the mRNA and protein levels of Col-1 had no significant difference between groups OW and SD+OW(<i>Pi>>0.05). Conclusion OW can reduce the content of Col-1 and elastic fibers in the extracellular matrix of arterial vessels,destroy the elastic lamina of vascular wall,up-regulate the expression of MMP-1 and MMP-2,thereby injuring arterial vessels.
Animals ; Collagen Type I ; Extracellular Matrix/metabolism* ; Matrix Metalloproteinase 1/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; RNA, Messenger/genetics* ; Rats ; Rats, Sprague-Dawley ; Tissue Inhibitor of Metalloproteinase-1/metabolism*

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.
Actins ; genetics ; Animals ; Cells, Cultured ; Collagen ; biosynthesis ; genetics ; Coumarins ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Mice ; Myocardium ; cytology ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Receptor, Transforming Growth Factor-beta Type I ; Receptors, Transforming Growth Factor beta ; genetics ; Signal Transduction ; drug effects ; Smad Proteins ; genetics ; Transforming Growth Factor beta1 ; genetics

OBJECTIVETo study the effect of total flavonoids of Astmgali Radix (TFA) on liver cirrhosis induced with dimethylnitrosamine (DMN) in rats, and the effect on peroxisome proliferator-activated receptor γ (PPARγ), uncoupling protein 2 (UCP2) and farnesoid X receptor (FXR).

METHODSFifty-three Sprague-Dawley rats were randomly divided into a control group (10 rats) and a DMN group (43 rats). Rats in the DMN group were given DMN for 4 weeks and divided randomly into a model group (14 rats), a low-dosage TFA group (14 rats) and a high-dosage TFA group (15 rats) in the 3rd week. Rats were given TFA for 4 weeks at the dosage of 15 and 30 mg/kg in the low- and high-TFA groups, respectively. At the end of the experiment blood and liver samples were collected. Serum liver function and liver tissue hydroxyproline content were determined. hematoxylin-eosin (HE), Sirus red and immunohistochemical stainings of collagen I, smooth muscle actin (α-SMA) was conducted in paraffinembedded liver tissue slices. Real time polymerase chain reaction (PCR) was adopted to determine PPARγ, UCP2 and FXR mRNA levels. Western blot was adopted to determine protein levels of collagen I, α-SMA, PPARγ, UCP2 and FXR.

RESULTSCompared with the model group, TFA increased the ratio of liver/body weight (low-TFA group P<0.05, high-TFA group P<0.01), improved liver biochemical indices (P<0.01 for ALT, AST, GGT in both groups, P<0.05 for albumin and TBil in the high-TFA group) and reduced liver tissue hydroxproline content (P<0.01 in both groups) in treatment groups significantly. HE staining showed that TFA alleviated liver pathological changes markedly and Sirus red staining showed that TFA reduced collagen deposition, alleviated formation and extent of liver pseudolobule. Collagen I and α-SMA immunohistochemical staining showed that staining area and extent markedly decreased in TFA groups compared with the model group. TFA could increase PPARγ, it regulated target UCP2, and FXR levels significantly compared with the model group (in the low-TFA group all P<0.05, in the high group all P<0.01).

CONCLUSIONTFA could improve liver function, alleviate liver pathological changes, and reduce collagen deposition and formation of liver pseudolobule in rats with liver cirrhosis. The antifibrotic effect of TFA was through regulating PPARγ signal pathway and the interaction with FXR.

Actins ; metabolism ; Animals ; Blotting, Western ; Body Weight ; drug effects ; Collagen Type I ; metabolism ; Dimethylnitrosamine ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Flavonoids ; pharmacology ; therapeutic use ; Hydroxyproline ; metabolism ; Liver ; drug effects ; pathology ; Liver Cirrhosis ; blood ; drug therapy ; genetics ; pathology ; Male ; Organ Size ; drug effects ; PPAR gamma ; genetics ; metabolism ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Uncoupling Protein 2 ; genetics ; metabolism

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.
Animals ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Female ; Heart Rate ; drug effects ; Hyperplasia ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Myocardial Infarction ; drug therapy ; pathology ; Myocardium ; metabolism ; pathology ; Rabbits ; Transforming Growth Factor beta ; genetics ; metabolism

OBJECTIVETo study the effect of ligustrazine nanoparticles nano spray (LNNS) on transforming growth factor β (TGF-β)/Smad signal protein of rat peritoneal mesothelial cells (RPMC) induced by tumor necrosis factor α (TNF-α), and the anti-adhesion mechanism of LNNS in the abdominal cavity.

METHODSThe primary culture and subculture of rat peritoneal mesothelial cells (RPMC) was processed by trypsin digestion method in vitro. The third generation was identifified for experiment and divided into 5 groups: a blank group: RPMC without treatment; a control group: RPMC stimulated with TNF-α; RPMC treated by a low-dosage LNNS group (2.5 mg/L); RPMC treated by a medium-dosage LNNS group (5 mg/L); and RPMC treated by a high-dosage LNNS group (10 mg/L). Reverse transcription-polymerase chain reaction was applied to test the expression of fifibronectin, collagen I (COL-I), TGF-β mRNA, and Western blot method to test the Smad protein 7 expression of RPMC.

RESULTSCompared with the blank group, a signifificant elevation in fifibronectin (FN), COL-I and TGF-β mRNA expression of RPMC were observed in the control group (P<0.05). Compared with the control group, LNNS suppressed the expressions of FN, COL-I and TGF-β mRNA in a concentrationdependent manner (P<0.05). The expression of Smad7 protein of RPMC was down-regulated by TNF-α stimulation, and up-regulated with the increase of LNNS dose (P<0.05).

CONCLUSIONSTNF-α may induce changes in RPMC's viability, leading to peritoneal injury. LNNS could reverse the induction of fifibrosis related cytokine FN, COL-I and TGF-β, up-regulating the expression of Smad7 by TNF-α in RPMC, thus attenuate peritoneal injury by repairing mesothelial cells.

Animals ; Collagen Type I ; genetics ; metabolism ; Epithelium ; drug effects ; metabolism ; Fibronectins ; metabolism ; Male ; Nanoparticles ; chemistry ; ultrastructure ; Particle Size ; Peritoneal Cavity ; cytology ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo explore the interaction between P311 and transforming growth factor beta 1 (TGF-β1) in murine fibroblasts and its effect on the function of fibroblasts.

METHODSSkin fibroblasts obtained from five neonatal P311 wild-type C57BL/6 mice and P311 gene knock-out C57BL/6 mice were cultured. The second passage of fibroblasts were used in the following experiments. All experiments were repeated for 3 times. (1) The fibroblasts of P311 wild-type mice were divided into blank control group and P311 over-expression group according to the random number table (the same grouping method below), with 36 wells in each group. Fibroblasts in blank control group were transfected with 10 μL control vector, and fibroblasts in P311 over-expression group were transfected with equal efficiency P311 expression adenovirus vector. After being cultured for 48 hours, the mRNA expression level of P311, and the mRNA and protein expression levels of TGF-β1, α-smooth muscle actin (α-SMA), and collagen type I of fibroblasts in both groups were determined with real-time fluorescent quantitative RT-PCR and Western blotting (the same detection methods below), respectively. (2) After cultured reaching the cell density of 80%-90%, the mRNA and protein expression levels of TGF-β1, α-SMA, and collagen type I of the fibroblasts of P311 wild-type mice and P311 gene knock-out mice, with 4 flasks in each type of fibroblasts, were determined. (3) The fibroblasts of P311 wild-type mice were divided into blank control group and 5, 10, 15, 20, and 25 ng/mL TGF-β1 groups after being starved treatment with DMEM medium containing 1% FBS for 3 hours, with 2 flasks in each group. Fibroblasts in blank control group were routinely cultured, while fibroblasts in the latter five groups were treated with 5, 10, 15, 20, and 25 ng/mL TGF-β1, respectively. After being cultured for 48 hours, the mRNA expression levels of P311 in fibroblasts of the six groups were determined. Another fibroblasts of P311 wild-type mice were divided into blank control group and 10 ng/mL TGF-β1 group, with 6 wells in each group, and the protein expression levels of P311 in both groups were determined by immunofluorescence staining. (4) The fibroblasts of P311 wild-type mice were divided into blank control group and 10 ng/mL TGF-β1 group after being starved treatment as above, with 2 flasks in each group, and fibroblasts in blank control group were routinely cultured, while fibroblasts in the latter group were treated with 10 ng/mL TGF-β1. After being cultured for 48 hours, the mRNA and protein expression levels of α-SMA and collagen type Ⅰ were determined. The fibroblasts of P311 gene knock-out mice were grouped and treated as above, and the mRNA and protein expression levels of α-SMA and collagen type I were determined. Data were processed with one-way analysis of variance and t test.

RESULTS(1) The mRNA expression level of P311 of fibroblasts in P311 over-expression group was increased nearly 300 000-fold compared with that in blank control group (t=9.942, P<0.001). The mRNA expression levels of TGF-β1, α-SMA, and collagen type I of fibroblasts in P311 over-expression group, and the protein expression levels of pro-TGF-β1, activated TGF-β1, α-SMA, and collagen type I of fibroblasts in P311 over-expression group were significantly higher than those in blank control group (with t values from 8.192 to 49.090, P values below 0.01). (2) The mRNA expression levels of TGF-β1, α-SMA, and collagen type I in fibroblasts of P311gene knock-out mice were significantly lower than those in fibroblasts of P311 wild-type mice (with t values from 8.157 to 22.270, P values below 0.01). The protein expression levels of pro-TGF-β1, α-SMA, and collagen type I in fibroblasts of P311 gene knock-out mice were significantly lower than those in fibroblasts of P311 wild-type mice (with t values from 2.995 to 12.600, P<0.05 or P<0.01), and the protein expression levels of active TGF-β1 were similar in two types of fibroblasts (t=1.070, P>0.05). (3) The mRNA expression levels of P311 of fibroblasts in blank control group and 5, 10, 15, 20 and 25 ng/mL TGF-β1 groups were 1.28 ± 0.44, 3.61 ± 0.91, 6.64 ± 0.92, 6.58 ± 1.04, 1.79 ± 0.31, 0.16 ± 0.06, respectively. Compared to the mRNA expression level of P311 of fibroblasts in the blank control group, the mRNA expression levels of P311 of fibroblasts in 5 and 20 ng/mL TGF-β1 groups were similar (with t values respectively 2.302 and 0.955, P values above 0.05), while they were significantly higher in 10 and 15 ng/mL TGF-β1 groups (with t values respectively 5.630 and 4.710, P values below 0.001), and they were significantly lower in 25 ng/mL TGF-β1 group (t=2.509, P<0.01). The protein expression level of P311 of fibroblasts in 10 ng/mL group was higher than that in blank control group. (4) The mRNA and protein expression levels of α-SMA and collagen type I of fibroblasts of P311 wild-type mice in 10 ng/mL TGF-β1 group were significantly higher than those in blank control group (with t values from 3.523 to 14.290, P<0.05 or P<0.01). The mRNA and protein expression levels of α-SMA and collagen type I of fibroblasts of P311 gene knock-out mice in 10 ng/mL TGF-β1 group were significantly higher than those in blank control group (with t values from 4.895 to 14.870, P<0.05 or P<0.01).

CONCLUSIONSThe interaction between P311 and TGF-β1 in murine fibroblasts exists and it may enhance the differentiation of fibroblasts in combination.

Actins ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; metabolism ; Fibroblasts ; cytology ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Nerve Tissue Proteins ; genetics ; metabolism ; RNA, Messenger ; Transfection ; Transforming Growth Factor beta1 ; metabolism