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Objective To evaluate the effect and mechanism of terminal fucosylation inhibitor 2-deoxy-D-galactose (2-D-gal) on ciclosporin (CsA)-induced renal epithelial-mesenchymal transition (EMT). Methods Fifteen male C57BL/6 mice aged 8-10 weeks were randomly and evenly divided into the control group (Ctrl group), CsA group and CsA+2-D-gal group (n=5). The expression levels of fucosyltransferase 1 (FUT1), EMT-associated proteins including E-cadherin, Vimentin, α-smooth muscle actin (α-SMA) in the kidney tissues of the Ctrl and CsA groups were detected by Western blot. The expression levels of terminal fucose in the kidney tissues of Ctrl and CsA groups were determined by immunofluorescence. The renal fibrosis of mice in each group was evaluated by Masson staining. The blood urea nitrogen and serum creatinine levels of mice in each group were detected. The in vitro EMT model of renal tubular epithelial cell HK2 was induced by CsA. HK2 cells were stimulated with 0, 2.5, 5.0 and 10.0 μmol/L CsA for 24 h, respectively. In addition, HK2 cells were divided into the Ctrl, 2-D-gal, CsA and CsA+2-D-gal groups. The morphology of HK2 cells after stimulation with different concentrations of CsA and in each group was observed. The expression levels of FUT1, E-cadherin, Vimentin and α-SMA in HK2 cells after stimulation with different concentrations of CsA and in each group were detected by Western blot. The expression level of terminal fucose in HK2 cells of the Ctrl and CsA groups was measured by immunofluorescence. Results Compared with the Ctrl group, the relative expression of E-cadherin protein was down-regulated, those of FUT1, Vimentin and α-SMA proteins were up-regulated (all P < 0.05), and that of terminal fucose in the mouse kidney tissues was up-regulated in the CsA group. Compared with the Ctrl group, the blood urea nitrogen and serum creatinine levels in the CsA and CsA+2-D-gal groups were up-regulated (all P < 0.05). Compared with the CsA group, the blood urea nitrogen and serum creatinine levels in the CsA+2-D-gal group were down-regulated (both P < 0.05). Compared with the Ctrl group, the collagen fiber deposition was increased and the relative expression of α-SMA protein was up-regulated in the mouse kidney tissues of CsA and CsA+2-D-gal groups (all P < 0.05). Compared with the CsA group, the collagen fiber deposition was decreased and the relative expression of α-SMA protein in the mouse kidney tissues was down-regulated in the CsA+2-D-gal group (both P < 0.05). With the increase of CsA concentration, the morphology of HK2 cells gradually became longer and thinner from original normal cobblestone shape, the relative expression levels of FUT1, Vimentin and α-SMA protein in HK2 cells were up-regulated, and that of E-cadherin protein was down-regulated in a concentration-dependent manner. Compared with the Ctrl group, the expression level of terminal fucose of HK2 cells was up-regulated in the CsA group. After CsA treatment combined with 2-D-gal intervention, the morphology of HK2 cells in the CsA+2-D-gal group was restored to resemble that of normal HK2 cells. Compared with the CsA group, the relative expression of E-cadherin protein in HK2 cells was up-regulated, whereas those of Vimentin and α-SMA proteins were down-regulated in the CsA+2-D-gal group (all P < 0.05). Conclusions CsA may induce EMT both in vivo and in vitro, and the terminal fucosylation is increased. 2-D-gal may inhibit CsA-induced EMT by suppressing the terminal fucosylation.
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Objective:To observe the effect of Jianpi Xiaoai prescription on the activation of normal human embryonic lung fibroblasts （HFL1） into tumor-associated fibroblasts （CAFs） induced by human colon cancer cells （HCT116） derived exosomes. Method:SD rats were gavaged with 13.1 g·kg-1 of Jianpi Xiaoai prescription to prepare drug-containing serum， and HCT116 cell exosomes-containing 10% exosomes-free serum and 20% Jianpi Xiaoai prescription drug serum were isolated by ultra-high speed centrifugation. The particle size distribution of exosomes were detected by Nanoparticle tracking analyzer （Zetaview）， and the exosomes' marker proteins apoptotic transfer gene 2 interaction protein X （Alix）， heat shock protein 70 （HSP70）， and tumor-susceptibility gene 101 （TSG101） were identified by Western blot， and the uptake of exosomes labeled with cell membrane staining kit （PKH67） by HFL1 was observed by fluorescence microscope. HFL1 cells were divided into six groups： the blank group， the transforming growth factor-β1 （TGF-β1） group， the TGF-β1 combined with HCT116 exosomes of 2 mg·L-1 group， the TGF-β1 combined with HCT116 exosomes of 4 mg·L-1 group， the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 2 mg·L-1 group， and the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 4 mg·L-1 group， and all groups were cultivated for 48 h. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to determine the protein and mRNA expressions of α-smooth muscle actin （α-SMA）. Result:The particle size distribution detected by Zetaview was mainly between 50-100 nm， and the exosomes were verified based on the expressions of marker proteins Alix， HSP70 and TSG101. After co-incubation of HFL1 cells with exosomes， a large number of exosomes were absorbed by HFL1 cells under fluorescence microscope. Compared with the blank control group， the protein and mRNA expressions of α-SMA in the TGF-β1 group and TGF-β1 combined with HCT116 exosome groups were increased （P<0.01）. Compared with the TGF-β1 combined with HCT116 exosome groups， the protein and mRNA expressions of α-SMA were decreased in the TGF-β1 combined with Jianpi Xiaoai prescription exosome groups （P<0.01）. Conclusion:Human colon cancer cell exosomes combined with TGF-β1 can induce the activation of HFL1 into CAFs， and Jianpi Xiaoai prescription can reduce the activation of HFL1 by affecting the expressions of α-SMA， thus antagonizing the lung metastasis of colon cancer.
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Objective::To investigate the effects of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts (GNC) on the protein expression of α-smooth muscle actin (α-SMA) and runt-related transcription factor2(Runx2) after high glucose-induced vascular aging in mice, and elucidate the protective mechanism of GNC in delaying vascular aging. Method::Totally 130 male C57BL/6 mice were randomly divided into normal control group and high glucose group. The mice in high glucose group were intraperitoneally injected with streptozotocin (STZ). After successful modeling, the mice received high-fat diet for 7 months, and then they were randomly divided into model group, GNC low-dose and high-dose groups (0.819, 1.638 g·kg-1), and metformin group (150 mg·kg-1). The drug was given by intragastric administration once a day for 9 weeks. Seven days before tissues collection, a new batch of 4-week-old male C57BL/6 mice were purchased and fed normally for 1 week as a youth group. The general condition of the mice was observed. Morphological changes of the common carotid artery in mice were determined by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Masson trichromatic staining was used to observe the fibrosis of common carotid artery in mice. The expression levels of matrix metalloproteinases-2 (MMP-2), cyclin-dependent kinase inhibitor 2A (p16), cyclic-dependent kinase inhibitor 1A (p21), α-SMA and Runx2 in the common carotid arteries of mice were detected by immunohistochemistry. Result::The results of HE, TEM and Masson showed that there was almost no change in the inimal and adventitial thickness, ultrastructure and relative contents of collagen and elastic fibers in the common carotid arteries of mice between the youth group and normal control group. As compared with the normal control group, the intima of the common carotid artery in the model group was not smooth, the endothelial cells were almost completely detached, the cytoplasm was lysed, the inner elastic membrane became thinner, fractured, or even detached, and the proliferating collagen fibers sneaked into the tunica media. The hyperplasia of tunica media and tunica adventitia was obvious and disordered (P<0.01). The vascular smooth muscle cells showed deformations, protuberances, bifurcations, and even fragmentation, and focal necrosis was observed. There were significantly more vacuoles, lysosomes, and obvious autophagy vesicles. The relative content of collagen and elastic fibers in vascular walls increased significantly (P<0.01). Compared with the model group, the above situation was relieved in each administration group (P<0.01). The results of immunohistochemistry showed that high glucose induced high expression of MMP-2, p16, p21 and Runx2 in the common carotid arteries(P<0.01), low expression of α-SMA(P<0.01), and the protein expression tended to be normal after drug intervention(P<0.05, P<0.01). Conclusion::High glucose can induce the aging of common carotid artery in mice and change the expression of α-SMA and Runx2 proteins. The Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts can delay vascular aging by regulating the protein expression of α-SMA and Runx2.
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Objective: To explore the protective effect and mechanism of modified Dahuang Zhechong Wan on renal interstitial fibrosis in rats with obstructive nephropathy. Method: The unilateral ureteral ligation (UUO)-induced renal interstitial fibrosis model was adopted, 50 SD rats were randomly divided into 5 groups:sham operation group, model group, enalapril group (0.001 g·kg-1), and high and low-dose modified Dahuang Zhechong Wan group (19, 9.5 g·kg-1). Rats in each group were put to death on the 15th day after operation. The serum levels of serum creatinine (SCr) and urea nitrogen (BUN) were collected by enzyme method. The 24-hour urine was collected for 24-hour urinary protein quantity(24 h-Upro) by pyrogallol red molybdenum end point. The kidney tissue was removed from the ligated side. Hematoxylin-eosin (HE) staining and Masson staining were performed; the expressions of transforming growth factor-β1 (TGF-β1), fibronectin (FN) and α-smooth actin (α-SMA) were determined by immunohistochemistry (IHC). Expressions of TGF-β1, p38 mitogen-activated protein kinase (p38 MAPK) and phosphorylated p38 MAPK (p-p38 MAPK) were detected by Western blot. Result: Compared with Sham group, UUO group showed a significant increase in 24 h-Upro, SCr, and BUN (Pβ1, FN, and α-SMA were increased obviously (Pβ1, p38 MAPK, and p-p38 MAPK were increased obviously (PPβ1, FN and α-SMA decreased obviously (Pβ1 and p-p38 decreased obviously (PConclusion: Modified Dahuang Zhechong Wan may improve renal interstitial fibrosis by reducing the high expressions of FN and α-SMA, down-regulating the expressions of p-p38 MAPK and TGF-β1 in p38 MAPK signaling pathway, and decreasing extracellular matrix over deposition and renal cell damage.
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Objective To explore the relationship of hydrogen sulfide (H2S) and the extracellular matrix of the diabetic nephropathic rats' glomerulars.Methods Twenty-one male rats(Sprague-Dawley) were randomly divided into Control group(n =7),DN group (n =7) and DN + H2 S group (n =7).Diabetes was induced in the rats except Control group rats by STZ (60mg/kg).The rats of the DN + H2S group were injected with NaHS every day,and rats of other groups were injected with NS.Eight weeks later,three groups were compared in proteinuria of 24 hours,KW/BW,the proportion of the BrdU positive cell and the distribution of α-SMA in glomerulars.Results Compared with Control group,DN group rats were significantly increased in proteinuria of 24 hours and KW/BW (P ＜ 0.01).Kidney biopsy in DN groups rats were typical DN pathological manifestation.In treatment with NaHS,the DN rats in the DN + H2S group were decreased in proteinuria of 24 hours,KW/BW,the proportion of the BrdU positive cell and the distribution of α-SMA in glomerulars (P ＜ 0.01),and was similar with the control group in the pathology of renal.Conclusion The H2 S control the multiplication of myofibroblast,and were decreased in the distribution of α-SMA in glomerulars.The H2S treatment may put off the progress of the extracellular matrix in the DN rats' glomerulars.
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Objective To investigate the expression of α-smooth muscle actin (α-SMA)in patients with valvular atrial fibrillation and study the relationship betweenα-SMA and atrial fibrosis in patients with valvular atrial fibrillation (AF).Methods For this study we enrolled 84 consecutive patients with rheumatic heart disease who were to receive cardiac surgery.The patients were divided into AF group (AF,n=39)and sinus rhythm group (SR, n=45).Their clinical data including baseline demographics,routine laboratory test and echocardiographics were collected before surgery.The right atrial tissue (0 .3-0 .5 cm3 )was disserted during the surgery.Right atrial fibrosis was observed by Masson staining.The mRNA expression ofα-SMA in atrial tissue were determined by Real-time quantitative PCR.Western blot was used to measure the protein expression ofα-SMA in atrial tissue.Results The two groups did not significantly differ in sex ratio,age,blood pressure,blood biochemical indicators or other aspects of medical history (P>0.05).However,left and right atrium diameters in AF group were significantly larger than those in SR group (P<0 .05 ).Masson staining suggested that collagen volume fraction and collagen content were significantly higher in AF group than in SR group (P<0 .05 ).The mRNA and protein expressions ofα-SMA in right atrial tissue were obviously higher in AF group than in SR group (coefficients P<0 .05 ).The mRNA and protein expressions ofα-SMA from right atrial tissue in the 84 patients were positively correlated with collagen content (coefficients of 0.587 and 0.607;P=0.029,0.014,respectively).Conclusion There is significant atrial fibrosis in patients with valvular atrial fibrillation,which is closely related to up-regulated expression ofα-SMA gene.
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Objective To evaluate the validity of botulinum toxin type A (BTXA) injections for the treatment of scar contracture.Methods 26 patients with scar contracture were randomly assigned into BTXA group and triamcinolone acetonide (TAC) group.Pinpoint tattooing was performed on each side of each scar in the plane of its longest axis.A template was used to ensure consistent length.These two tattoo points were measured to assess scar contraction at baseline,at every month for a total of 6 months.Histological analysis was conducted to study the physiological environment and immunohistochemistry to detect the expression of α-SMA and myosin-Ⅱ at different groups.Results Scar contraction was more relaxed in BTXA group than that in TAC group after 1 month (P＜0.05),especially in the 6th month (the D value in BTXA group and TAC group was (1.23±0.42) cm,and (0.56±0.33) cm respectively).For immunohistochemistry,the expression of α-SMA and myosin-Ⅱ also decreased in BTXA group (P＜0.05).Conclusions The treatment of scar contracture by suitable BTXA injections is safe and effective.