Dahuang zhechong pills regulate ASICla/VEGF pathway and alleviate hepatic fibrosis / 中国药理学通报

Aim To examine the therapeutic effects of DHZCP on carbon tetrachloride(CCl4)-induced chemical hepatic fibrosis model in rats and the mechanism of acid-sensitive ion channels 1a(ASIC1a)and vascular endothelial growth factor(VEGF)-related mechanisms.Methods The rats were injected intraperitoneally with CCl4 vegetable oil mixture to establish hepatic fibrosis model,and randomly divided into six groups:control group,hepatic fibrosis model group,DHZCP low dose group,DHZCP medium dose group,DHZCP high dose group and colchicine(Col)positive control group.HE staining was used to observe the pathological changes of hepatic structures in each group,Masson staining to view the production of collagen fibers in each group,and immunohistochemistry,Western blot,q-PCR to investigate the expression level of ASIC1a,CaMKKβ,VEGF,α-SMA,Collagen-I proteins.Results In model group,serum ALT and AST levels were obviously up-regulated,liver tissue structure was severely damaged,and ASIC1a,CaMKKβ,VEGF,α-SMA,Collagen-I gene and protein expression levels were significantly elevated.Compared with model group,each treatment group of DHZCP could markedly alleviate the pathological changes of liver fibrosis caused by CCl4,significantly reduce the serum ALT and AST levels,and dose-dependently down-regulate the gene and protein expression levels of ASIC1a,CaMKKβ,VEGF,α-SMA,Collagen-I,etc.Conclusions DHZCP ameliorates hepatic fibrosis in rats,and its mechanism of action may be associated with the regulation of ASIC1a/VEGF.

Effect and mechanism of terminal fucosylation inhibitor on ciclosporin-induced renal epithelial-mesenchymal transition / 器官移植

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.

Expression and distribution of human dermal reticular fibroblasts in keloid tissues / 中华皮肤科杂志

Objective:To investigate the expression and distribution of human dermal papillary fibroblasts (Fp) , reticular fibroblasts (Fr) , and myofibroblasts (MFB) in keloid tissues.Methods:Keloid tissues were collected from 15 outpatients (including 8 males and 7 females) aged 20-50 years, who were diagnosed in the Department of Dermatology, Renmin Hospital of Wuhan University from May to December 2019. Normal skin tissues were taken from 15 age-matched women who underwent mammoplasty, and served as controls. The distribution of fibroblast activation protein (FAP) , CD90 and alpha-smooth muscle actin (α-SMA) was observed in the keloid tissues and normal skin tissues by dual immunofluorescence staining. Furthermore, fibroblasts were isolated from 3 normal skin and 3 keloid tissue samples, and subjected to primary culture. Subsequently, the fibroblasts were treated with 10 ng/ml transforming growth factor-β1 (TGF-β1) for 48 hours in vitro, during which, changes in fibroblast phenotypes were observed in the 2 groups. Fluorescence-based quantitative RT-PCR and Western blot analysis were performed to determine the mRNA and protein expression of FAP, CD90 and α-SMA. Measurement data were compared between 2 groups by using t test. Results:Immunofluorescence staining of the normal skin tissues revealed that FAP +/CD90 - fibroblasts were predominantly distributed in the superficial dermis, FAP -/CD90 + fibroblasts in the deep dermis, and CD90 + cells hardly expressed α-SMA; however, a large number of FAP + fibroblasts and CD90 + fibroblasts were observed in the deep keloid tissues, and many CD90 + fibroblasts also expressed α-SMA. Dual immunofluorescence staining showed that normal tissue-derived fibroblasts hardly expressed α-SMA, and keloid-derived fibroblasts expressed α-SMA. The fluorescence intensity of α-SMA + cells significantly increased in the normal tissue-and keloid-derived fibroblasts after 24-hour treatment with TGF-β1 (21.058 ± 0.709, 27.112 ± 0.097, respectively) compared with that in the corresponding untreated fibroblasts (11.312 ± 0.636, 21.306 ± 0.464, t=22.430, 13.370, respectively, both P < 0.05) . RT-PCR and Western blot analysis showed that the mRNA and protein expression of FAP, CD90 and α-SMA significantly increased in the keloid-derived fibroblasts after 48-hour treatment with TGF-β1 (mRNA: 92.610 ± 3.667, 1.366 ± 0.105, 3.240 ± 0.141; protein: 0.652 ± 0.073, 1.046 ± 0.119, 0.946 ± 0.117, respectively) compared with the untreated keloid-derived fibroblasts (all P < 0.05) . Conclusion:CD90 + Fr aberrantly proliferated in the deep dermis of keloid tissues, suggesting that directional intervention in aberrantly proliferating FAP -/CD90 + Fr in the deep dermis may promote the efficacy for keloids.

Mechanism of total C-21 steroidal glucosides from root of Cynanchum auriculatum in activation of human hepatic stellate cells via TGF-β / 中国药理学通报

Aim To investigate the inhibitory effect of total C-21 steroidal glucosides (TCSG) from the root of Cynanchum auriculatum on activation of human hepatic stellate cells and the underlying mechanism. Methods The fibrosis model in vitro was established by treating LX-2 cells with TGF-β

Effect of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma Extracts on Expression of α-SMA and Runx2 Protein After High Glucose-induced Vascular aging in Mice / 中国实验方剂学杂志

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.

Effect of Jianpi Xiaoai Prescription on Activation of HFL1 into CAFs Induced by Exosomes in Colon Cancer / 中国实验方剂学杂志

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.

Anti-fibrotic mechanism of Sedum sarmentosum total flavanones in inhibiting activation of HSC by regulating Smads / 中国中药杂志

This paper was aimed to observe the interventional effect of Sedum sarmentosum total flavanones on hepatic fibrosis and its possible mechanism through the subcutaneous injection of CCl_4 in rats. Sixty male SD rats were randomly divided into normal control group, model group, low-dose, medium-dose, high-dose S. sarmentosum total flavanones groups(100, 200, 400 mg·kg~(-1)) and silymarin group(200 mg·kg~(-1)). The model of liver fibrosis was established by subcutaneous injection of rats with 40% CCl_4. After the modeling, the drug groups were intragastrically administered with corresponding drugs once a day for consecutively five weeks, while the normal group and the model group were given 0.9% sodium chloride solution during the same period. After the experiment, the general conditions of rats and the pathological changes of liver tissues were observed, and the contents of serum ALT, AST, HA and LN were measured. Besides, the expressions of the protein and relevant mRNA of Smad2/3, Smad4 and α-SMA in rats were detected. Compared with model group, S. sarmentosum total flavanones could significantly increase the rats' body weight, inhibit the increase of liver and spleen index in rats of liver fibrosis, reduce the levels of ALT, AST, HA and LN, and alleviate pathological changes. Meanwhile, compared with the model group, the protein expressions of Smad2/3, Smad4 and α-SMA as well as relevant mRNA expressions in S. sarmentosum total flavanones group were obviously decreased, while Smad7 expression was markedly increased. As a result, S. sarmentosum total flavanones could significantly alleviate CCl_4-induced liver fibrosis, and its anti-hepatic fibrosis mechanism may be related to intervention with Smads pathway, so as to inhibit the activation of HSC.

Anti-fibrosis effects of polysaccharide of Prismatomeris tetrandra on silicosis rats / 中草药

Objective: To investigate the anti-fibrosis effects of polysaccharide of Prismatomeris tetrandra on the silicosis rats and the underlying mechanism. Methods: The experimental silicosis rat model was established by oropharyngeal aspiration method. Forty male adult Wistar rats were randomly divided into two groups: saline group and modeling group. The experimental fibrosis of silicosis was induced by dripping 1 mL of 50 mg/mL SiO2 onto the oropharynx of rats of the modeling group. After modeling, the modeling group were randomly divided into one model group with SiO2 and three polysaccharide groups with SiO2 plus low, medium, and high dose of polysaccharide of P. tetrandra, eight rats per group. One day after modeling, rats in three polysaccharide groups were administered with the polysaccharide at a dose of 125, 250, and 500 mg/kg, respectively, daily for 56 d, meanwhile, the saline group and the model group were given the same amount of saline daily for 56 d. The rats were then sacrificed at the 56th day of the experiment and lung tissues were collected. The pathological changes and fibrosis of the lung tissues were observed by HE staining and Masson staining. Real-time quantitative PCR method was used to detect the mRNA expression of epithelial calcium adhesion protein (E-cadherin) and alpha smooth muscle actin (α-SMA) in lung tissue; Western blotting method was used to measure the levels of E-cadherin, α-SMA, and Vimentin in lung tissue. Results: There were no significant differences in body weight between groups. Compared with the saline group, the alveolar inflammation and the pulmonary fibrosis were aggravated, the lung/body coefficient was increased, the levels of α-SMA mRNA and α-SMA protein were significantly increased, the mRNA and protein expression level of E-cadherin was decreased markedly, and the level of Vimentin was increased significantly in the model group. Compared with the model group, the alveolar inflammation and the pulmonary fibrosis were alleviated, the lung/body coefficient was decreased, the levels of E-cadherin mRNA and protein were increased markedly, the levels of α-SMA mRNA and protein expression were decreased sharply, and the levels of Vimentin were decreased significantly in the polysaccharide groups. Conclusion: The polysaccharide of P. tetrandra exhibited the anti-fibrosis effects on silicosis by reducing silicosis-induced lung injury and slowing down the process of pulmonary fibrosis.

Effect of Qingguang'an on collagen fiber, alpha-SMA and FN in scar of filter tract after glaucoma surgery / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To observe the effects on four effective components of Qingguang'an on collagen fibers, α-smooth muscle actin(α-SMA)and fibronectin(FN)in rabbits after glaucoma surgery.<p>METHODS: Apply four kinds of effective components of Qingguang'an and Qingguang'an Chinese medicine suspension to D, E, F, G, H groups after filtration surgery, and pass with group A(blank control group)and group B(model group)Compared with group C(Mitomycin C group), the effects of four effective components of Qingguang'an and Qingguang'an traditional Chinese medicine suspension on collagen fibers, α-SMA and FN in the scar tissue of glaucoma after filtration were observed.<p>RESULTS: Compare to B group, the ratio of collagen fiber area to E, F, H group, the expressions of α-SMA and the expressions of FN were different(<i>P</i><0.05). <p>CONCLUSION: Qingguang'an effective components 2, Qingguang'an effective components 3, mitomycin C and Qingguang'an suspension reduce the proliferation of myofibroblasts and fibroblasts by inhibiting the expression of collagen fibers, α-SMA and FN, and showed obvious anti-glaucoma staining scar after surgery.

Protective Effect and Mechanism of Modified Dahuang Zhechong Wan on Renal Interstitial Fibrosis in Rats with Obstructive Nephropathy / 中国实验方剂学杂志

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.

Effect of Yangxinkang Tablets in Regulating Autophagy on Myocardial Fibrosis in Mice After Chronic Heart Failure / 中国实验方剂学杂志

Objective: To study the effect of Yangxinkang tablets on myocardial fibrosis in mice after heart failure, and to explore its mechanism.Method: The model of chronic heart failure in mice was established by thoracic aorta constriction (TAC). After successful modeling, mice were randomly divided into sham operation group, model group, 3-methyladenine(3-MA,15 mg·kg-1) autophagy inhibitor group, Yangxinkang tablets high, medium, and low dose groups (1 170,585,390 mg·kg-1).The sham operation group received equal volume of distilled water. After 30 days, cardiac ultrasound was performed to collect hemodynamic parameters. Cardiac paraffin slices were stained with Masson to observe the morphological changes and fibrosis of cardiomyocytes. Western blot was used to detect lysosome-associated membrane protein(LAMP), microtubule-associated protein light chain 3 (LC3), Beclin-1 autophagyportein, α-smooth muscle activin(α-SMA),Collagen Ⅰ,Collagen Ⅲ protein expression.Result: As compared with normal group, the left ventricular ejection fraction (LVEF) and fractional shortening(FS) were significantly decreased(PPPα-SMA, Collagen Ⅰ, Collagen Ⅲ, LAMP, LC3, and Beclin-1 were significantly increased in model group (PPPα-SMA,Collagen Ⅰ,Collagen Ⅲ,LAMP,LC3 and Beclin-1 were decreased in 3-MA group, Yangxinkang high and medium dose groups(PConclusion: Yangxinkang tablets can reduce myocardial fibrosis and improve cardiac function in mice with heart failure probably by down-regulating autophagy.

Activation of hepaticstellate cells cocultured with hepatoma carcinoma cells in vitro and in vivo / 西安交通大学学报(医学版)

Objective: To investigate the activation of hepatic stellate cells (HSCs) cocultured with hepatoma carcinoma cells. Methods: MHCC97H cells and HSCs were cocultured by cell-cell contact method, fibrinogen-thrombin paste technique, conditioned media from MHCC97H(MHCC97H-CM) and Transwell coculture technique, respectively. MHCC97H cells and HSCs were inoculated s. c. into nude mice. The expression of α-SMA in HSCs was assessed by immunocytochemical staining and Western blotting. Proliferation and migration of HSCs was determined using Cell-Counting Kit-8 (CCK-8) and wound healing and Transwell technique, respectively. Results: The activation of HSCs was significantly increased in the all cocultured system. The expression of α-SMA was up-regulated by cell-cell contact method, MHCC97H-CM, Transwell coculture technique in vitro and in cancer-bearing mice in vivo. The increased chemotaxis of MHCC97H cells and HSCs was observed by cell-cell contact method, fibrinogen-thrombin paste technique and in cancer-bearing mice. The proliferation and migration abilities of HSCs were significantly enhanced. Conclusion: Hepatoma carcinoma cells can promote the activation, proliferation and migration of HSCs under the cocultured system in vitro and in vivo.

Effect of Dilong on expression of fibrogenic factors TGF-β1 and α-SMA in lung tissue of mice with pulmonary fibrosis / 中国中药杂志

The aim of this paper was to investigate the effect of Dilong( geosaurus) on the expressions of fibrotic factors TGF-β1 and α-SMA in bleomycin-induced pulmonary fibrosis mice. The binding ability of Dilong to fibrotic factor TGF-β1 was initially detected by Biacore technology and verified by in vivo pharmacodynamics. A total of 60 SPF C57 mice were randomly divided into 6 groups. Except the blank group( injecting 0. 08 m L·kg-1 sodium chloride in the trachea),the other five groups were given bleomycin( 4 mg·kg-1) to replicate the pulmonary fibrosis model. After 14 days of drug treatment,the expressions of TGF-β1 and α-SMA were detected by Masson staining,immunohistochemistry and RT-PCR. The results of Biacore experiment showed that the extract of Dilong was well bound to TGF-β1 protein in vitro,and the binding value reached 619. 3. Compared with the model group,Masson's results showed that cellulose deposition in high-dose,medium-dose and low-dose Dilong groups decreased to varying degrees. RT-PCR results showed that different doses of Dilong could reduce protein and mRNA expressions of TGF-β1 and α-SMA to a certain extent in a dose-dependent manner. In conclusion,Dilong could delay the process of pulmonary fibrosis by binding to target protein TGF-β1 and inhibiting the expression of α-SMA.

The role of c-Jun amino terminal kinase pathway in the differentiation of anaerobic renal tubular epitheli-al cells / 实用医学杂志

Objective To explore the role of c-Jun N-terminal protein kinase(JNK)pathway in renal tubular epithelial cells(RTEC)transdifferentiation by detecting the expression of JNK pathway in the injury of hypoxic RTEC. Methods In vitro cultured rat RTEC were randomly divided into four groups of normal control, hypoxia,inhibitors,dimethyl sulfoxide(DMSO). The groups of hypoxia,inhibitor and DMSO were placed into a vacuum tank to establish hypoxia model. The cells were harvested 6,12 and 24 h after hypoxia started. RT-PCR and Western blot test were used to detect the mRNA expressions of alpha smooth muscle actin(α- SMA)and protein expressions of JNK,pJNK and alpha SMA,respectively,in all the groups. Results Hypoxia induced significantly increased expressions of α-SMA mRNA and proteins,and JNK and pJNK proteins as well in RTEC (all P<0.05).After addition of inhibitors,the expression of α-SMA mRNA and proteins,the protein expression of pJNK in RTEC were significantly decreased(all P < 0.05)and the expression of JNK protein was significantly increased(P < 0.05). There was no significant difference in the expressions of JNK,pJNK,α-SMA in RTECs between hypoxia group and DMSO group(all P>0.05).There was a significant positive correlation between pJNK protein and α-SMA protein expression(P < 0.01). Conclusion In hypoxic RETC injury in rats,JNK pathway may be involved in the RTEC phenotype transdifferentiation.

The inhibitory effect of Euonymus alatus against mouse hepatic fibrosis induced by CCl4 / 中国药理学通报

Aim To explore the inhibitory effect of Euonymus alatus on hepatic fibrosis induced by carbon tetrachloride (CCl4) in mice and its mechanism. Methods Eighty C57BL/6 male mice were randomly divided into eight groups: normal group, CCl4model group, Euonymus alatus(EA) ethanol extracts groups in early stage(EAE), EA ethanol extracts groups in later stage(EAL),two drug groups with low/medium/high dose(EAE-L/M/H, EAL-L/M/H), with 10 mice in each group. Fibrosis model was established by injecting CCl4in peritoneal cavity,and the study lasted for 30 days. Different doses of drugs were given from 1 st day to 15 th day in EAE while from 16 th day to 30 th day in EAL,then all mice were sacrificed to for the observation of the morphological changes and collage-nous fiber by HE and Masson staining. Liver index, ALT,AST and TNF-α were tested by ELISA. The ex-pressions of α-SMA and CollagenⅠwere measured by immunohistochemistry and Western blot. Results Compared to normal group, liver index, ALT, AST, TNF-α, α-SMA and CollagenⅠ in EA groups were lower than those in model group in a dose-dependent manner(P<0.01 or P<0.05). Liver morphology and collagenous fiber in EAE and EAL were better than those in model group in a dose-dependent manner. The effect of EAE were superior to that of the EAL in HE, Masson, α-SMA, Collagen Ⅰ indexes(P <0.05). Conclusions Euonymus alatus may inhibit the process of hepatic fibrosis in mice with dose-effect de-pendence, and drug treatment in early stage performs better,which may be related to the decrease of TNF-α that affects the expression of α-SMA and Collagen Ⅰ.

Effect of diabetes on CCl4-induced rats liver fibrosis / 中国药理学通报

Aim To observe the effect of diabetes on carbon tetrachloride (CCl4)-induced rats chemical liv-er injury and liver fibrosis by establishing diabetes mer-ged with liver fibrosis rat model (double model). Methods High fat feeding combined with streptozoto-cin (STZ) was used to induce diabetes rat model,and liver fibrosis rat model was induced by CCl4. HE stai-ning was used to observe the rat liver pathological changes, and Western blot and q-PCR were used to detect liver fibrosis related factor genes α-SMA and Collagen Ⅰ expression. Results Compared with con-trol group, diabetes group, liver fibrosis group and double model group all had different levels of liver damage,especially double model group. Rat liver tis-sues of α-SMA and CollagenⅠexpression from differ-ent model groups also increased, especially those from double model group, and significant differences were detected compared to diabetes and liver fibrosis group. Conclusions Diabetes can cause liver damage and in-crease the occurrence and development of CCl4-in-duced rats liver fibrosis, and the mechanism may be related to the formation and/or degradation of extracel-lular matrix.

Effects of Single Dose of Cisplatin on Renal Interstitial Fibrosis Indicators in Rats / 中国药房

OBJECTIVE: To discuss the effects of single dose of cisplatin on renal interstitial fibrosis indicators in rats dynamically. METHODS: 72 SD rats were randomly divided into normal group and cisplatin group, with 36 rats in each group. Normal group and cisplatin group were given equal volume of normal saline and cisplatin 5 mg/kg intraperitoneally on the first day, respectively. Each 6 rats were sacrificed on 8th, 14th, 30th, 50th, 60th, 90th day. The serum levels of blood urea nitrogen (BUN) and creatinine (Cr) were determined, and the degree of renal tubulointerstitial injury and relative area of renal tubulointerstitial fibrosis were evaluated. The expression of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Col Ⅰ) and transforming growth factor β1 (TGF-β1) were determined in renal tissue. RESULTS: Compared with normal group, the serum levels of BUN and Cr, renal tubulointerstitial injury indexes, relative area of renal tubulointerstitial fibrosis, and the expression of α-SMA, Col Ⅰ and TGF-β1 in renal tissue were increased significantly (P<0. 05 or P<0. 01). In cisplatin group, within the 8th-90th days, serum level of BUN in rats had no significant change; serum level of Cr, renal tubulointerstitial injury indexes, renal tubulointerstitial fibrosis, the expression of a-SMA, Col Ⅰ and TGF-β1 in renal tissue increased first and then decreased. CONCLUSIONS: A single dose of clinical dose of cisplatin can induce renal interstitial fibrosis in rats, and its mechanism may be related to the expression of TGF-β1 in renal tissue.

Estimation on Formation Time of Thrombus / 法医学杂志

OBJECTIVES@#To observe the changes of the formation time of venous thrombus in rats, and to provide new ideas and methods for the estimation on thrombus formation time of the forensic cases died from thrombosis.@*METHODS@#Totally 80 rats were randomly divided into 10 groups （0 h, 3 h, 6 h, 12 h, 1 d, 3 d, 1 week, 2 weeks, 3 weeks and 4 weeks after operation）. A vein thrombosis model was established by the "narrow" method. The processes of thrombosis, organization, recanalization and the features of change on hemosiderin and calcium salt were observed by HE stain, Perls stain and Von Kossa stain. The expression changes of CD61, α-SMA and CD34 were observed by immunohistochemical staining technique.@*RESULTS@#Platelets adhered to the exposed blood vessel intima 3 h after operation, and platelet trabeculae were formed by the repeated accumulation of platelets 1 d after operation. The thrombus organization formed through the fibroblasts from vessel wall that grew into the interior of the thrombus 3 d after operation. Endothelial cells covered the surface of thrombus and then the new blood vessels were reformed, and the vessels were reconstructed. The expression of CD61 upregulated at the stages of the thrombus formation （3 h） and thrombus reformation （4 weeks）, and reached the peak 1 d after thrombus formation. The release of hemosiderin and the initial expression of α-SMA were detected 3 d later. Calcium deposit and expression of CD34 were observed 1 week later.@*CONCLUSIONS@#The hemosiderin, calcium salt, CD61, α-SMA and CD34 show time-dependent changing characteristics, which is expected to provide a reference for the estimation on thrombus formation time of the forensic cases died from thrombosis.

The effect of astragaloside on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice / 重庆医学

Objective To study the effect of astragaloside on TGF-β1,SMAD2/3,and α-SMA expression in the kidney tissue of diabetic KKAy mice,and evaluate its potential role in renal interstitial fibrosis.Methods 20 type 2 diabetic KKAy mice were randomly divided into model group and astragaloside group,while 10 male C57BL/6J mice were selected as the control.Astragaloside at 40 mg · kg-1 · d-1 was given when the KKAy mice fed with high-fat diet to 14 weeks old.The mice in the control and model group received normal saline at 40 mg · kg-1 · d-1.Blood glucose meter was used to detect the blood glucose value of each mice at 16th,20th and 24th week.The mice were killed at 24 weeks old and the kidney tissue samples were collected.Pathology morphological changes were observed.Results (1) blood glucose value:cmpared with the control group,the blood glucose value of KKAy mice at 14 week increased significantly,and that of model group also increased significantly at 16th,20th and 24th week (P＜0.05);the blood glucose value of astragaloside group decreased compared with control group (P＜0.05).(2) Morphology of kidney:in the control group,the glomerular and tubular had clear structure,there was no renal interstitial fibrosis;in the model group,the renal glomerular mesangial matrix had broaden,mesangial cell had increased,renal tubular epithelial cell cytoplasm showed vacuole degeneration,renal interstitial inflammatory cell had increaised.In astragaloside group,there were few renal tubular epithelial cell cytoplasm,and there was no obvious fibrosis.(3)TGF-β1,SMAD2/3,and α-SMA expression levels of the kidney issuse:compared with control group,mice in model group up-regulated TGF-β1,SMAD2/3 and α-SMA expression (P＜ 0.05).TGF-β1,SMAD2/3,and α-SMA expression levels in astragaloside group were significantly lower than those in the model group (P＜0.05).There was few phosphorylated SMAD2/3 expression in renal tubular and glomerular nuclei,while that of model group increased (P＜0.01),and compared with model group,that of the astragaloside group decreased (P＜0.05).Conclusion Astragaloside can delay the renal fibrosis process in diabetic mice by influencing the TGF-β/SMADS signaling pathway and down-regulating TGF-β1 and α-SMA expression,thus to relieve renal fibrosis in diabetic mice.

Study of apoptosis in aveolar epithial A549 cells exposed to high-dose paraquat / 中华急诊医学杂志

Objective To We Used high-dose paraquat to induce apoptosis of A549 cells,and observed the expression of E-cadherin,α-SMA,caspase-8,caspase-3 and caspase-9.Then explored the possible mechanisms of apoptosis.Methods A549 cells were treated with various concentrations of PQ (0.1-1.0 mmol/L) for 24 and 48 hours,then observe the cell morphology and measured cell survival rate respectively determined by MTT method.A549 cells were treated with various concentrations of PQ (0.1,0.2,0.3 mmol/L) for 24 hours.The expressions of the mRNAs for α-SMA、E-cadherin,caspase-8,caspase-3 and caspase-9 were analysed by quantitative real-time PCR.Exposed to various concentrations of PQ (0.1,0.2,0.3mmol/L) for 24 hours,we analysed the protein expressions of α-SMA、E-cadherin、caspase-8、caspase-3 and caspase-9 by Western blot analysis.Results Treated with various concentrations of PQ (0.1-1.0mmol/L) for 24 hours,the A549 cells survival rate significantly reduced by MTT method.For 48 hours,the survival rate was lower.So,in subsequent experiments,we used 0.1,0.2 and 0.3 mmol/L concentration of paraquat to A549 cells,trained for 24 hours.After high-dose (0.1,0.2 and 0.3 mmol/L) exposure to PQ,a decrease in E-cadherin was observed while a decrease in α-SMA was also detected of the mRNAs.While the expressions of the mRNAs for caspase-8、caspase-3 and caspase-9 wereincrease.The protein expressions of α-SMA and E-cadherin were decrease with the increase of concen trations by Western blot analysis,and the levels of caspase-8、caspase-3 and caspase-9 were increase.Conclusions After high-dose short-time exposure to PQ,the survival rate of A549 cells is decreased obviously,cell morphology changed significantly,and the expressions of α-SMA and E-cadherin were decrease.But the expressions of caspase-8、caspase-3 and caspase-9 were increase.