Exploring the Potential Mechanism of Guchang Zhixie Wan for Treating Ulcerative Colitis by Comprehensive Network Pharmacological Approaches and Molecular Docking Validation as Well as Cell Experiments.

Guchang Zhixie Wan (GZW) is a commonly used Chinese medicine for the treatment of ulcerative colitis (UC). This research explored the potential pharmacological mechanism of GZW in UC. The active ingredients, potential targets, and UC-related genes of GZW were retrieved from public databases. The pharmacological mechanisms including key components, potential targets and signal pathways were determined through bioinformatics analysis. The results of this study were verified through virtual molecular docking and cell experiments. Network analysis revealed that 26 active GZW compounds and 148 potential GZW target proteins were associated with UC. Quercetin, kaempferol and ß-sitosterol were identified as the core active ingredients of GZW. IFNG, IL-1A, IL-1B, JUN, RELA, and STAT1 were indicated as key targets of GZW. These key targets have a strong affinity for quercetin, kaempferol, and ß-sitosterol. GO and KEGG enrichment analysis showed that GZW target proteins are highly enriched in inflammatory, immune, and oxidative stress-related pathways. This study confirmed the therapeutic effect and revealed potential molecular mechanism of GZW on UC. And the protective effects of GZW on inflammatory bowel disease pathway were also revealed through STAT3/NF-κB/IL-6 pathway. The findings of this study enhanced our understanding of GZW in the treatment of UC and provided a feasible method for discovering potential drugs from traditional Chinese medicine formulations.

Co-Culture of Adipose-Derived Stem Cells and Chondrocytes With Transforming Growth Factor-Beta 3 Promotes Chondrogenic Differentiation.

Tissue engineering cartilage is a promising strategy to reconstruct the craniofacial cartilaginous defects. It demands plenty of chondrocytes to generate human-sized craniofacial frameworks. Partly replacement of chondrocytes by adipose-derived stem cells (ADSCs) can be an alternative strategy.The study aimed at evaluating the chondrogenic outcome of ADSCs and chondrocytes in direct co-culture with transforming growth factor-beta (TGF-ß3). Porcine ADSCs and chondrocytes were obtained from abdominal wall and external ears. Four groups: ADSCs or chondrocytes monocultured in medium added with TGF-ß3; ADSCs and ACs co-cultured with or without TGF-ß3. Cell growth rate was performed to evaluate the cell proliferation. Morphological, histologic and real-time polymerase chain reaction analysis were performed to characterize the chondrogenic outcome of pellets. ADSCs had favorable multi-lineage differentiation potential. Further, when ADSCs were co-cultured with chondrocytes in medium added with TGF-ß3, the cell proliferation was promoted and the chondrogenic differentiation of ADSCs was enhanced. We demonstrate that pellet co-culture of ADSCs and chondrocyte with TGF-ß3 could construct high quantity cartilages. It suggests that this strategy might be useful in future cartilage repair.

[Factors affecting the safety and efficacy of peroral endoscopic myotomy for achalasia].

OBJECTIVE: To identify the factors that affect the safety and efficacy of peroral endoscopic myotomy (POEM) for treatment of achalasia. METHODS: Data of consecutive patients undergoing POEM for confirmed achalasia between December, 2010 and December, 2015 were collected, including the procedure time, approach of tunnel entry incision, approach of myotomy, complications and follow-up data. RESULTS: Among the total of 439 patients enrolled, the overall complication rate was 28.7% (126/439). Treatment success (Eckardt score≤3) was achieved in 94.5% of 364 patients followed up for a median of 6 months (1-48 months), and the mean score was reduced significantly from 6.7∓1.5 before treatment to 1.2∓1.1 after the treatment (P<0.05). Logistic regression revealed that the year when POEM was performed and the approach of entry incision were two significant factors contributing to complications: with the year 2015 as the reference, the odds ratio (OR) was 9.454 (95% CI: 2.499-35.76) for the years before 2011, 2.177 (95% CI: 0.794-5.974) for 2012, 3.975 (95% CI: 1.904-8.298) for 2013, and 1.079 (95% CI: 0.601-1.940) for 2014; with the longitudinal entry incision as the reference, the OR was 0.369 (95% CI: 0.165-0.824) for inverted T entry incision and 0.456 (95% CI: 0.242-0.859) for transverse entry incision. The approach of myotomy was the significantly associated with symptomatic relapse: with full-thickness myotomy combined with indwelling an anti-reflux belt as the reference, the OR was 0.363 (95% CI: 0.059-2.250) for gradual full-thickness myotomy, 2.137 (95% CI: 0.440-10.378) for circular muscle myotomy, and 4.385 (95% CI: 0.820-23.438) for circular muscle myotomy in combination with balloon shaping; the recurrence rate was 0 with a full-thickness myotomy. CONCLUSION: The complication rates of POEM appears to decrease over time, and an inverted T entry incision is the best choice for controlling the complications. Gradual full-thickness myotomy is an excellent approach for treatment of achalasia in terms of the relapse rate, procedure time and the incidence of reflux esophagitis.

[Preliminary Investigation of Medicinal Plant Resources in Kangle County, Gansu Province].

OBJECTIVE: To study the medicinal plant resources and their diversity in Kangle County, Gansu Province, and to provide scientific basis on utilization and protection of the medicinal plant resources of the county. METHODS: By field survey, sample collection, taxonomic identification and data verification methods. RESULTS: There were 258 species, 65 families in existing medicinal plants, of which 43 species, 39 genera and 24 families were national protection medicinal plants. Dominant families were mainly Asteraceae and Rosaceae. In this area,the plants used whole herbs and roots ( or rhizomes) as medicinal materials represented 40.31% and 25.19% respectively, and antipyretic and rheumatism medicine accounted for 28.68% and 12.79% respectively. 12 medicinal plants were cultivated and the cultivated area was 3,000 hectares. CONCLUSION: However, the reserves of most medicinal plants are less enough and the resources are diminishing increasingly in recent years. So we should accelerate the research progress as well as developing and utilizing rationally on the premise of protection.

[Screen and validation of differentially expressing genes related to silicotic pulmonary fibrosis in rats].

OBJECTIVE: To screen the differentially expressing genes between silicotic lung tissue and normal lung tissue, to identify the differentially expressing genes of matrix metalloproteinase-12 (MMP-12) and Cathepsin E and to explore the roles of those genes in silicosis development. METHODS: Thirty male SD rats were divided randomly into two groups: control group (6 rats) and exposure group (24 rats) which was exposed to SiO2 by intra-tracheal perfusion. On the 30 th, 60 th and 90 th days after exposure, 8 rats in model group and 2 rats in control group were executed and the lung tissues were obtained. The morphologic changes of lung tissues were observed with HE staining and VG staining under a light microscope. The gene microarrays were used to identify differentially expressing genes of lung tissues in rats exposed to SiO2 for 60 days. Two significantly up-regulated genes, MMP-12 and Cathepsin E, were validated using RT-PCR, immunohistochemistry and Western Blot assay. RESULTS: A total of 338 differentially expressing genes were identified from the 26 962 genes between silicotic rats and normal rats, including 267 up-regulated genes and 71 down-regulated genes. The results of RT-PCR showed that in the lung tissues of exposure group on the 30 th, 60 th and 90 th days, the mRNA expression levels of MMP-12 were 4.306, 5.338, 6.713 times higher than those in the control group, the mRNA expression levels of Cathepsin E were 1.434, 2.974, 3.889 times higher than those in the control group, respectively. The results of immunohistochemical showed that in the lung tissues of exposure group on the 30th, 60th and 90th days, the mRNA expression levels of MMP-12 were 1.435, 1.746, 2.069 times higher than those in the control group, the mRNA expression levels of Cathepsin E were 1.372, 1.663, 2.103 times higher than those in the control group, respectively. The results of immunohistochemical showed that in the lung tissues of exposure group on the 30th, 60th and 90th days, the expression levels of MMP-12 protein were 1.214, 1.531, 1.959 times higher than those in the control group, the expression levels of Cathepsin E protein were 1.262, 1.828, 1.907 times higher than those in the control group, respectively. Compared with the control group, the mRNA and protein expression levels of MMP-12 and Cathepsin E in lung tissues of exposure group were significantly up-regulated (P < 0.05). CONCLUSION: The differentially expressing genes in rat lung tissues screened by gene chip were validated, which suggested that a complex gene regulatory network may be contributed to occurrence of silicosis. MMP-12 and Cathepsin E genes may be involved in the development of silicotic pulmonary fibrosis by degrading the basement membrane of alveolar wall and participating in the immune response.

[Effects of silicotic alveolar macrophages exposed to SiO2 on the expression of type III collagen and type III procollagen in human lung fibroblasts].

UNLABELLED: Effects of silicotic alveolar macrophages exposed to SiO2 on the expression of type III collagen and type OBJECTIVE: To study the effects of supernatant of alveolar macrophages (AM) exposed to SiO2 on the expression of type III collagen and type III procollagen in human lung fibroblasts (HELF) and the intervention effects of anti-TGF-beta1 antibody. METHODS: AMs collected from a silicotic by bronchoalveolar lavage were divided into 2 parts, one part was exposed to SiO2 and other part served as control. The supernatant was obtained from AMs cultured for 18 h. HELF were divided into (1) exposure group, which was added with supernatant from AMs exposed to SiO2; (2) control group, which was added with the supernatant from AMs not exposed to SiO2; (3) blank control group, which was added with DMEM; (4) exposure group plus anti-TGF-beta1 antibody (10 microg/ml); (5) control group plus anti-TGF-beta1 antibody (10 microg/ml). (1)-(3) groups were cultured for 6, 12, 18, 24, 36, 48h, respectively. (4)-(5) groups were cultured for 18, 24, 36, respectively. Immunocytochemical test and Western blot assay were used to detect pC III expression levels in HELF and C III expression levels in the supernatant of HELF culture, respectively. RESULTS: The pC III expression levels of exposure group were 0.1423 +/- 0.0107, 0.1624 +/- 0.0011, 0.1925 +/- 0.0050, 0.2421 +/- 0.0097 and 0.2103 +/- 0.0103, respectively, which were significantly higher than those (0.1212 +/- 0.0079, 0.1414 +/- 0.0058, 0.1620 +/- 0.0081, 0.1965 +/- 0.0103, 0.1715 +/- 0.0116) of control group (P < 0.05 or P < 0.01). The C III levels of exposure group were (0.2559 +/- 0.0061, 0.3249 +/- 0.0110, 0.4171 +/- 0.0193, 0.5441 +/- 0.0452, 0.4751 +/- 0.0252), respectively, which were significantly higher than control group (0.2296 +/- 0.0121, 0.2778 +/- 0.0116, 0.3367 +/- 0.0269, 0.3722 +/- 0.0214). The pC III and C III expression levels of exposure plus anti-TGF-beta1 antibody group were significantly lower than those of control plus anti-TGF-beta1 antibody group (P < 0.05 or P < 0.01). CONCLUSION: AMs exposed to SiO2 can induce the elevated pC IIII and C III expression levels in HELF by TGFbetaP1 to some extent.

[Effect of human silicotic alveolar macrophages on expression of matrix metalloproteinase, tissue inhibitor of metalloproteinase and collagen in human lung fibroblasts].

OBJECTIVE: To study the effect of culture supernatant of alveolar macrophage alveolar macrophages (AM) stimulated by SiO2 on the expression of matrix metalloproteinases (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagen of fibroblast human embryonic lung fibroblasts (HELF) in the development of silicosis fibrosis. METHODS: AMs were collected from a silicotic patient by bronchoalveolar lavage and exposed to SiO2, cultured human embryo lung fibroblast were allocated into a treated group, a control group, a positive group, and a blank group. HELF was incubated with the cultured supernatant of AMs for 6, 12, 18, 24, 36, 48 h. Immunocytochemical and Western blot technology were used to detect MMP-1 and TIMP-1 expressions in HELF and collagen expression in supernatant of HELF respectively. RESULTS: The supernatant of AM exposed to SiO2 significantly decreased the expressions of MMP-1 (0.0605 +/- 0.0201, 0.0519 +/- 0.0117, 0.0412 +/- 0.0105 and 0.0213 +/- 0.0106 in the treated group at 18, 24, 36 and 48 h) compared with the control group and the blank group (P < 0.05, P < 0.01) but stimulated expressions of TIMP-1 and collagen (P < 0.05, P < 0.01). The ratio of TIMP-1 to MMP-1 increased. The ratio of TIMP-1 to MMP-1 was positively correlated with the expression of collagen III (r = 0.88, P < 0.01). CONCLUSION: Through AM mediation SiO2 can accelerate the expression of TIMP-1 and collagen, and inhibit the expression of MMP-1. The imbalance between the expression of TIMP-1 and that of MMP-1 is related with the abnormal increase in collagen III.

[Effect of SiO(2) on expression of platelet derived growth factor in human silicotic alveolar macrophages and human embryonic lung fibroblasts].

OBJECTIVE: To study the effect of SiO(2) on the expression of platelet derived growth factor (PDGF) in human silicotic alveolar macrophages (AM) and human embryonic lung fibroblasts (HELF). METHODS: Human alveolar macrophages were collected from a silicotic patient by bronchoalveolar lavage and exposed to SiO(2) for 3, 6, 12, 18, 24 and 36 h. The cultured supernatant at 24 h was incubated with human embryonic lung fibroblasts for 6, 12, 18, 24, 36 and 48 h. The immunocytochemistry and Western blot were used to detect the level of expression of PDGF in lung fibroblasts and their supernatant respectively. (3)H-proline was used to detect the synthesis and secretion of collagen in HELF. RESULTS: The expression of the PDGF in the supernatant of alveolar macrophages exposed to SiO(2) increased significantly and reached the peak at 24 h (average optical density: 0.282 +/- 0.019 vs 0.214 +/- 0.014, P < 0.01) with ELISA. The expression of PDGF in lung fibroblasts and their supernatant increased at different time (6, 12, 18, 24, 36 and 48 h) with immunocytochemistry and Western blot respectively when incubated with the cultured supernatant of silicotic AM exposed to SiO(2). The expression of PDGF was significantly different from the control group (P < 0.05). The synthesis and secretion of collagen in FB were increased markedly when incubated with the cultured supernatant of AM stimulated by SiO(2) compared with the control group. CONCLUSION: SiO(2) may affect the expression of PDGF and synthesis of collagen through AM mediation and participate in the formation of lung fibrosis.

[Construction and primary identification of a naive Fab fragment phage library].

OBJECTIVE: To construct a naive human Fab fragment phage display library, provide a platform for human antibody preparation and a new therapy for the malignant tumors. METHODS: Peripheral blood lymphocytes were isolated from 200 ml blood, which was obtained from a healthy blood donor. The heavy chain Fd fragments and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E. coli XL1-Blue. The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody of Fabs. The phagemids abstracted from amplified E. coli were cut with endonucleases such as Sac I, Xba I, Spe I and Xho I and amplified by PCR to monitor the insertion of the light chain or heavy chain Fd genes. Human albumin and interleukin-2 were utilized as antigens to conduct respectively three rounds of panning to the original Fab antibody library. RESULTS: By combination of light chain and heavy chain genes, an antibody library containing 1.2 x 10(6) clones was obtained, and both the cutting of enzymes and PCR showed that there were the light chain or heavy chain Fd genes of the phagemids. After the original antibody library having been panned respectively by two kinds of antigen proteins, it gained enrichment in different degree. CONCLUSION: Utilizing the technology of phage surface display, special antibody can be gained from the human naive Fab phage display library, which can be used as a new therapy for tumors.

[Effect of SiO2 on the expression of MMP-9 and TIMP-1 in human alveolar macrophages in vitro].

OBJECTIVE: To study the effect of SiO(2) on the expression of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) in human alveolar macrophages (AMs) associated with the pathogenesis of silicotic fibrosis. METHODS: AMs were collected from a silicotic patient by bronchoalveolar lavage, and exposed to SiO(2) (50 microg/ml), and cultured in DMEM without serum for different time (2, 6, 12, 18, 24, 36 h). Immunocytochemical method was used to detect the level of expression of MMP-9 and TIMP-1 in AMs. RESULTS: The expression of MMP-9 in AMs exposed to silica was up-regulated, and reached the peak at 18 h [average optical density: (0.440 +/- 0.021) vs (0.390 +/- 0.011), P < 0.05]. After that, the expression reduced markedly. However, the expression of TIMP-1 of AMs were not significantly different from the control group [average optical density: (0.175 +/- 0.019) vs (0.162 +/- 0.044), P > 0.05]. CONCLUSION: SiO(2) could induce up-expression of MMP-9 in AMs. Degradation of basement membrane by MMP-9 produced by AMs at early stage of lung injury may associate with the immigration of various cells including lung fibroblasts into the injured region.

[Effect of human silicotic alveolar macrophages on the expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 in human lung fibroblasts].

OBJECTIVE: To study the effect of the cultured supernatant of human silicotic alveolar macrophages (AM) on the expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in human lung fibroblasts (FB). METHODS: Human alveolar macrophages were collected from a silicotic patients by bronchoalveolar lavage and exposed to SiO(2), then the cultured supernatant were incubated with human fetal lung fibroblasts for 6, 12, 18, 24, 36, 48 h. The immunocytochemical method was used to detect the level of expression of MMP-1 and TIMP-1 in lung fibroblasts. RESULTS: The expression of MMP-1 in FB in 24 h incubation was lower in cultured supernatant of silicotic AM unexposed to SiO(2) than in blank control [integrated OD (IOD)]: 0.103 +/- 0.014 vs 0.133 +/- 0.023), while the expression of TIMP-1 was higher (IOD: 0.108 +/- 0.012 vs 0.065 +/- 0.006). The expression of MMP-1 in FB in cultured supernatant of AM exposed to SiO(2) for 24 h was further decreased (IOD: 0.062 +/- 0.008 vs 0.133 +/- 0.023), while that of TIMP-1 was further increased (IOD: 0.143 +/- 0.015 vs 0.065 +/- 0.006). CONCLUSION: SiO(2) may affect the expression of MMP-1 and TIMP-1 system through AM mediation and participate in the formation of lung fibrosis.