BACKGROUND AND AIMS:Real-world evidence on ustekinumab for the treatment of perianal fistulizing Crohn's disease (PFCD) remains limited. This study assessed the long-term efficacy of ustekinumab in PFCD. METHODS:A total of 143 CD patients with perianal fistulas who initiated ustekinumab therapy were enrolled. Clinical remission was defined as the absence of pain and drainage from fistula tracts, as assessed by physicians. Deep remission was defined as concurrent clinical remission and radiological healing. RESULTS:The study population comprised 143 patients, with a mean age of 28.6 years and a mean number of prior perianal surgeries of 2.5. Overall, 77.6 % of patients had complex fistulas, and 65.7 % experienced prior biologic failure. During a median follow-up of 136.0 (108-156) weeks, 22/143 (18.5 %) patients discontinued ustekinumab therapy. Among patients with active PFCD, clinical remission was achieved in 88/119 (73.9 %) patients. Deep remission was achieved in 40 of 100 evaluable patients (40.0 %), typically 32.1 weeks after clinical remission. Multivariate analysis identified prior exposure to biological agents (hazard ratio [HR] = 0.38, 95 % confidence interval [CI]: 0.20-0.73, P = 0.004) and ileocolonic lesions (HR = 0.47, 95 % CI: 0.23-0.96, P = 0.040) as negative predictors of deep remission. Recurrence was rare (6.3 %), with a 2-year recurrence-free survival rate of 93.4 %. CONCLUSIONS:Ustekinumab demonstrates remarkable efficacy in achieving sustained clinical and deep remission in PFCD. Prospective studies are warranted to confirm these findings.

The effect of long intergenic noncoding RNA 01315 (LINC01315) on colorectal cancer has widely been proved. Nevertheless, how LINC01315 functions in the stemness of colorectal cancer and whether LINC01315 exists in colorectal cancer stem-like cell-derived exosomes remain dim, which are thus investigated in this research. CD133(+)/CD44(+) colorectal cancer stem cells were sorted and verified through flow cytometry. Exosomes derived from CD133(+)/CD44(+) colorectal cancer stem cells were collected. The viability, proliferation, stemness and migration of CD133(+)/CD44(+), CD133(-)/CD44(-), and colorectal cancer cells after transfection or the co-culture with exosomes were detected by MTT, colony formation, spheroid, and wound healing assays, respectively. Expressions of LINC01315, BCL-2, Bax, cleaved caspase-3, MMP-9, E-cadherin, and vimentin in cells or exosomes were analyzed using western blot or qRT-PCR. Genes interacted with LINC01315 in colorectal cancer were predicted by bioinformatics analysis. The results showed that LINC01315 was high-expressed in CD133(+)/CD44(+) colorectal cancer stem cells and exosomes. Compared with colorectal cancer cells, the viability, proliferation, stemness, and migration of CD133(+)/CD44(+) cancer cells were stronger, while these of CD133(-)/CD44(-) cancer cells were weaker. Besides, LINC01315 silencing decreased the viability, proliferation, stemness, and migration of CD133(+)/CD44(+) cancer cells, while sh-LINC01315 inhibited the promotive effects of CD133(+)/CD44(+) cancer cell-derived exosomes on the viability, proliferation, stemness, and migration of colorectal cancer cells. LINC01315 was also found to be correlated with DPEP1, KRT23, ASCL2, AXIN2, and DUSP4 in colorectal cancer. In conclusion, colorectal cancer stem cell-derived exosomal LINC01315 promotes the proliferation, migration, and stemness of colorectal cancer cells.

Objective To explore the medication laws of Chinese medicine compounds in fumigation-washing treatment of external hemorrhoid and analyze new prescriptions. Methods The papers on the fumigation-washing treatment of external hemorrhoid with Chinese medicine were retrieved from China Science Periodical Database（ CSPD）, VIP database（ VIP）, and China Biology Medicine disc（ CBMdisc） from the establishment of databases to August 2021. The data analysis platform（ Traditional Chinese Medicine Inheritance Auxiliary Platform） was used to analyze the medication laws of the prescriptions. Results Through the analysis by the platform, 84 Chinese medicine compound prescriptions were screened out, including 111 Chinese medicines. The cold property was dominant in four properties, and the bitter flavor was dominant in five flavors. Liver meridian was the main meridian. The main drugs were qi-regulating drugs and blood-activating drugs. Conclusion The fumigation-washing treatment of external hemorrhoid with Chinese medicine compound preparations takes promoting blood circulation, relieving pain, astringent, and detumescence as the basic principle, which provides certain references for clinical treatment.

Long non-coding RNA long intergenic non-protein coding RNA 01315 (LncRNA LINC01315) has been found to be implicated in various cancers, but its role and functions in colorectal cancer (CRC) remain to be addressed. Data on LINC01315 expression in CRC were gathered using bioinformatics analysis, and cancer stem cells (CSCs) were sorted by aldehyde dehydrogenase (ALDH) assay and flow cytometry. Migration, invasion, and stemness of CSCs isolated from CRC cells after transfection were determined by scratch, Transwell, and sphere-formation assays, respectively. Tumor xenograft model was constructed. Target genes and potential-binding sites were predicted using online databases and further confirmed via dual-luciferase reporter assay. Relative factors expressions were determined via quantitative real-time polymerase-chain reaction and Western blot as needed. LINC01315 was high-expressed in CRC and ALDH+ cells. LINC01315 silencing suppressed the migration, invasion, and sphere formation of CRC cells and tumor growth, and downregulated expressions of CSC molecules (ALDH, cluster of difference 44 (CD44), Prominin, and sex determining region Y-box 2 (SOX2)), Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Vimentin but upregulated E-Cadherin expression. MiR-484 could competitively bind with LINC01315, and LINC01315 silencing promoted miR-484 expression. The level of Delta Like Non-Canonical Notch Ligand 1 (DLK1), the target gene of miR-484, was enhanced by overexpressed LINC01315 yet was suppressed by LINC01315 silencing. Also, DLK1 silencing reversed the effects of downregulated miR-484 on migration, invasion, sphere formation, and CSC molecules expressions in CRC cells. LINC01315 silencing modulated CSC properties and epithelial-to-mesenchymal transition via miR-484/DLK1 axis.

PURPOSE:Targeting cancer stem cells (CSCs) may be an efficacious strategy against cancer. We were devoted to exploring the role of neogambogic acid in characteristics and growth of colorectal CSCs.METHODS:SW480 and HCT116 cells were treated with neogambogic acid at different concentrations and transfected with siDLK1 and pcDNA3.1-DLK1 plasmids. The effect of neogambogic acid on the viability of SW480 and HCT116 cells was assessed by MTT assay. Spheroid formation assay was adopted to enrich colorectal CSCs from SW480 and HCT116 cells. The effect of neogambogic acid on colony number, aldehyde dehydrogenase (ALDH) level, apoptosis and cell cycle of SW480 and HCT116 CSCs was detected by colony formation and flow cytometry assays. The expressions of CSC markers, proliferation marker (proliferation nuclear antigen (PCNA)), apoptosis markers (cleaved caspase-3, cleaved caspase-9), Wnt/β-catenin pathway markers (P-GSK3β, GSK3β, β-catenin and Wnt) and DLK1 were determined by qRT-PCR or Western blot.RESULTS:Neogambogic acid suppressed viability, the spheroid formation ability and the levels of CSC markers in colorectal cancer (CRC) cells, accompanied with inhibition of colony-formation and ALDH level, apoptosis induction and G0/G1 phase arrest. Furthermore, neogambogic acid inhibited expressions of PCNA, P-GSK3β, P-GSK3β/GSK3β, β-catenin and Wnt, but promoted those of cleaved caspase-3, cleaved caspase-9 and GSK3β in colorectal CSCs. DLK1 silencing caused opposite results. DLK1 overexpression abrogated the effects of neogambogic acid on colorectal CSCs.CONCLUSION:Neogambogic acid could be an efficacious natural compound targeting colorectal CSCs via inhibition of DLK1 and Wnt/β-catenin pathway. Thus, neogambogic acid may be an attractive agent against CRC.

目的 通过观察枳术方对慢传输型便秘(STC)大鼠结肠组织中5-羟色胺4受体(5-HT4R)、钙调蛋白(CaM)、肌球蛋白轻链激酶(MLCK)的影响,探讨其作用机制.方法 将大鼠随机分为正常组、模型组、枳术方组及阳性药(普芦卡必利)组.采用复方地芬诺酯片建立STC模型,枳术方组随后灌胃枳术方,阳性药组灌胃普芦卡必利.检测各组大鼠粪便含水量,肠道炭末推进率,结肠组织中5-HT4R、CaM、MLCK的mRNA及蛋白相对表达水平.结果 与正常组相比,模型组大鼠粪便含水量和炭末推进率明显降低(P＜0.05,P＜0.01),结肠组织中5-HT4R、CaM、MLCK的mRNA和蛋白相对表达水平也明显降低(P＜0.01).与模型组相比,枳术方组和阳性药组大鼠粪便含水量和炭末推进率明显升高(P＜0.05),结肠组织中5-HT4R、CaM、MLCK的mRNA和蛋白相对表达水平明显升高(P＜0.05,P＜0.01).枳术方组与阳性药组比较,各项数据差异无统计学意义(P＞0.05).结论 枳术方能改善STC大鼠的便秘症状,可能与上调大鼠结肠组织中5-HT4R表达并激活下游CaM-MLCK信号通路有关.

Fructus has motivation effect on gastrointestinal tract. Hesperidin is extracts of Fructus, and we attempted to prove its effects on improving the gastrointestinal transmission function and determine the possible mechanisms by a loperamide-induced slow transit constipation (STC) model. Constipation phenotypes were measured in rats with Lop-induced constipation after treatment with hesperidin. The amounts and water content of stool were significantly higher in the hesperidin-treated group than the loperamide-induced model group, whereas food intake was maintained at constant levels. Moreover, intestinal transit rate was increased in the treatment group of hesperidin. Histological alteration was detected by H&E staining, we found that the colon smooth muscle cells and neuron cells of the rats were increased, and the infiltration of inflammatory cells was decreased in the hesperidin-treated group compared with the loperamide-induced model group. 5-Hydroxytryptamine (5-HT) receptor4 fluorescence intensity and intracellular-free calcium ions in colon tissue were increased, and relative protein of cAMP/PKA pathway and p-cAMP response component-binding protein (CREB) pathway were upregulated in the hesperidin-treated group compared with the loperamide-induced model group. Further, SMCs from colon tissue of rats were cultured and identified. We found hesperidin could significantly promote tegaserod-induced increase of 5-HTR4 fluorescence intensity, intracellular calcium ions, relative protein of cAMP/PKA pathway and p-CREB pathway, and cell proliferation and inhibit GR113808-induced decrease of 5-HTR4 fluorescence intensity, 5-HTR4 pathway-related proteins (ADCY3, cAMP, PKA, and p-CREB), intracellular calcium ions, and cell proliferation. The analysis of our data suggested that hesperidin could obviously improve the gastrointestinal transmission function in loperamide-induced STC rat model via increasing the 5-HTR4 and intracellular-free calcium ions to enhance the expression of relative protein of cAMP/PKA pathway and p-CREB pathway. Hesperidin could be used in the treatment of STC, and our data not only provide experimental basis for the treatment of STC in hesperidin but also provides a theoretical reference for clinical treatment.

目的:探讨藤黄酸对不同发生途径的人结直肠癌细胞凋亡的影响.方法:采用Annexin V-FITC/PI双染法,检测藤黄酸给药后对HCT116及SW480细胞凋亡的影响.结果:0.25、0.5、1.0μg/mL浓度的藤黄酸对HCT116细胞的总凋亡率分别为25.98％、31.08％、37.94％,各浓度的藤黄酸对HCT116细胞的总凋亡率与空白对照组相比,差异有统计学意义(P＜0.05),并且呈浓度依赖性;0.25、0.5、1.0 μg/mL浓度的藤黄酸对SW480细胞的总凋亡率分别为8.36％、35.05％、38.93％,各浓度的藤黄酸对SW480细胞的总凋亡率与空白对照组相比,差异有统计学意义(P＜0.05),并且呈浓度依赖性.结论:藤黄酸对人结肠癌细胞有较好的促凋亡作用.

BACKGROUND/AIM:The aim of the present study was to investigate the efficacy of an ethanolic extract of gamboge (EEG), a traditional Chinese medicine (TCM), both in vitro on colon cancer cells and in vivo in an orthotopic mouse model of human colon cancer.MATERIALS AND METHODS:The in vitro cytotoxicity of EEG on colon cancer cells was determined with the CCK8 proliferation assay and the Annexin V-PE/7-AAD apoptosis assay. Efficacy of EEG in vivo was evaluated in an orthotopic mouse model of human colon cancer implated with the green fluorescent protein-expressing human colon cancer cell line SW480-GFP. The tumor-bearing mice were treated with vehicle (0.2 ml/dose normal saline, po, daily), irinotecan (50 mg/kg/dose, ip, twice a week), 5-FU (15 mg/kg/dose, ip, every other day) as positive controls or EEG at doses of 12.5, 25 and 50 mg/kg/dose, po, daily. Real-time fluorescence imaging was performed to determine tumor inhibition in each treated group compared to the untreated controls. The protein expression of β-catenin, MMP-7, cyclin D1 and E-cadherin in the tumors was analyzed by immunohistochemistry.RESULTS:EEG significantly induced proliferation inhibition and apoptosis of SW480 colon cancer cells in vitro in a dose-dependent manner. Tumor growth in the colon-cancer orthotopic model was significantly inhibited by irinotecan, 5-FU and all three doses of EEG. The efficacy of EEG was comparable to irinotecan and 5-FU. Irinotecan, 5-FU and 50 mg/kg EEG significantly decreased the protein expression of β-catenin and MMP-7. Cyclin D1 expression was decreased and E-cadherin expression was increased by irinotecan, 5-FU and all three doses of EEG.CONCLUSION:The present study demonstrates anti-tumor efficacy of EEG on colon cancer both in vitro and in vivo through inducing proliferation inhibition and apoptosis of SW480 colon cancer cells and inhibiting tumor growth, respectively. EEG exerts anti-tumor activity at least partly via down-regulation of the Wnt/β-catenin signaling pathway.

Objective To explore the therapeutic effects of soluble cytokines secreted by mesenchymal stem cells (MSCs) on acute liver failure (ALF).Methods MSCs isolated from Sprague-Dawley rats were determined by FACS analysis.Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray.SD rats were divided into 3 groups:(1) ALF + dulbecco's modified eagle medium (DMEM) group:1 ml DMEM was injected into SD rats after D-Gal administration;(2) ALF + MSCs group:1 ml MSCs (1 × 106) was injected into SD rats after D-Gal administration;(3) ALF + MSCs-CM group:1 ml MSCs-CM was injected into SD rats after D-Gal administration.Biochemical indicators,survival rate,histology and inflammatory factors were studied.Exogenous recombinant rat IL-10,antirat IL-10 antibody and AG490 (STAT3 signaling pathway inhibitor) were administrated to explore the therapeutic mechanism of MSCs-CM.Results The respective serum biochemical indexes of ALF + DMEM group,ALF + MSCs group,and ALF + MSCs-CM group were:ALT (1 709.8 ± 372.1,865.5 ± 52.8,964.7 ± 414.6 U/L),AST (4234.0 ± 807.3,2440.8 ± 511.9,2739.8 ± 587.3 U/L),andTBil (79.3 ± 10.9,43.8 ± 7.0,61.2 ± 6.7 μg/L).The survival rates of the three groups were 10.0％,80.0％,and 70.0％,respectively.The levels of inflammatory factors in each group were IFN-γ (69.8 ± 4.7,46.4 ± 4.3,54.6 ± 2.4pg/ml),IL-1β (58.5 ± 7.6,40.5 ± 6.9,44.1 ± 6.0pg/ml),IL-6 (71.9 ± 16.1,38.4 ± 7.7,45.3 ± 9.0),and IL-10 (38.3 ± 6.0,75.4 ± 11.1,59.6 ± 11.9 pg/ml).Protein microarray results suggested that MSCs-CM expresses a variety of inflammatory-related cytokines,with IL-10 levels being most pronounced.IL-10 (ALT 1 126.9 ± 419.3 U/L,AST2370.8 ± 561.2 U/L) alone significantly reduced transaminase levels compared with ALF group (ALT 1 709.8 ± 372.1 U/L,AST 4234.0 ± 807.3 U/L),while anti-IL-10 antibody (ALT 1 568.5 ± 325.4 U/L,AST4043.7 ± 819.0 U/L) neutralized the therapeutic effect of MSCs-CM (ALT 964.7 ± 414.6 U/L,AST 2 739.8 ± 587.3 U/L).IL-10 could significantly increase the level of pSTAT3 in ALF rats (0.93 ± 0.03 vs 0.68 ± 0.01),while STAT3 inhibitor AG490 (0.84 ± 0.04) could decrease the expression of pSTAT3 and reverse the therapeutic effect of IL-10.Conclusion The factors released by MSCs,especially IL-10,have the potential therapeutic effect on ALF,and STAT3 signaling pathway may mediate the anti-inflammation effects of IL-10.

BACKGROUND:Mesenchymal stem cells (MSCs) transplantation has been proven to have therapeutic potential for acute liver failure (ALF). However, the mechanism remains controversial. Recently, modulation of inflammation by MSCs has been regarded as a crucial mechanism. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects in ALF.METHODS:MSCs isolated from Sprague-Dawley rats were identified by fluorescence-activated cell sorting analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. MSCs and MSCs-CM were transplanted into rats with D-galactosamine-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, anti-rat IL-10 antibody, and AG490 (signal transducer and activator of transcription 3 [STAT3] signaling pathway inhibitor) were administered to explore the therapeutic mechanism of MSCs-CM. Statistical analysis was performed with SPSS version 19.0, and all data were analyzed by the independent-sample t-test.RESULTS:There are statistical differences of the survival curve between ALF+MSCs group and ALF+Dulbecco's modified Eagle's medium (DMEM) group, as well as ALF+MSCs-CM group and ALF+DMEM group (all P < 0.05). Serum alanine aminotransferase (ALT) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (865.53±52.80 vs. 1709.75±372.12 U/L and 964.72±414.59 vs. 1709.75±372.12 U/L, respectively, all P < 0.05); meanwhile, serum aspartate aminotransferase (AST) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (2440.83±511.94 vs. 4234.35±807.30 U/L and 2739.83±587.33 vs. 4234.35±807.30 U/L, respectively, all P < 0.05). Furthermore, MSCs or MSCs-CM treatment significantly reduced serum interferon-γ (IFN-γ), IL-1β, IL-6 levels and increased serum IL-10 level compared with DMEM (all P < 0.05). Proteome profile analysis of MSCs-CM indicated the presence of anti-inflammatory factors and IL-10 was the most distinct. Blocking of IL-10 confirmed the therapeutic significance of this cytokine. Phosphorylated STAT3 was upregulated after IL-10 infusion and inhibition of STAT3 by AG490 reversed the therapeutic effect of IL-10.CONCLUSIONS:The factors released by MSCs, especially IL-10, have the potential for therapeutic recovery of ALF, and the STAT3 signaling pathway may mediate the anti-inflammatory effect of IL-10.