Tong-Xie-Yao-Fang induces mitophagy in colonic epithelial cells to inhibit colitis-associated colorectal cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Based on the core pathogenesis of hepatosplenic disorder and qi transformation disorder in ulcerative colitis, Tong-Xie-Yao-Fang (TXYF) is a classical traditional Chinese medicine commonly used to treat ulcerative colitis. Our study revealed that it has the potential to prevent colitis-associated colorectal cancer, which embodies the academic concept in traditional Chinese medicine of treating the disease before it develops. AIM OF THE STUDY: This study was aimed at evaluating the therapeutic role of TXYF in treating colitis-associated colorectal cancer and exploring its possible underlying mechanisms. MATERIALS AND METHODS: A colitis-associated colorectal cancer model was established in mice using azoxymethane and dextran sulfate sodium salt to examine the therapeutic effect of TXYF. The mouse body weights were observed. Hematoxylin-eosin staining was used to evaluate mouse colon histopathology. Colon cancer cells and colon epithelial cells were used to explore the potential molecular mechanisms. The proliferation and apoptosis of cells were detected by CCK8 and cell colony assays, flow cytometry and western blotting. The epithelial-mesenchymal transition (EMT) and mitophagy markers were examined by immunohistochemistry, western blotting, quantitative real-time PCR and immunofluorescence staining. RESULTS: TXYF inhibited the tumorigenesis of mice with colitis-associated colorectal cancer and the growth of inflammatory colon cells. TXYF induced mitophagy in colon cancer cells through the PTEN-induced putative kinase 1 (PINK1)/Parkin pathway to reverse EMT, which was consistent with the results in mice with colitis-associated colorectal cancer. CONCLUSIONS: The results of the present study demonstrated that TXYF effectively inhibited the progression of colitis-associated colorectal cancer through the PINK1/Parkin pathway, which provides new evidence for prevention strategies for this disease.

Learning curve for flexible bronchoscope-guided orotracheal intubation for anesthesiology residents: A cumulative sum analysis.

BACKGROUND: Endotracheal intubation with a flexible bronchoscope is a well-recognized airway management technique that anesthesiologists must master. Skill acquisition and knowledge must reach an appropriate level before trainees perform independent practice on patients. There are a paucity of evidence-based outcome measures of trainee competence in performing flexible bronchoscopy. The objectives of this study were to 1) construct a learning curve for flexible bronchoscope-guided orotracheal intubation for anesthesiology residents using the CUSUM method and 2) determine the number of procedures required to achieve proficiency. METHODS: This study included 12 first-year anesthesiology residents with no previous experience with flexible bronchoscopic intubation. Trainees attended theoretical and simulation training and performed flexible bronchoscope-guided orotracheal intubation in adult patients with normal airways under general anesthesia. Number of intubation attempts, intubation success rate, time to intubation, and incidence of dental and mucosal injuries were recorded. The cumulative sum (CUSUM) method was used to evaluate the learning curve of flexible bronchoscope-guided orotracheal intubation. RESULTS: Trainees performed flexible bronchoscope-guided orotracheal intubation on 364 patients. First-attempt intubation success occurred in 317 (87.1%) patients. Second-attempt intubation success occurred in 23 (6.3%) patients. Overall, the flexible bronchoscope-guided orotracheal intubation success rate was 93.4% (range, 85.3% to 100%). The mean number of orotracheal intubation procedures per trainee was 31 ± 5 (range, 23 to 40). All trainees crossed the lower decision boundary (H0) after 15.1 ± 5.6 procedures (range, 8 to 25 procedures). There was a significant decrease in median intubation time [39s (IQR: 30, 50) vs. 76s (IQR: 54, 119)] (P < 0.001) after crossing the lower decision boundary (H0) compared to before. There were no dental, mucosa, arytenoid or vocal cord trauma events associated with intubation. CONCLUSIONS: Learning curves constructed with CUSUM analysis showed that all trainees (anesthesiologist residents) included in this study achieved competence (intubation success rates ≥ 80%) in flexible bronchoscope-guided orotracheal intubation. Trainees needed to perform 15 (range, 8 to 25) procedures to achieve proficiency. There was wide variability between trainees. TRIAL REGISTRATION: Trial registration: Chinese Clinical Trial Register, ChiCTR 2000032166.

Astragaloside IV promotes keratinocyte proliferation and migration through upregulating lncRNA H19 recruited ILF3 to enhance the stability of CDK4 mRNA.

Skin is the first line of the body to resist pathogen invasion. A potentially fatal infection may result from problems with wound healing. Small molecule drugs like astragaloside IV (AS-IV) show pro-healing activities, but the mechanisms are not fully understood. Using real-time quantitative PCR and a western blot assay, the amount of gene expression was evaluated. The proliferation and migration of keratinocytes were determined by MTS and wound healing assay, respectively. The binding of lncRNA H19 to RBP protein ILF3 and the binding of ILF3 protein to CDK4 mRNA were confirmed by RNA immunoprecipitation. Treatment with AS-IV enhanced the expression of lncRNA H19, ILF3, and CDK4 and improved the proliferation and migration of keratinocytes HaCaT. Additionally, apoptosis of keratinocytes was attenuated by AS-IV. Further studies showed that both lncRNA H19 and ILF3 were important for AS-IV-mediated keratinocyte growth and migration. In addition, lncRNA H19 recruited ILF3 to increase CDK4 mRNA level and enhanced cell proliferation. We discovered a lncRNA H19/ILF3/CDK4 axis that is activated by AS-IV to promote keratinocyte migration and proliferation. These results elucidate the mechanism of action of AS-IV and justify its application in further application in wound healing treatment.

Impact of adequate lymph nodes dissection on survival in patients with stage I rectal cancer.

Background and Aims: The NCCN guidelines recommended an assessment of ≥ 12 lymph nodes (LN) as an adequate LN dissection (LND) for rectal cancer (RC). However, the impact of adequate LND on survival in stage I RC patients remained unclear. Thus, we aimed to compare the survival between stage I RC patients with adequate and inadequate LND. Methods: A total of 1,778 stage I RC patients in the SEER database from 2010 to 2017 treated with radical proctectomy were identified. The association between ≥ 12 LND and survival was examined using the multivariate Cox regression and the multivariate competing risk model referenced to < 12 LND. Results: Stage I RC patients with ≥ 12 LND experienced a significantly lower hazard of cancer-specific death compared with those with < 12 LND in both multivariate Cox regression model (adjusted HR [hazard ratio], 0.44, 95% CI, 0.29-0.66; P < 0.001) and the multivariate competing risk model (adjusted subdistribution HR [SHR], 0.45, 95% CI, 0.30-0.69; P < 0.001). Further, subgroup analyses performed by pT stage. No positive association between ≥ 12 LND and survival was found in pT1N0 RC patients (adjusted HR: 0.62, 95%CI, 0.32-1.19; P = 0.149; adjusted SHR: 0.63, 95%CI, 0.33-1.20; P = 0.158), whereas a positive association between ≥ 12 LND and survival was found in pT2N0 RC patients (adjusted HR: 0.35, 95%CI, 0.21-0.58; P < 0.001; adjusted SHR: 0.36, 95%CI, 0.21-0.62; P < 0.001). Conclusions: The long-term survival benefit of adequate LND was not found in pT1N0 but in pT2N0 RC patients, which suggested that pT2N0 RC patients should be treated with adequate LND and those with inadequate LND might need additional therapy.

Bone marrow mesenchymal stem cells-derived exosomes containing miR-539-5p inhibit pyroptosis through NLRP3/caspase-1 signalling to alleviate inflammatory bowel disease.

BACKGROUND: Exosomes derived from bone mesenchymal stem cells (BMSCs) are potential candidates for inflammatory bowel disease (IBD) treatment. The present study investigated the therapeutic effect and potential mechanism of BMSCs-derived exosomes on pyroptosis in IBD. METHODS: We induced IBD in mice and cell models through dextran sulfate sodium (DSS) and LPS, respectively. The mRNA and protein expression levels were assessed by qRT-PCR, Western blotting, IF and IHC. The concentrations of IL-1ß, IL-18 and TNFα were assessed using ELISA. ROS levels were determined using DCFH-DA staining. Cell proliferation of mIECs was analysed using an MTT assay. In addition, a flow cytometry assay was performed to detect pyroptosis. Finally, the binding relationship between miR-539-5p and NLRP3 was verified by a dual luciferase reporter gene assay. RESULTS: Our results revealed that intraperitoneal injection of BMSCs-derived exosomes inhibited DSS-induced pyroptosis as well as IBD symptoms in mice. In addition, BMSCs-derived exosome treatment suppressed pyroptosis, ROS levels and the concentrations of proinflammatory cytokines (IL-1ß, IL-18 and TNFα) in LPS-treated mIECs in a miR-539-5p-dependent manner. Further research found that miR-539-5p suppressed NLRP3 expression in mIECs by directly targeting NLRP3. As expected, pyroptosis in LPS-treated mIECs was significantly reduced by NLRP3 knockdown. In addition, NLRP3 silencing restored the inhibitory effect of exosomes derived from BMSCs transfected with miR-539-5p inhibitor on pyroptosis in LPS-treated mIECs. CONCLUSION: The present study demonstrated that BMSCs-derived exosomal miR-539-5p suppresses pyroptosis through NLRP3/caspase-1 signalling to inhibit IBD progression.

Effects of wogonin on the growth and metastasis of colon cancer through the Hippo signaling pathway.

Wogonin is an effective component of Scutellaria baicalensis Georgi, which exhibits anti-tumor activity. The aim of this study was to explore the effects of wogonin on colon cancer (CC). Human CC cell lines, SW480 and HCT116, were cultured, and MTT assay was performed to detect cell survival. RT-qPCR and Western blotting were used to measure mRNA and protein expression, respectively. The migration and invasion abilities of the CC cells were determined by a transwell assay. Immunofluorescence staining was performed to determine the localization of IRF3. Xenograft mice were used to investigate the effects of wogonin on CC in vivo. Wogonin inhibited the survival and metastasis of CC cells. In addition, wogonin suppressed epithelial-mesenchymal transition (EMT). Furthermore, the protein expression of YAP1 and IRF3 was downregulated, and p-YAP1 was upregulated after wogonin treatment. Wogonin also suppressed IRF3 expression in the nuclei of CC cells and overexpression of YAP1 reversed the effects of wogonin in CC cells. Finally, wogonin inhibited the tumor growth in the mice and overexpression of YAP1 reversed the wogonin effects. Thus, these results showed that wogonin relieved the carcinogenic behaviors and EMT of CC cells via the IRF3-mediated Hippo signaling pathway.

Atractylenolide I inhibits antibiotic-induced dysbiosis of the intestinal microbiome.

BACKGROUND: Atractylenolide I (AT-I) is an active component that is isolated from Rhizoma Atractylodis macrocephalae and it exerts anti-apoptotic, anti-oxidant, and anti-coagulant properties, and has been widely applied in the treatment of cardiovascular and cerebrovascular diseases in China. This study aimed to investigate the effects and possible mechanism of AT-I on intestinal dysbacteriosis in a mouse model. METHODS: Mice dysbacteriosis models were established and treated with AT-I, and the intestinal microbiome of the mice were compared. Using antibiotics-induced bacterial elimination in an intestinal dysbacteriosis-associated xenograft model, the gut microbiota-mediated anti-tumor mechanism was investigated. RESULTS: The intestinal microbiome was changed in the dysbacteriosis mice compared to the control mice, and AT-I could affect the intestinal microbiome of the dysbacteriosis mice. Manipulation of gut bacteria in the intestines of the dysbacteriosis-associated xenograft model further confirmed that the inhibition of tumor progression by AT-I was mediated by the gut microbiota, and that the underlying mechanism involves down-regulation of TLR4/MyD88/NF-κB signaling. AT-I repressed the phosphorylation of p65-NF-κB as well as the downstream cytokines, IL-6 and IL-1ß, in dysbacteriosis mice. CONCLUSIONS: AT-I may inhibit dysbacteriosis by affecting the intestinal microbiome via the regulation of TLR4/MyD88/NF-κB signaling. The present study provides a basis for the application of AT-I as an alternative medication for treating gastrointestinal disorders.

Respiratory exposure to carbon black nanoparticles may induce testicular structure damage and lead to decreased sperm quality in mice.

Environmental carbon black nanoparticles (CBNPs) can enter into various organs including testes through the respiratory tract. However, there are few studies describing reproductive toxicity of CBNPs after respiratory exposure. In this study, male KM mice were exposed to CBNPs in their natural breathing state. Four-, 8-, and 12-week-old mice were exposed to 0, 9, 18 and 27 mg/m3 of CBNPs for 4 weeks in order to examine the relationship between CBNP exposure and age. Eight-week-old mice were exposed to CBNPs at the same four concentrations for 1-4 weeks in order to examine the effects of CBNP exposure time. After CBNP exposure, testicular oxidative stress and inflammation increased significantly, and these effects varied with exposure time. Seminiferous tubule diameter (STD), seminiferous epithelium height (SEH), the number of spermatogenic and Leydig cells, sperm motility, and sperm speed decreased significantly, and these effects varied with exposure dose. Data analyses suggested that increased oxidative stress and inflammation in testes damaged testicular morphology, spermatogenesis, and testosterone secretion, and decreased sperm quality. Morphological damage to the testes was also closely related to decreased the sperm quantity. These findings are of significance for evaluating the reproductive toxicity of CBNPs.

MiR-223 promotes pyroptosis of enteritis cells through activating NF-κB signalling pathway by targeting SNIP1 in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a common inflammation-related intestinal disease. Studies have shown that excessive pyroptosis of intestinal cells is involved in the development of IBD. However, the regulatory mechanism of pyroptosis in IBD remains unclear. Here, our study purposed to clarify the underlying regulatory mechanism of miR-223 to promote pyroptosis in IBD.MiR-223 and Smad Nuclear Interacting Protein 1 (SNIP1) expression in colon tissues collected from IBD patients and healthy volunteers were evaluated using qRT-PCR. Cell viability and pyroptosis were evaluated by CCK8 and flow cytometry assay, respectively. Pyroptosis-related proteins and nuclear factor κB (NF-κB) signals were determined by WB. Dual-luciferase reporter gene assay was employed to investigate the binding relationship between miR-223 and SNIP1.MiR-223 was significantly upregulated in IBD colon tissues and cell models, while SNIP1 was significantly decreased. Silence of miR-223 markedly enhanced cell viability and inhibited pyroptosis in the IBD cell model. MiR-223 could bind to 3'-UTR of SNIP1 and SNIP1 could activate NF-κB signalling pathway. Further rescued experiment found that knockdown of SNIP1 dramatically abolished the bio-effects mediated by miR-223 silence on the cell viability and pyroptosis of the IBD cell model. Likewise, the inactivation of NF-κB signalling markedly weakened the regulatory roles of SNIP1 downregulation in the IBD cell model. Besides, inhibition of NF-κB signalling attenuated the pyroptosis-promoting effect of overexpressing miR-223.Our data suggested that miR-223 activated the NF-κB pathway via targeting SNIP1, thus promoting the process of cell pyroptosis, and ultimately participating in the pathogenesis of IBD.

Correlation between PTEN and P62 gene expression in rat colorectal cancer cell.

OBJECTIVE: Autophagy is a cellular pathway that regulates the transportation and degradation of cytoplasmic macromolecules and organelles towards lysosome, which is often related to the tumorigenesis and tumor suppression. Here, we investigate the regulating effect of PTEN gene on autophagy-related protein P62 in rat colorectal cancer (CRC) cells and explore the application value of PTEN gene in clinic. METHODS: Rat colorectal cancer was induced by intraperitoneal injection of 1,2-dimethyl hydrazine in male ACI rats. A total of 20 rats were randomly selected from those successfully induced with CRC as the experimental group, while 10 healthy rats as control. The rat CRC cells were isolated and cultured. After transfecting the rat CRC cells with pEGFP-N1-PTEN plasmid, RT-PCR was adopted to examine that gene expression of p62 and PTEN, while Western blotting was used to detect the protein expression of p62 and PTEN. Also, the proliferation of CRC cells was measured by MTT assay. RESULTS: The expression of PTEN gene in the experimental group was significantly inhibited as compared with the control group, while the expression of P62 gene was significantly increased (p < 0.05). Western blotting demonstrated that the PTEN protein in the experimental group was lower, while the expression of P62 protein was higher. When the CRC cells were transfected with pEGFP-N1-PTEN plasmid, the PTEN expressions were elevated, while p62 was down-regulated. Also, the proliferation of CRC cells was inhibited. CONCLUSION: The expression of PTEN gene is negatively correlated with the expression of P62 gene in rat CRC cells. And the expression of PTEN gene can inhibit the occurrence and development of colorectal cancer, thus providing theoretical basis for future clinical treatment.

Astragaloside II alleviates the symptoms of experimental ulcerative colitis in vitro and in vivo.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory intestinal disease, and its morbidity is rising worldwide. Previous study indicated that astragaloside II (AS II), a monomeric compound, was used to treat bowel disease. However, the effects of AS II on UC remains unclear. Thus, this study aimed to investigate the therapeutic effects of AS II on experimental UC in vitro and in vivo. METHODS: CCD-18Co cells were stimulated by 1 µg/mL LPS to mimic UC in vitro. In addition, dextran sulfate sodium (DSS)-induced UC mouse model was established in vivo. CCK-8 assay was used to detect cell proliferation in vitro. Moreover, the concentrations of inflammatory factors interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), nitric oxide (NO), superoxide dismutase (SOD) and malondialdehyde (MDA) in CCD-18Co cells and colon tissues were determined by ELISA, respectively. Meanwhile, the expressions of hypoxia-inducible factor 1α (HIF-α), phospho-inhibitor of NF-κB (p-IκB) and phospho-NF-κB p65 (p-p65) were detected by western blotting in vitro and in vivo, respectively. RESULTS: In this study, the levels of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 were significantly increased in lipopolysaccharide (LPS)-stimulated CCD-18Co cells. However, LPS-induced inflammatory response was markedly alleviated by AS II. In addition, LPS-induced HIF-α, p-IκB and p-p65 proteins increases were markedly ameliorated by AS II treatment. Moreover, AS II reduced disease activity index (DAI) scores and increased the colon lengths in DSS-treated mice. Meanwhile, AS II decreased the levels of IL-6, TNF-α, IL-1ß, NO, MPO and MDA, and increased the level of SOD in colon of DSS-treated mice. Furthermore, AS II downregulated the expressions of HIF-α, p-IκB and p-p65 in DSS-induced UC in mice. CONCLUSION: Our findings indicated that AS II could alleviate inflammatory response in LPS-induced CCD-18Co cells and in DSS-induced UC in mice. In conclusion, AS II may serve as a potential agent for the treatment of UC.

MicroRNA-362 Inhibits Cell Proliferation and Invasion by Directly Targeting SIX1 in Colorectal Cancer.

PURPOSE: Colorectal cancer (CRC) is the third most common cancer in China and poses high morbidity and mortality. In recent years, increasing evidence has indicated that microRNAs played important functions in the occurrence and development of tumors. The purpose of this study was to identify the biological mechanisms of miR-362 in CRC. MATERIALS AND METHODS: Quantitative real-time PCR was carried out to assess the expression of miR-362 and SIX1. The Kaplan-Meier method was employed to evaluate the 5-year overall survival of CRC patients. The proliferative and invasive abilities of CRC cells were assessed by MTT and transwell assays. RESULTS: miR-362 was significantly decreased in CRC tissues and cell lines, compared to the normal tissues and normal cells. A significant connection was confirmed between the overall survival of 53 CRC patients and low expression of miR-362. Downregulation of miR-362 inhibited the proliferation and invasion through binding to the 3'-UTR of SIX1 mRNA in CRC. Additionally, we discovered that SIX1 was a direct target gene of miR-362 and that the expression of miR-362 had a negative connection with SIX1 expression in CRC. SIX1 could reverse partial functions in the proliferation and invasion in CRC cells. CONCLUSION: miR-362 may be a prognostic marker in CRC and suppress CRC cell proliferation and invasion in part through targeting the 3'-UTR of SIX1 mRNA. The newly identified miR-362/SIX1 axis provides insight into the progression of CRC.

Curcumin and resveratrol suppress dextran sulfate sodium­induced colitis in mice.

Curcumin and resveratrol are two natural products, which have been described as potential anti­inflammatory, anti­tumor, and anti­oxidant molecules. The aims of the present study were to investigate the protective effect of curcumin and resveratrol on dextran sulfate sodium (DSS)­induced ulcerative colitis (UC) in mice, in addition to understanding the underlying molecular mechanisms. In order to accomplish this, BALB/c mice received drinking water containing 3.5% DSS. Curcumin (50 mg/kg/day) or resveratrol (80 mg/kg/day) were administered orally for 7 days. Survival rate, body weight, disease activity index score, colon length, pro­inflammatory cytokines, and the expression autophagy­associated proteins, and mechanistic target of rapamycin (mTOR) and sirtuin 1 (SIRT1) were measured. Curcumin or resveratrol treatment prolonged the survival of mice with UC, reduced body weight loss and attenuated the severity of the disease compared with the DSS­treated mice. This effect was associated with a substantial clinical amelioration of the disruption of the colonic architecture and a significant reduction in pro­inflammatory cytokine production. Furthermore, curcumin or resveratrol significantly downregulated the expression of autophagy­related 12, Beclin­1 and microtubule­associated protein light chain 3 II, and upregulated the expression of phosphorylated mTOR and SIRT1 in the colon tissue, compared with those in the DSS­treated group. These results suggest that curcumin and resveratrol exert protective effects on DSS­induced UC, partially through suppressing the intestinal inflammatory cascade reaction, reducing autophagy and regulating SIRT1/mTOR signaling.

Safety and therapeutic effect of mesenchymal stem cell infusion on moderate to severe ulcerative colitis.

One of the primary targets of the clinical treatment of ulcerative colitis (UC) is to repair the damaged colonic mucosa. Mesenchymal stem cells (MSCs) have therapeutic potential in regenerative medicine due to their differentiation capacity and their secretion of numerous bioactive molecules. The present study describes a clinical trial (trial registration no. NCT01221428) investigating the safety and therapeutic effect of MSCs derived from human umbilical cord on moderate to severe UC. Thirty-four patients with UC were included in group I and treated with MSC infusion in addition to the base treatment, and thirty-six patients were in group II and treated with normal saline in addition to the base treatment. One month after therapy, 30/36 patients in group I showed good response, and diffuse and deep ulcer formation and severe inflammatory mucosa were improved markedly. During the follow up, the median Mayo score and histology score in group I were decreased while IBDQ scores were significantly improved compared with before treatment and group II (P<0.05). Compared with group II, there were no evident adverse reactions after MSC infusion in any of the patients in group I, and no chronic side effects or lingering effects appeared during the follow-up period. In conclusion, MSC infusion might be a useful and safe therapy for treating UC.