Combination of Pirfenidone and Andrographolide Ameliorates Hepatic Stellate Cell Activation and Liver Fibrosis by Mediating TGF-ß/Smad Signaling Pathway.

Background: Biliary atresia (BA) is a devastating congenital disease characterized by inflammation and progressive liver fibrosis. Activation of hepatic stellate cells (HSCs) plays a central role in the pathogenesis of hepatic fibrosis. Our study aimed to investigate the pharmacological effect and potential mechanism of pirfenidone (PFD) and andrographolide (AGP) separately and together on liver fibrosis of BA. Materials and Methods: The bile ducts of male C57BL/6J mice were ligated or had the sham operation. The in vivo effects of PFD and/or AGP on liver fibrosis of BA were evaluated. Human hepatic stellate cells (LX-2) were also treated with PFD and/or AGP in vitro. Results: PFD and/or AGP ameliorates liver fibrosis and inflammation in the mice model of BA, as evidenced by significant downregulated in the accumulation of collagen fibers, hepatic fibrosis markers (α-SMA, collagen I, and collagen IV), and inflammatory markers (IL-1ß, IL-6, and TNF-α). Moreover, compared with monotherapy, these changes are more obvious in the combined treatment of PFD and AGP. Consistent with animal experiments, hepatic fibrosis markers (α-SMA, collagen I, and CTGF) and inflammatory markers (IL-1ß, IL-6, and TNF-α) were significantly decreased in activated LX-2 cells after PFD and/or AGP treatment. In addition, PFD and/or AGP inhibited the activation of HSCs by blocking the TGF-ß/Smad signaling pathway, and the combined treatment of PFD and AGP synergistically inhibited the phosphorylation of Smad2 and Smad3. Conclusion: The combined application of PFD and AGP exerted superior inhibitive effects on HSC activation and liver fibrosis by mediating the TGF-ß/Smad signaling pathway as compared to monotherapy. Therefore, the combination of PFD and AGP may be a promising treatment strategy for liver fibrosis in BA.

ZEB2 alleviates Hirschsprung's-associated enterocolitis by promoting the proliferation and differentiation of enteric neural precursor cells via the Notch-1/Jagged-2 pathway.

BACKGROUND: Hirschsprung's-associated enterocolitis (HAEC) is a prevalent complication of Hirschsprung's disease (HSCR). Zinc finger E-box binding homeobox 2 (ZEB2) and Notch-1/Jagged-2 are dysregulated in HSCR, but their role in HAEC progression remains poorly understood. We aimed to explore the role and underlying mechanism of enteric neural precursor cells (ENPCs) and the ZEB2/Notch-1/Jagged-2 pathway in HAEC development. METHODS: Colon tissues were collected from HSCR and HAEC patients. ENPCs were isolated from the HAEC group and stimulated by lipopolysaccharide (LPS). The expressions of ZEB2/Notch-1/Jagged-2 were measured using RT-qPCR and Western blot. Immunofluorescence and cell counting kit-8 assays were performed to assess the differentiation and proliferation of ENPCs. Inflammatory factors were measured by ELISA kits. Co-immunoprecipitation and bioinformatic analysis were used to explore the interaction between ZEB2 and Notch-1. Small interfering RNA and overexpression vectors were used to investigate the role and mechanism of ZEB2 and Notch-1 in regulating ENPCs' proliferation and differentiation during HAEC progression. RESULTS: We observed increased LPS in the colon tissues of HAEC, with downregulated ZEB2 expression and upregulated Notch-1/Jagged-2 expression. ZEB2 interacts with Notch-1. LPS treatment downregulated ZEB2 expression, upregulated Notch-1/Jagged-2 expression, and induced proliferation and differentiation disorders in ENPCs, which were reversed by the knockdown of Notch-1. Furthermore, overexpression of ZEB2 inhibited Notch-1/Jagged-2 signaling and ameliorated inflammation and dysfunction in LPS-induced ENPCs. Notch-1 overexpression enhanced LPS-induced dysfunction, but this effect was antagonized by the overexpression of ZEB2. CONCLUSION: Overexpression of ZEB2 ameliorates LPS-induced ENPCs' dysfunction via the Notch-1/Jagged-2 pathway, thus playing a role in HAEC.

ALKBH5 facilitates the progression of infantile hemangioma by increasing FOXF1 expression in a m6A-YTHDF2 dependent manner to activate HK-2 signaling.

Alkylation repair homolog protein 5 (ALKBH5) is reported to participate in infantile hemangioma (IH) progression. However, the underlying mechanism of ALKBH5 in IH remains unclear. Using qRT-PCR and Western blotting, ALKBH5, forkhead box F1 (FOXF1) and hexokinase 2 (HK-2) expressions in IH tissues and IH-derived endothelial cells XPTS-1 were assessed. The Me-RIP assay was used to analyze FOXF1 m6A level. CCK8, colony formation, flow cytometry and transwell assays were employed to determine IH cell viability, proliferation, apoptosis, migration and invasion. The interactions between YTH (YT521-B homology) domain 2 (YTHDF2), FOXF1 and HK-2 were analyzed by RIP, dual luciferase reporter gene assay and/or ChIP assay. The in vivo IH growth was evaluated in immunocompromised mice. FOXF1 was overexpressed in IH tissues, and its silencing inhibited IH cell proliferation, migration and invasion whereas promoting cell apoptosis in vitro. ALKBH5 upregulation facilitated FOXF1 mRNA stability and expression in IH cells in a m6A-YTHDF2-dependent manner. FOXF1 downregulation reversed the impact of ALKBH5 upregulation on IH cellular phenotypes. It also turned out that FOXF1 positively regulated HK-2 expression in IH cells through interacting with the HK-2 promoter. HK-2 upregulation abolished FOXF1 knockdown's inhibition on IH cell aggressive behaviors. ALKBH5 or FOXF1 silencing suppressed IH tumor development via HK-2 signaling in immunocompromised mice. ALKBH5 promoted FOXF1 expression m6A-YTHDF2 dependently, which in turn elevated HK-2 expression, thereby accelerating IH development.

ß-Carotene Attenuates Apoptosis and Autophagy via PI3K/AKT/mTOR Signaling Pathway in Necrotizing Enterocolitis Model Cells IEC-6.

Background: Necrotizing enterocolitis (NEC) is a devastating disease affecting the gastrointestinal tract in the newborn period. In recent years, the role of apoptosis and autophagy in intestinal mucosal barrier dysfunction has come into prominence in research regarding the pathogenesis of NEC. ß-Carotene is a well-known vitamin A precursor, and its content in breast milk is relatively high, especially in the colostrum. In the present study, we investigated the protective effect of ß-carotene on necrotizing enterocolitis model cells IEC-6 induced by lipopolysaccharide (LPS). Methods: CCK-8 assay was performed to evaluate cell viability. The Annexin V-FITC/PI method was used to detect apoptosis. Western blotting was utilized to measure the expression levels of proteins. Immunofluorescence analysis was used to assess the autophagy of IEC-6 cells. Results: Our findings indicated that ß-carotene inhibited the apoptosis of IEC-6 cells by downregulating cleaved caspase-3 levels and Bax levels and upregulating Bcl-2 levels, reducing cell autophagy via downregulating LC3II/I ratio and upregulating p62 levels. In addition, the expression of p-PI3K, p-AKT, and p-mTOR was upregulated after ß-carotene treatment. Interestingly, these changes induced by ß-carotene were partially reversed by rapamycin and voxtalisib. Conclusion: In conclusion, our findings indicated that ß-carotene can attenuate apoptosis and autophagy of IEC-6 cells induced by LPS via activating the PI3K/AKT/mTOR signaling pathway. Therefore, ß-carotene may be a promising drug used in the clinical treatment of NEC.

ALKBH5 promotes the progression of infantile hemangioma through regulating the NEAT1/miR-378b/FOSL1 axis.

Our work aims to investigate long non-coding RNA (lncRNA) N6-methyladenosine (m6A) modification and its role in infantile hemangioma (IH). The mRNA and protein expression levels were assessed using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. Me-RIP assay was performed to evaluate lncRNA NEAT1 m6A levels. Cell proliferation, migration and invasion were evaluated using cell counting kit-8 assay, transwell migration and invasion assay, respectively. Photo-activatable ribonucleoside-enhanced crosslinking and immunoprecipitation assay was conducted to verify the binding relationship between lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) and ALKBH5 (an RNA demethylase). The binding relationship between lncRNA NEAT1, microRNA (miR)-378b and FOS-like antigen 1 (FOSL1) was verified using dual-luciferase reporter gene assay and/or RNA immunoprecipitation assay. ALKBH5, lncRNA NEAT1 and FOLS1 expression was elevated in IH tissues, while miR-378b was downregulated. ALKBH5 knockdown suppressed cell proliferation, migration and invasion of IH cells, while promoting cell apoptosis. ALKBH5 promoted lncRNA NEAT1 expression by reducing the m6A modification of lncRNA NEAT1. In addition, miR-378b was the target of lncRNA NEAT1, and its overexpression reversed the promotion effect of lncRNA NEAT1 overexpression on IH cell tumor-like behaviors. Moreover, FOLS1 was the target of miR-378b, and its overexpression reversed the inhibitory effect of miR-378b overexpression on IH cell tumor-like behaviors in vitro. ALKBH5 might have great potential as therapeutic target for IH, since ALKBH5 silencing suppressed IH progression by regulation of the NEAT1/miR-378b/FOSL1 axis.

Circ-ITCH overexpression promoted cell proliferation and migration in Hirschsprung disease through miR-146b-5p/RET axis.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital intestinal disease caused by the abnormal proliferation and migration of enteric nerve cells (ENCC). Research suggested critical roles for circular RNA (circRNA) itchy E3 ubiquitin protein ligase (ITCH) in gastrointestinal malignancies progression. However, the function of circ-ITCH in HSCR remains poorly defined. METHODS: The related genes expression in 30 HSCR patients and 30 controls without HSCR were detected using qRT-PCR. Cell proliferation was assessed by CCK-8 assay and EdU assay. Cell migration was detected with wound-healing assay and transwell assay. The interactions among circ-ITCH, miR-146b-5p, and RET were confirmed by Dual luciferase reporter assay. RESULTS: Circ-ITCH and RET expressions were downregulated in HSCR patients and cells, while the miR-146b-5p expression was upregulated. Circ-ITCH overexpression facilitated cell proliferation, migration, and activated MAPK pathway, which were reversed by circRNA-ITCH knockdown. Circ-ITCH negatively regulated miR-146b-5p expression. MiR-146b-5p overexpression abolished the promoting effects of circ-ITCH overexpression on cell proliferation and migration. MiR-146b-5p inhibited RET expression. RET overexpression eliminated the inhibitory effects of miR-146b-5p overexpression on cell proliferation and migration. CONCLUSION: Circ-ITCH overexpression facilitated cell proliferation and migration in HSCR by regulating miR-146b-5p/RET/MAPK axis. IMPACT: The expressions of Circ-ITCH and RET were markedly reduced in HSCR, while miR-146b-5p expression was increased in HSCR. Circ-ITCH overexpression enhanced the proliferative and migratory abilities of SH-SY5Y and 293T cells. Circ-ITCH negatively regulated miR-146b-5p expression.

[Effect of enhancer of zeste homolog 2 on the expression of glial cell line-derived neurotrophic factor family receptor α-1 in the colon tissue of children with Hirschsprung's disease].

OBJECTIVE: To study the expression levels of glial cell line-derived neurotrophic factor family receptor α-1 (GFRα1) and enhancer of zeste homolog 2 (EZH2) in the intestinal tissue of children with Hirschsprung's disease (HSCR), as well as the role of EZH2 in the regulation of GFRα1 gene expression and the pathogenesis of HSCR. METHODS: The samples of colon tissue with spasm from 24 children with HSCR after radical treatment of HSCR were selected as the experimental group, and the samples of necrotized colon tissue from 18 children with neonatal necrotizing enterocolitis after surgical resection were selected as the control group. Real-time PCR and Western blot were used to measure the expression levels of GFRα1 and EZH2 in colon tissue in both groups. Human neuroblastoma SH-SY5Y cells were divided into an EZH2 over-expression group and a negative control group. The cells in the EZH2 over-expression group were transfected with pCMV6-EZH2 plasmid, and those in the negative control group were transfected with pCMV6 plasmid. The expression levels of EZH2 and GFRα1 were measured after transfection. RESULTS: Compared with the control group, the experimental group had significant reductions in the mRNA and protein expression levels of GFRα1 and EZH2 in colon tissue (P<0.05), and the protein expression of EZH2 was positively correlated with that of GFRα1 (r=0.606, P=0.002). Compared with the negative control group, the EZH2 over-expression group had significant increases in the expression levels of EZH2 and GFRα1 after SH-SY5Y cells were transfected with EZH2 over-expression plasmid (P<0.05). CONCLUSIONS: Low expression of EZH2 in the colon tissue of children with HSCR may be one of the causes of inadequate expression of GFRα1 and onset of HSCR.