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Background Alteration of polyol pathway is a critical event in serials of disease, including inflammatory bowel disease (IBD). Shikonin, an inhibitor of PKM2, has been reported to exert a wide range of biological and pharmacological properties. However, the function of shikonin-mediated polyol pathway in IBD remained to be addressed. Methods We conducted WB and qPCR to analyze the effect of shikonin on intestinal epithelial cell differentiation. IF, IP, luciferase assay and ChIP were employed to demonstrate the mechanism that shikonin regulated cell differentiation through polyol pathway. Elisa was used to measure the serum sorbitol in subjects enrolled in this study, and the clinical sample was collected to detect PKM2, AR and NRF2 expression. Results Herein, we showed that shikonin treatment in HT-29 and CaCO2 cells not only contributed to CDX2 and its targets expression, including MUC2 and villin, leading to cell differentiation, but also induced NRF2-mediated AKR1B1 and AKR1B10 expression, resulting in increasing AR activity. A synergistic effect on CDX2-mediated cell differentiation was observed in CaCO2 cells in response to cotreatment with shikonin and AR agonist bortezomib for 24 hours, while aldose reductase inhibitor fidarestat treatment in CaCO2 cells reversed the enhanced CDX2 expression caused by shikonin. Mechanistically, PKM2 interacted with NRF2, and this interaction was enhanced in response to shikonin stimulation, resulting in NRF2 phosphorylation and nuclear translocation, which further increased AKR1B1 and AKR1B10 transactivation. Moreover, both AKR1B1 and AKR1B10 expression were decreased, while PKM2 was increased in intestinal epithelial cells labeled with E-cadherin in patients with IBD, and a negative clinical relationship was observed between PKM2 and AKR1B1/AKR1B10, respectively. What's more, the serum sorbitol level was decreased in patients with IBD. Conclusion These results suggested that shikonin could promote intestinal cell differentiation through NRF2-mediated polyol pathway, implying that shikonin may be a promising therapeutic drug for the treatment of IBD.

Periodic post-embryonic changes in insects, including growth, development and metamorphosis, are strictly controlled by many compounds, including steroid hormones. The biosynthesis and clearance of 20-hydroxyecdysone (20E), the major active form of the insect steroid hormone ecdysone, result in titer fluctuations that help control insect development. The inactivation of 20E in the silkworm Bombyx mori is highly tissue-specific, with CYP18A1 and ecdysone oxidase controlling 20E inactivation specifically in the mid-silk gland and midgut, respectively. Here, we characterized silkworm 3-dehydroecdysone 3α reductase (Bm3DE3α) and 3-dehydroecdysone 3β reductase (Bm3DE3β), two enzymes involved predominantly in the C-3-mediated catalysis of 20E in fat bodies. The ubiquitous and silk gland-specific overexpression of Bm3DE3α decreased the 20E titer, resulting in larval lethality and larval-pupal transition failure, respectively. In contrast, the ubiquitous and mid-silk gland-specific overexpression of Bm3DE3β increased the 20E titer, resulting in larval growth delays and lethality at the mid-fifth larval stage, respectively. Thus, Bm3DE3α and Bm3DE3β mediate fat body-specific steroid hormone metabolism in B. mori, indicating that highly diversified 20E metabolism-related mechanisms exist in different insect species.

NLRP3 inflammasome has emerged as a crucial regulator of inflammatory bowel disease (IBD) characterized by a chronic inflammatory disease of the gastrointestinal tract. The expression of MCT4 is significantly increased in intestinal mucosal tissue of IBD, which has been identified to regulate intestinal barrier function. However, the function of MCT4 in cell pyroptosis remained unknown. In this study, we have established a stable cell line with MCT4 overexpression in HT-29 and CaCO2 cells, respectively. Functional analysis revealed that ectopic expression of MCT4 in CaCO2 cells contributed to cell pyroptosis as evidenced by LDH assay, which is largely attributed to Caspase-1-mediated canonical pyroptosis, but not Caspase-4 and Caspase-5, leading to cleave pro-IL-1β and IL-18 into mature form and release mediated by cleaved GSDMD. Mechanically, MCT4 overexpression in HT-29 and CaCO2 cell triggered the phosphorylation of ERK1/2 and NF-κB p65, while inhibition of MCT4 by MCT inhibitor α-Cyano-4-hydroxycinnamic acid (α-CHCA) in HT-29 and CaCO2 cells led to a significant downregulation of ERK1/2 and NF-κB activity. What’s more, blockade of ERK1/2-NF-κB pathway could reverse the promotion effect of MCT4 on IL-1β expression. Importantly, both MCT4 and Caspase-1, GSDMD were significantly increased in patients with IBD, and a positive clinical correlation between MCT4 and Caspase-1 expression was observed (p < 0.001). Taken together, these findings suggested that MCT4 promoted Caspase-1-mediated canonical cell pyroptosis to aggravate intestinal inflammation in intestinal epithelial cells (IECs) through the ERK1/2-NF-κB pathway.

Insect growth and development are precisely controlled by hormone homeostasis. The prothoracicotropic hormone (PTTH) receptor, Torso, is a member of the receptor tyrosine kinase family in insects. Activation of Torso by PTTH triggers biosynthesis and release of the steroid hormone in the prothoracic gland (PG). Although numbers of genes functioned in steroid hormone synthesis and metabolism have been identified in insects, the PTTH transduction pathway via its receptor Torso is poorly understood. In the current study, we describe a loss-of-function analysis of Torso in the silkworm, Bombyx mori, by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori Torso (BmTorso) did not eventually affect larval ecdysis and metamorphosis processes. Instead, BmTorso-deficiency resulted in significant extension of developing time during larval and pupal stages with increased pupa and cocoon sizes. The ecdysteriod titers in the haemolymph of BmTorso mutants declined sharply. Transcriptional levels of genes involved in ecdysone biosynthesis and ecdysteroid signalling pathways were significantly reduced in BmTorso-deficient animals. Additionally, RNA-Seq analysis revealed that genes involved in the longevity pathway and protein processing in the endoplasmic reticulum pathway were affected after BmTorso deletion. These results indicate that Torso is critical for maintaining steroid hormone homeostasis in insects. This article is protected by copyright. All rights reserved.

Insect ion transport peptides (ITPs) are important regulators of many physiological processes and they exert their functions by interacting with their receptors (ITPRs). In the current study, we comprehensively investigated the physiological functions of ITPR in the lepidopteran model insect, the silkworm (Bombyx mori), using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing technique. Mutations in silkworm ITPR (BNGR-A2) resulted in a prolongnation of the larval stage by 3.5-day as well as failure in wing expansion of moths. The BNGR-A2 mutation accelerated food transition throughout the digestive tract, which is 1.55-fold that of wild type (WT) insects. Excretion was 1.56-fold of WT insects during the larval stage, resulting in the loss of body water content. Loss of BNGR-A2 function induced significant upregulation of nitric oxide synthase (NOS) enzyme activity and nitric oxide (NO) content, as well as downstream Ca2+/NO/cGMP signaling pathways. Key genes in insulin and ecdysone signaling pathways were also affected by BNGR-A2 disruption. Our data show that ITPR plays key roles in regulating insect water homeostasis and development.

The dysregulation of glycolysis leads to serials of disease. Rabeprazole is a representative of proton pump inhibitors and widely used in anti-ulcer treatment. However, the function of Rabeprazole on glycolysis in gastric epithelial cells remained to be identified. In this study, 30（Helicobacter pylori）H. pylori-negative cases and 26H. pylori-positive cases treated with Rabeprazole were recruited. The qPCR and Western blotting results showed that Rabeprazole suppressed cell proliferation by inhibition of HK2-mediated glycolysis in BGC823 cells, leading to decrease glucose uptake and lactate production in a dose-dependent way. Furthermore, the phosphorylation of signal transducer and activator of transcription 3 (STAT3) was drastically reduced in response to Rabeprazole stimulation, leading to attenuate STAT3 nuclear translocation. Luciferase and Chromatin immunoprecipitation (ChIP) analysis showed that Rabeprazole treatment led to a significant inhibition of the binding of STAT3 to the promoter of the HK2 gene, repressing transcriptional activation of HK2. Moreover, the ectopic expression of STAT3 in BGC823 cells resulted in recovery of HK2 transactivation and cell proliferation in Rabeprazole-treated cells. Most importantly, HK2 expression was significantly increased in H. pylori-infected gastric mucosa. These findings suggested that Rabeprazole inhibited cell proliferation by targeting STAT3/HK2 signaling-mediated glucose metabolism in gastric epithelial cells. Therefore, targeting HK2 is an alternative strategy in improving the treatment of patients with H. pylori infection.