Gentisic acid prevents colorectal cancer metastasis via blocking GPR81-mediated DEPDC5 degradation.

BACKGROUND: Metastasis driven by epithelial-mesenchymal transition (EMT) remains a significant contributor to the poor prognosis of colorectal cancer (CRC), and requires more effective interventions. GPR81 signaling has been linked to tumor metastasis, while lacks an efficient specific inhibitor. PURPOSE: Our study aimed to investigate the effect and mechanism of Gentisic acid on colorectal cancer (CRC) metastasis. STUDY DESIGN: A lung metastasis mouse model induced by tail vein injection and a subcutaneous graft tumor model were used. Gentisic acid (GA) was administered by an intraperitoneal injection. HCT116 was treated with lactate to establish an in vitro model. METHODS: MC38 cells with mCherry fluorescent protein were injected into tail vein to investigate lung metastasis ability in vivo. GA was administered by intraperitoneal injection for 3 weeks. The therapeutic effect was evaluated by survival rates, histochemical analysis, RT-qPCR and live imaging. The mechanism was explored using small interfering RNA (siRNA), Western blotting, RT-qPCR and immunofluorescence. RESULTS: GA had a therapeutic effect on CRC metastasis and improved survival rates and pathological changes in dose-dependent manner. GA emerged as an GPR81 inhibitor, effectively suppressed EMT and mTOR signaling in CRC induced by lactate both in vivo and in vitro. Mechanistically, GA halted lactate-induce degradation of DEPDC5 through impeding the activation of Chaperone-mediated autophagy (CMA). CONCLUSION: CMA-mediated DEPDC5 degradation is crucial for lactate/GPR81-induced CRC metastasis, and GA may be a promising candidate for metastasis by inhibiting GPR81 signaling.

Vanillic acid restores homeostasis of intestinal epithelium in colitis through inhibiting CA9/STIM1-mediated ferroptosis.

The damage of integrated epithelial epithelium is a key pathogenic factor and closely associated with the recurrence of ulcerative colitis (UC). Here, we reported that vanillic acid (VA) exerted potent therapeutic effects on DSS-induced colitis by restoring intestinal epithelium homeostasis via the inhibition of ferroptosis. By the CETSA assay and DARTS assay, we identified carbonic anhydrase IX (CAIX, CA9) as the direct target of VA. The binding of VA to CA9 causes insulin-induced gene-2 (INSIG2) to interact with stromal interaction molecule 1 (STIM1), rather than SREBP cleavage-activating protein (SCAP), leading to the translocation of SCAP-SREBP1 from the endoplasmic reticulum (ER) to the Golgi apparatus for cleavage into mature SREBP1. The activation of SREBP1 induced by VA then significantly facilitated the transcription of stearoyl-CoA desaturase 1 (SCD1) to exert an inhibitory effect on ferroptosis. By inhibiting the excessive death of intestinal epithelial cells caused by ferroptosis, VA effectively preserved the integrity of intestinal barrier and prevented the progression of unresolved inflammation. In conclusion, our study demonstrated that VA could alleviate colitis by restoring intestinal epithelium homeostasis through CA9/STIM1-mediated inhibition of ferroptosis, providing a promising therapeutic candidate for UC.

Endoplasmic reticulum stress in skin aging induced by UVB.

Aging is a normal and complex biological process. Skin is located in the most superficial layer of the body, and its degree of aging directly reflects the aging level of the body. Endoplasmic reticulum stress refers to the aggregation of unfolded or misfolded proteins in the endoplasmic reticulum and the disruption of the calcium ion balance when cells are stimulated by external stimuli. Mild endoplasmic reticulum stress can cause a series of protective mechanisms, including the unfolded protein response, while sustained high intensity stimulation leads to endoplasmic reticulum stress and eventually apoptosis. Photoaging caused by ultraviolet radiation is an important stimulus in skin aging. Many studies have focused on oxidative stress, but increasing evidence shows that endoplasmic reticulum stress plays an important role in photoaging. This paper reviews the development and mechanism of endoplasmic reticulum stress (ERS) in skin photoaging, and provides research directions for targeting the ERS pathway to slow aging.

Vascular Endothelial Growth Factor A (VEGFA) Regulates Hepatic Lipid and Glycogen Metabolism in Schizothorax prenanti.

Vascular endothelial growth factor A (VEGFA) plays important roles in angiogenesis, inflammatory response as well as energy metabolism in mammals. However, its effect on glycolipid metabolism in fish has not been reported. In this study, we cloned and characterized the vegfa gene of Schizothorax prenanti (S. prenanti). vegfa expression was significantly higher in liver and muscle than that in other tissues. Then, the VEGFA recombinant protein was expressed in Escherichia coli and obtained after purification. VEGFA i.p. injection significantly increased the serum glucose and TG content compared with the control group. Moreover, VEGFA protein aggravated the glycogen and lipid deposition in the liver of S. prenanti. In addition, we found that VEGFA treatment increased hepatocyte glycogen and lipid droplet content and increased the levels of pAMPKα (T172). Furthermore, AMPKα inhibition attenuated the ability of VEGFA to induce TG and glycogen accumulation. These results demonstrate that VEGFA regulates hepatic lipid and glycogen metabolism through AMPKα in S. prenanti, which may contribute to a better understanding of VEGFA functions in the glycolipid metabolism of fish.

Inhibiting sorting nexin 10 promotes mucosal healing through SREBP2-mediated stemness restoration of intestinal stem cells.

Intestinal stem cell (ISC) is a promising therapeutic target for inflammatory bowel disease. Cholesterol availability is critical for ISC stemness. Low plasma cholesterol is a typical feature of Crohn's disease (CD); however, its impact on mucosal healing remains unclear. Here, we identified an essential role of sorting nexin 10 (SNX10) in maintaining the stemness of ISCs. SNX10 expression in intestinal tissues positively correlates with the severity of human CD and mouse colitis. Conditional SNX10 knockout in intestinal epithelial cells or ISCs promotes intestinal mucosal repair by maintaining the ISC population associated with increased intracellular cholesterol synthesis. Disassociation of ERLIN2 with SCAP by SNX10 deletion enhances the activation of SREBP2, resulting in increased cholesterol biosynthesis. DC-SX029, a small-molecule inhibitor of SNX10, was used to verify the druggable potential of SNX10 for the treatment of patients with CD. Our study provides a strategy for mucosal healing through SREBP2-mediated stemness restoration of ISCs.

Gas-Mediated Intestinal Microbiome Regulation Prompts the Methanol Extract of Schizonepetae Spica to Relieve Colitis.

Intestinal dysbiosis plays an important role in the pathogenesis of colitis (UC). Schizonepetae Herba can achieve anti-inflammatory effects as a medicine and food homologous vegetable. Luteolin, eriodictyol, fisetin, and kaempferol are the main anti-inflammatory active compounds obtained through mass spectrometry from the methanol extract of Schizonepetae Spica (JJSM). JJSM intervention resulted in attenuated weight loss, high disease-activity-index score, colon length shortening and colonic pathological damage in DSS-induced colitis mice. Interestingly, hydrogen sulfide (H2S) was inhibited remarkably, which is helpful to elucidate the relationship between active substance and intestinal flora. Furthermore, JJSM administration improved intestinal flora with down-regulating the abundance of harmful bacteria such as Clostridiales and Desulfovibrio and up-regulating the abundance of beneficial bacteria such as Muribaculaceae and Ligolactobacillus and enhanced the production of SCFAs. It is worth noticing that Desulfovibrio is related to the production of intestinal gas H2S. The elevated levels of Desulfovibrio and H2S will hasten the onset of colitis, which is a crucial risk factor for colitis. The results displayed that JJSM could considerably ameliorate colitis by rebuilding H2S-related intestinal flora, which provides a new therapeutic strategy for Schizonepetae Spica to be utilized as a functional food and considered as an emerging candidate for intestinal inflammation.

Dynamic Transcriptomic Profiling During Liver Development in Schizothorax Prenanti.

Liver is an important organ for glucose and lipid metabolism, immunity, and detoxification in fish. However, the gene regulatory network of postnatal liver development still remains unknown in teleost fish. In this study, we performed transcriptome analysis on the liver of S. prenanti at three stages. A total of 1692 differentially expressed genes (DGEs) were identified across three liver developmental stages. The oil red O staining and PAS staining revealed that the lipid content of liver was increased and the glycogen content of liver was decreased during liver development. The fatty acids biosynthesis related genes were upregulated in adult and young stages compared with juvenile stage, while lipid degradation related genes were downregulated. The genes related to glycolysis, gluconeogenesis and glycogenolysis were upregulated in juvenile or young stages compared with adult stage. Further pathway analysis indicated that the CYP450 pathway, cell cycle and amino acid metabolic pathway were induced in the process of liver maturation. Our study presents the gene expression pattern in different liver development stages of S. prenanti and may guide future studies on metabolism of S. prenanti liver.

Paeoniflorin prevents aberrant proliferation and differentiation of intestinal stem cells by controlling C1q release from macrophages in chronic colitis.

The pathological features of inflammatory bowel disease necessitate therapeutic strategies aimed at restoring intestinal mucosal barrier function in addition to controlling inflammation. Paeoniflorin, a bioactive herbal constituent isolated from the root of Paeonia albiflora Pall, has been reported to protect against acute colitis in mice. However, the direct molecular target of paeoniflorin in preventing colitis remains elusive. Here, we evaluated the therapeutical effects of Paeoniflorin using IL-10-/- chronic colitis model, and explored the precise mechanism of action involved. Our results demonstrated that intragastric administration of Paeoniflorin significantly ameliorated inflammatory response and restored the aberrant intestinal proliferation and differentiation in IL-10-/-colitis mice. By utilizing a chemical biology approach, we identified C1qa, a crucial component of C1q, is the direct target of Paeoniflorin. Binding of Paeoniflorin to C1qa prevented the cleavage of C1q on macrophages, resulting in the aggregation of surface membrane-anchored C1q and the diminished C1q secretion. The excessive surface membrane-anchored C1q significantly enhanced the phagocytic capability of macrophages and promoted the elimination of infiltrated bacteria and inflammatory cells in mouse colon. The reduced C1q secretion conferred by Paeoniflorin dampened Wnt/ß-catenin signaling activation, thereby rectifying the aberrant proliferation and differentiation of intestinal stem cells (ISCs). In summary, our study demonstrates that Paeoniflorin can orchestrate mucosal healing and intestinal inflammation elimination through C1q-bridged macrophage-ISCs crosstalk, highlighting a novel strategy to treat chronic colitis by restoring mucosal homeostasis via targeting C1q.

The Methanol Extract of Polygonatum odoratum Ameliorates Colitis by Improving Intestinal Short-Chain Fatty Acids and Gas Production to Regulate Microbiota Dysbiosis in Mice.

The potential impacts of methanol extract from Polygonatum odoratum on (YZM) colonic histopathology, gut gas production, short-chain fatty acids (SCFAs), and intestinal microbiota composition were evaluated with dextran sulfate sodium (DSS)-induced colitis mice in this study. These results indicated that YZM increased colon length and ameliorated colonic histopathology in DSS-induced colitis mice. Moreover, YZM administration reversed intestinal microbiota compositions leading to the inhibition of H2S-related bacteria (e.g., Desulfovibrionaceae) and the lower level of H2S and higher contents of SCFA-related bacteria (e.g., Muribaculaceae). Taken together, the effects of methanol extract from Polygonatum odoratum are studied to provide new enlightenment and clues for its application as a functional food and clinical drug. Our study first revealed the relationship between intestinal gas production and key bacteria in ulcerative colitis.

Mechanism of N-Methyl-N-Nitroso-Urea-Induced Gastric Precancerous Lesions in Mice.

Early diagnosis and treatment of gastric precancerous lesions (GPL) are key factors for reducing the incidence and morbidity of gastric cancer. The study is aimed at examining GPL in mice induced by N-methyl-N-nitroso-urea (MNU) and to illustrate the underlying mechanisms of tumorigenesis. In this study, we utilized an in vivo MNU-induced GPL mouse model, and histopathological changes of the gastric mucosa were observed by hematoxylin and eosin (H&E-stain) and alcian blue (AB-PAS-stain). The level of miR-194-5p in the gastric mucosa was determined by real-time polymerase chain reaction. We used transmission electron microscopy to observe the effects of MNU on gastric chief cells and parietal cells. We performed immunohistochemical detection of HIF-1α, vWF, Ki-67, and P53, while the changes in the protein expression of key genes in LKB1-AMPK and AKT-FoxO3 signaling pathways were detected by western blot analysis. We demonstrated that the miR-194-5p expression was upregulated under hypoxia in GPL gastric tissues, and that a high miR-194-5p expression level closely related with tumorigenesis. Mechanistically, miR-194-5p exerted the acceleration of activities related to metabolic reprogramming through LKB1-AMPK and AKT-FoxO3 pathways. Furthermore, similar to miR-194-5p, high expression levels of AMPK and AKT were also related to the metabolic reprogramming of GPL. Moreover, we revealed the correlation between the expression levels of miR-194-5p, p-AMPKα, p-AKT, and FoxO3a. These findings suggest that miR-194-5p/FoxO3 pathway is important for the reversal of metabolic reprogramming in GPL. Thus, exploring strategies to regulate the miR-194-5p/FoxO3a pathway may provide an efficient strategy for the prevention and treatment of GPL.

Molecular and functional characterization of the retinol-binding protein 4 (RBP4) in hepatocytes of Schizothorax prenanti in response to palmitic acid.

Retinol-binding protein 4 (RBP4) protein is a kind of adipokines synthesized and secreted by the liver, which has been verified to play important roles in liver metabolism and energy homeostasis. However, the effects of RBP4 on hepatic lipid accumulation are still elusive in fish. In the present study, we cloned and characterized the RBP4 gene in Schizothorax prenanti (S. prenanti). RBP4 gene was specifically expressed in the liver and abdominal adipose tissue. Palmitic acid (PA; 400 µM) can significantly increase lipid deposition in primary hepatocytes after 12 h of treatment. Furthermore, RBP4 knockdown can relieve the excessive lipid deposition and endoplasmic reticulum stress in the hepatocytes caused by PA. The inhibition of RBP4 abolished the ability of PA to induce the expression of genes involved in lipogenesis and endoplasmic reticulum stress. These results demonstrate that RBP4 inhibition attenuated PA-induced lipid deposition and endoplasmic reticulum stress in hepatocytes of S. prenanti. This study could contribute to improve the understanding of RBP4 functions in the PA-induced lipid deposition in hepatocytes of fish.

Methanol extract of Inonotus obliquus improves type 2 diabetes mellitus through modifying intestinal flora.

Type 2 diabetes mellitus (T2DM) poses a significant risk to human health. Previous research demonstrated that Inonotus obliquus possesses good hypolipidemic, anti-inflammatory, and anti-tumor properties. In this research, we aim to investigate the potential treatment outcomes of Inonotus obliquus for T2DM and discuss its favourable influences on the intestinal flora. The chemical composition of Inonotus obliquus methanol extracts (IO) was analyzed by ultra-high-performance liquid chromatography-Q extractive-mass spectrometry. IO significantly improved the blood glucose level, blood lipid level, and inflammatory factor level in T2DM mice, and effectively alleviated the morphological changes of colon, liver and renal. Acetic acid, propionic acid, and butyric acid levels in the feces of the IO group were restored. 16S rRNA gene sequencing revealed that the intestinal flora composition of mice in the IO group was significantly modulated. Inonotus obliquus showed significant hypoglycemic and hypolipidemic effects with evident anti-inflammatory activity and improved the morphological structure of various organs and cells. Inonotus obliquus increased the levels of short-chain fatty acids in the environment by increasing the population of certain bacteria that produce acid, such as Alistipes and Akkermansia, which are beneficial to improve intestinal flora disorders and maintain intestinal flora homeostasis. Meanwhile, Inonotus obliquus further alleviated T2DM symptoms in db/db mice by down-regulating the high number of microorganisms that are dangerous, such as Proteobacteria and Rikenellaceae_RC9_gut_group and up-regulating the abundance of beneficial bacteria such as Odoribacter and Rikenella. Therefore, this study provides a new perspective for the treatment of T2DM by demonstrating that drug and food homologous active substances could relieve inflammation via regulating intestinal flora.

SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade.

Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram-negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase-5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase-5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E-cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC-SX029, a novel SNX10 inhibitor, rescues OMV-induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase-5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment.

BIG1 mediates sepsis-induced lung injury by modulating lipid raft-dependent macrophage inflammatory responses.

Sepsis is a systemic inflammatory response syndrome with high mortality. It has been reported that brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) is involved in the pathogenesis of sepsis. However, the mechanism is not fully elucidated. In the present study, we explored the role of BIG1 in mediating lipid raft-dependent macrophage inflammatory response and its impact on lung injury in murine sepsis. In vitro studies revealed that BIG1 deficiency reduces the upregulation and secretion of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1ß and inhibits the activation of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88-dependent nuclear factor kappa-B signaling pathway induced by the lipopolysaccharide (LPS) treatment. Further experiments revealed that the inhibitory effects of BIG1 deficiency on LPS-induced inflammation are due to the upregulation of adenosine triphosphate-binding cassette transporter A1. This promotes the free-cholesterol efflux from lipid rafts and results in the reduction of lipid raft TLR4 content. The decrease in TLR4 content in lipid raft thereby inhibits the LPS-induced inflammatory response. Furthermore, using the cecal ligation and puncture-induced polymicrobial sepsis mouse model, we found that conditional knockout (cKO) of the myeloid cell BIG1 significantly reduced the serum concentrations of TNF-α, IL-6, and IL-1ß, and downregulated their mRNA expressions in the lungs. Pathological analysis confirmed that the BIG1 cKO alleviated the sepsis-induced lung injury. These results revealed the crucial new role of BIG1 in mediating lipid raft-dependent macrophage inflammatory response. Hence, BIG1 may be a potential promising therapeutic target for the treatment of septic lung injury.

Total glucosides of Paeony restores intestinal barrier function through inhibiting Lyn/Snail signaling pathway in colitis mice.

BACKGROUND: Inflammatory bowel disease (IBD) is an autoimmune disease. The pathogenesis of IBD is complicated and intestinal mucosal barrier damage is considered as the trigger factor for the initiation and recurrence of IBD. Total Glucosides of Paeony (TGP) has shown good inhibitory effects on immune-inflammation in clinic studies. However, its effect and mechanism on IBD are largely unknown. PURPOSE: The purpose of this study is to evaluate the effect and mechanism of TGP on IBD. STUDY DESIGN: DSS-induced colitis mouse model was used. TGP was given by gavage. Caco-2 cells were stimulated by outer membrane vesicles (OMV) to establish an in vitro model. METHODS: C57BL/6 mice were divided into normal control group, model group, mesalazine group, paeoniflorin (PA) group, high-dose group of TGP, and low-dose group of TGP. The model was induced with 2.5% DSS for 7 days, and TGP was intragastrically administered for 10 days. The therapeutic effect of TGP was evaluated by symptoms, histochemical analysis, RT-qPCR and ELISA. The mechanism was explored by intestinal permeability, Western blot and immunofluorescence in vivo and in vitro. RESULTS: Our results showed that TGP could significantly improve the symptoms and pathological changes, with reduced levels of TNF-α, IL-17A, IL-23 and IFN-γ in the colon tissues and serum under a dose-dependent manner. TGP also reduced the intestinal permeability and restored the protein expression of tight junction and adherens junction proteins of intestinal epithelial cells in vivo and in vitro. Furthermore, TGP could inhibit the expression of p-Lyn and Snail and prevent Snail nuclear localization, thereby maintaining tight and adherens junctions. CONCLUSION: TGP effectively improves the symptoms of DSS-induced colitis in mice, protects the intestinal epithelial barrier by inhibiting the Lyn/Snail signaling pathway, and maybe a promise therapeutic agent for IBD treatment.

Allicin inhibits mouse colorectal tumorigenesis through suppressing the activation of STAT3 signaling pathway.

Allicin is the major biologically active compounds of freshly crushed garlic. It has been reported to inhibit the proliferation and promote the apoptosis of multiple colorectal cancer cells. However, the anti-colorectal cancer effect of Allicin has not been verified by in vivo studies. In the present study, we investigated the effect of Allicin on azoxymethane/dextran sodium sulfate (AOM/DSS) colorectal cancer mouse model and explore the underlying possible mechanism. Our result showed that Allicin could inhibit colonic tumorigenesis of AOM/DSS mice in vivo. In vitro study showed that Allicin promoted the apoptosis and suppressed the survival and proliferation of HCT116 cells. The molecular mechanism is related to the suppression of STAT3 signaling activation. Thus, our data provide further support for Allicin as a potential favorable supplement for human colorectal cancer.

Sorting nexin 10 controls mTOR activation through regulating amino-acid metabolism in colorectal cancer.

Amino-acid metabolism plays a vital role in mammalian target of rapamycin (mTOR) signaling, which is the pivot in colorectal cancer (CRC). Upregulated chaperone-mediated autophagy (CMA) activity contributes to the regulation of metabolism in cancer cells. Previously, we found that sorting nexin 10 (SNX10) is a critical regulator in CMA activation. Here we investigated the role of SNX10 in regulating amino-acid metabolism and mTOR signaling pathway activation, as well as the impact on the tumor progression of mouse CRC. Our results showed that SNX10 deficiency promoted colorectal tumorigenesis in male FVB mice and CRC cell proliferation and survival. Metabolic pathway analysis of gas chromatography-mass spectrometry (GC-MS) data revealed unique changes of amino-acid metabolism by SNX10 deficiency. In HCT116 cells, SNX10 knockout resulted in the increase of CMA and mTOR activation, which could be abolished by chloroquine treatment or reversed by SNX10 overexpression. By small RNA interference (siRNA), we found that the activation of mTOR was dependent on lysosomal-associated membrane protein type-2A (LAMP-2A), which is a limiting factor of CMA. Similar results were also found in Caco-2 and SW480 cells. Ultra-high-performance liquid chromatography-quadrupole time of flight (UHPLC-QTOF) and GC-MS-based untargeted metabolomics revealed that 10 amino-acid metabolism in SNX10-deficient cells were significantly upregulated, which could be restored by LAMP-2A siRNA. All of these amino acids were previously reported to be involved in mTOR activation. In conclusion, this work revealed that SNX10 controls mTOR activation through regulating CMA-dependent amino-acid metabolism, which provides potential target and strategy for treating CRC.