Intrauterine arsenic exposure induces glucose metabolism disorders in adult offspring by targeting TET2-mediated DNA hydroxymethylation reprogramming of HNF4α in developing livers, an effect alleviated by ascorbic acid.

Exposure to arsenic during gestation has lasting health-related effects on the developing fetus, including an increase in the risk of metabolic disease later in life. Epigenetics is a potential mechanism involved in this process. Ten-eleven translocation 2 (TET2) has been widely considered as a transferase of 5-hydroxymethylcytosine (5hmC). Here, mice were exposed, via drinking water, to arsenic or arsenic combined with ascorbic acid (AA) during gestation. For adult offspring, intrauterine arsenic exposure exhibited disorders of glucose metabolism, which are associated with DNA hydroxymethylation reprogramming of hepatic nuclear factor 4 alpha (HNF4α). Further molecular structure analysis, by SEC-UV-DAD, SEC-ICP-MS, verified that arsenic binds to the cysteine domain of TET2. Mechanistically, arsenic reduces the stability of TET2 by binding to it, resulting in the decrease of 5hmC levels in Hnf4α and subsequently inhibiting its expression. This leads to the disorders of expression of its downstream key glucose metabolism genes. Supplementation with AA blocked the reduction of TET2 and normalized the 5hmC levels of Hnf4α, thus alleviating the glucose metabolism disorders. Our study provides targets and methods for the prevention of offspring glucose metabolism abnormalities caused by intrauterine arsenic exposure.

Inhibition of ACLY overcomes cancer immunotherapy resistance via polyunsaturated fatty acids peroxidation and cGAS-STING activation.

Adenosine 5'-triphosphate citrate lyase (ACLY) is a cytosolic enzyme that converts citrate into acetyl-coenzyme A for fatty acid and cholesterol biosynthesis. ACLY is up-regulated or activated in many cancers, and targeting ACLY by inhibitors holds promise as potential cancer therapy. However, the role of ACLY in cancer immunity regulation remains poorly understood. Here, we show that ACLY inhibition up-regulates PD-L1 immune checkpoint expression in cancer cells and induces T cell dysfunction to drive immunosuppression and compromise its antitumor effect in immunocompetent mice. Mechanistically, ACLY inhibition causes polyunsaturated fatty acid (PUFA) peroxidation and mitochondrial damage, which triggers mitochondrial DNA leakage to activate the cGAS-STING innate immune pathway. Pharmacological and genetic inhibition of ACLY overcomes cancer resistance to anti-PD-L1 therapy in a cGAS-dependent manner. Furthermore, dietary PUFA supplementation mirrors the enhanced efficacy of PD-L1 blockade by ACLY inhibition. These findings reveal an immunomodulatory role of ACLY and provide combinatorial strategies to overcome immunotherapy resistance in tumors.

As3MT via consuming SAM is involved in arsenic-induced nonalcoholic fatty liver disease by blocking m6A-mediated miR-142-5p maturation.

Arsenic, a common environmental hazard, is a risk factor for nonalcoholic fatty liver disease (NAFLD). However, the mechanism remains unclear. Here, we found that chronic exposure to environmental-related doses of arsenic disturbed fatty acid and methionine metabolism in mice, caused liver steatosis, increased arsenic (3) methyltransferase (As3MT), sterol regulatory element binding protein 1 (SREBP1) and lipogenic gene levels, and decreased N6-methyladenosine (m6A) and S-adenosylmethionine (SAM) levels. Mechanistically, arsenic blocks m6A-mediated miR-142-5p maturation by consuming SAM via As3MT. miR-142-5p was involved in arsenic-induced cellular lipid accumulation by targeting SREBP1. SAM supplementation or As3MT deficiency blocked arsenic-induced lipid accumulation by promoting the maturation of miR-142-5p. Moreover, in mice, folic acid (FA) and vitamin B12 (VB12) supplementation blocked arsenic-induced lipid accumulation by restoring SAM levels. Arsenic-exposed heterozygous As3MT mice showed low liver lipid accumulation. Our study demonstrates that SAM consumption caused by arsenic, through As3MT, blocks m6A-mediated miR-142-5p maturation, thereby elevating the levels of SREBP1 and lipogenic genes, leading to NAFLD, which provides a new mechanism and biological insights into the therapy of NAFLD induced by environmental factors.

[Mechanism of Buyang Huanwu Decoction glycosides against atherosclerotic inflammation through NF-κB signaling pathway].

This study aims to explore the effect of Buyang Huanwu Decoction glycosides on the inflammatory response of apolipoprotein E~(-/-)(ApoE~(-/-)) mice and RAW264.7 cells through nuclear factor kappa-B(NF-κB) signaling pathway. In the in vivo experiment, ApoE~(-/-) mice were fed with high-fat diets for 12 weeks to induce the animal model of atherosclerosis, and 75 µg·mL~(-1) oxidized low-density lipoprotein(Ox-LDL) incubated RAW264.7 cells for 24 h to establish the atherosclerosis cell model. Automatic biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay(ELISA), Western blot, and droplet digital polymerase chain reaction(PCR) were used to determine the blood lipid levels, aortic intimal thickness, inflammatory factor content, NF-κB pathway-related proteins, and mRNA expression levels, and evaluate arterial atherosclerotic lesions and anti-atherosclerotic mechanisms of the drug. The model of atherosclerosis was successfully established in ApoE~(-/-) mice after 12 weeks of feeding with high-fat diets. In the model group, the plasma levels of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-C) were increased(P<0.01), the intima of the blood vessels was thickened, the levels of inflammatory factors tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were increased, and the protein and mRNA expressions of NF-κB and inhibitor of NF-κB(IκBα) were significantly increased as compared with the control group. Compared with the model group, the high-dose Buyang Huanwu Decoction glycoside group decreased the plasma levels of TC, TG, and LDL-C, reduced the plaque area and thickness and the content of inflammatory factor TNF-α, and inhibited the protein and mRNA expressions of NF-κB and IκBα, with the effect same as Buyang Huanwu Decoction. In the in vivo experiment, 75 µg·mL~(-1) Ox-LDL stimulated RAW264.7 cells for 24 h to successfully establish a foam cell model. As compared with the control group, the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα in the model group increased. Compared with the model group, the middle-dose and high-dose Buyang Huanwu Decoction glycoside groups decreased the nuclear amount of NF-κB and the protein and mRNA expressions of IκBα. The above results show that the glycosides are the main effective substances of Buyang Huanwu Decoction against atherosclerosis, which inhibit the NF-κB pathway and reduce the inflammatory response, thus playing the role against atherosclerotic inflammation same as Buyang Huanwu Decoction.

SIRT1/PGC-1α is involved in arsenic-induced male reproductive damage through mitochondrial dysfunction, which is blocked by the antioxidative effect of zinc.

Exposure to arsenic poses threats to male reproductive system, including impairing the testes and sperm quality. Although an association regarding arsenic exposure and male reproductive damage has been reported, the undergoing molecular mechanisms and interventions for prevention remain unclear. For the present work, male mice were exposed to 0, 2.5, 5, or 10 ppm sodium arsenite (NaAsO2) for 8 months. The results showed that arsenic-exposed mice had reduced fertility with abnormalities in the testes, epididymides, and sperm. Exposure of mice to arsenic caused a redox imbalance, decreased SIRT1 and PGC-1α levels, and affected mitochondrial biogenesis and proteins related to mitochondrial dynamics. For immortalized spermatogenic (GC-2) cells, arsenic caused apoptosis and oxidative stress, reduced SIRT1/PGC-1α levels and ATP production, inhibited mitochondrial respiration, and changed the mitochondrial membrane potential (MMP). Mitochondrial biogenesis and dynamics were also impaired. However, by reducing mitochondrial damage in GC-2 cells, upregulation of SIRT1 or zinc (Zn) supplementation reversed the apoptosis induced by arsenic. For mice, Zn supplementation blocked arsenic-induced oxidative stress, the decreases of SIRT1 and PGC-1α levels, and the impairment of mitochondrial function, and it reversed the damage to testes, low sperm quality, and low litter size. Collectively, these results suggest that arsenic causes excessive production of ROS, inhibits the SIRT1/PGC-1α pathway, and causing mitochondrial dysfunction by mediating impairment of mitochondrial biogenesis and dynamics, which results in germ cells apoptosis and male reproductive damage, processes that are blocked by Zn via an antioxidative effect. Our study contributes to understanding of the mechanisms for arsenic-induced male reproductive damage and points to the therapeutic significance of Zn.

As3MT-mediated SAM consumption, which inhibits the methylation of histones and LINE1, is involved in arsenic-induced male reproductive damage.

Studies have demonstrated that arsenic (As) induces male reproductive injury, however, the mechanism remains unknown. The high levels of arsenic (3) methyltransferase (As3MT) promote As-induced male reproductive toxicity. For As-exposed mice, the germ cells in seminiferous tubules and sperm quality were reduced. Exposure to As caused lower S-adenosylmethionine (SAM) and 5-methylcytosine (5 mC) levels, histone and DNA hypomethylation, upregulation of long interspersed element class 1 (LINE1, or L1), defective repair of double-strand breaks (DSBs), and the arrest of meiosis, resulting in apoptosis of germ cells and lower litter size. For GC-2spd (GC-2) cells, As induced apoptosis, which was prevented by adding SAM or by reducing the expression of As3MT. The levels of LINE1, affected by SAM content, were involved in As-induced apoptosis. Furthermore, folic acid (FA) and vitamin B12 (VB12) supplements restored SAM, 5 mC, and LINE1 levels and blocked impairment of spermatogenesis and testes and lower litter size. Exposed to As, mice with As3MT knockdown showed less impairment of spermatogenesis and testes and greater litter size compared to As-exposed wild-type (WT) mice. Thus, the high As3MT levels induced by As consume SAM and block histone and LINE1 DNA methylation, elevating LINE1 expression and evoking impairment of spermatogenesis, which causes male reproductive damage. Overall, we have found a mechanism for As-induced male reproductive damage, which provides biological insights into the alleviation of reproductive injury induced by environmental factors.

Mechanism of electroacupuncture and herb-partitioned moxibustion on ulcerative colitis animal model: A study based on proteomics.

BACKGROUND: Ulcerative colitis (UC) is a chronic, nonspecific intestinal inflammatory disease. Acupuncture and moxibustion is proved effective in treating UC, but the mechanism has not been clarified. Proteomic technology has revealed a variety of biological markers related to immunity and inflammation in UC, which provide new insights and directions for the study of mechanism of acupuncture and moxibustion treatment of UC. AIM: To investigate the mechanism of electroacupuncture (EA) and herb-partitioned moxibustion (HM) on UC rats by using proteomics technology. METHODS: Male Sprague-Dawley rats were randomly divided into the normal (N) group, the dextran sulfate sodium (DSS)-induced UC model (M) group, the HM group, and the EA group. UC rat model was prepared with 3% DSS, and HM and EA interventions at the bilateral Tianshu and Qihai acupoints were performed in HM or EA group. Haematoxylin and eosin staining was used for morphological evaluation of colon tissues. Isotope-labeled relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry were performed for proteome analysis of the colon tissues, followed by bioinformatics analysis and protein-protein interaction networks establishment of differentially expressed proteins (DEPs) between groups. Then western blot was used for verification of selected DEPs. RESULTS: The macroscopic colon injury scores and histopathology scores in the HM and EA groups were significantly decreased compared to the rats in the M group (P < 0.01). Compared with the N group, a total of 202 DEPs were identified in the M group, including 111 up-regulated proteins and 91 down-regulated proteins, of which 25 and 15 proteins were reversed after HM and EA interventions, respectively. The DEPs were involved in various biological processes such as biological regulation, immune system progression and in multiple pathways including natural killer cell mediated cytotoxicity, intestinal immune network for immunoglobulin A (IgA) production, and FcγR-mediated phagocytosis. The Kyoto Encyclopedia of Genes and Genomes pathways of DEPs between HM and M groups, EA and M groups both included immune-associated and oxidative phosphorylation. Network analysis revealed that multiple pathways for the DEPs of each group were involved in protein-protein interactions, and the expression of oxidative phosphorylation pathway-related proteins, including ATP synthase subunit g (ATP5L), ATP synthase beta subunit precursor (Atp5f), cytochrome c oxidase subunit 4 isoform 1 (Cox4i1) were down-regulated after HM and EA interventions. Subsequent verification of selected DEPs (Synaptic vesicle glycoprotein 2A; nuclear cap binding protein subunit 1; carbamoyl phosphate synthetase 1; Cox4i1; ATP synthase subunit b, Atp5f1; doublecortin like kinase 3) by western blot confirmed the reliability of the iTRAQ data, HM and EA interventions can significantly down-regulate the expression of oxidative phosphorylation-associated proteins (Cox4i1, Atp5f1) (P < 0.01). CONCLUSION: EA and HM could regulate the expression of ATP5L, Atp5f1, Cox4i1 that associated with oxidative phosphorylation, then might regulate immune-related pathways of intestinal immune network for IgA production, FcγR-mediated phagocytosis, thereby alleviating colonic inflammation of DSS-induced UC rats.

Glycosides from Buyang Huanwu Decoction inhibit atherosclerotic inflammation via JAK/STAT signaling pathway.

BACKGROUND: Buyang Huanwu Decoction (BYHWD) has been used to treat or prevent cardiovascular disease. The prescription and its glycosides have the effects of protecting blood vessels, and resisting atherosclerosis. However, their protective mechanism of anti-atherosclerosis remains unclear. PURPOSE: This study aims to explore whether glycosides are the main effective components of BYHWD in anti-atherosclerotic inflammation and whether their mechanism is related to the classical JAK/STAT inflammatory signaling pathway. METHODS: UPLC-MSMS method was used to determine the main components of BYHWD and its glycosides. Network pharmacological analysis and molecular docking were used to predict the potential therapeutic targets of glycosides. Atherosclerosis model was prepared by feeding HFD in ApoE-/- mice. The effects of glycosides on atherosclerosis were detected by blood lipids measurement, Masson staining, immunohistochemistry, immunofluorescence, western-blot and droplet digital PCR. RAW264.7 cells were used to establish foam cells model. The mechanism of glycosides anti-atherosclerotic inflammation was detected by measuring intracellular lipids, Oil Red O staining, ELISA, western-blot and droplet digital PCR. RESULTS: 1. Glycosides were absorbed into the blood through oral administrations and existed in the blood in the form of glycosides structures. 2. Glycosides attenuated hyperlipidemia, alleviated atherosclerotic lesions and inhibited inflammatory reaction. They could regulate blood lipids by decreasing TC, TG, LDL-c, increasing HDL-c level in ApoE-/- mice, alleviating intimal area and thickness, and inhibiting atherosclerotic plaque formation, which were similar to BYHWD. 3. Glycosides anti-atherosclerotic inflammation was related to JAK/STAT signaling pathway by network pharmacology analysis. Interactions between glycosides (astragaloside IV, paeoniflorin and amygdalin) and JAK/STAT pathway-related proteins by molecular docking. 4. Glycosides alleviated atherosclerotic inflammation by decreasing the release of pro-inflammatory factors and adhesions molecules, inhibiting the activation of JAK/STAT pathway in vivo. 5. Glycosides reduced the number of foam cells and intracellular lipid content. It also prevented the inflammation of macrophages by decreasing the levels of pro-inflammatory factors, reducing the phosphorylation of JAK2, STAT1 and STAT3 in vitro. CONCLUSION: This study demonstrated that glycosides were the main active components of BYHWD, and they could inhibit atherosclerosis by alleviating atherosclerotic inflammation. the mechanism is inhibiting the activation of JAK/STAT signaling pathway.

[Therapeutic effect of Shengjiang Powder on experimental autoimmune encephalomyelitis mice through IL-6/JAK2/STAT3 signaling pathway:based on HPLC].

A high performance liquid chromatography(HPLC) method was established to analyze the components in Shengjiang Powder(SJP) such as emodin and curcumin and explore its therapeutic effect on experimental autoimmune encephalomyelitis(EAE) mice. To be specific, HPLC was performed to determine the content of compounds in SJP such as emodin and curcumin. A total of 72 female SPF C57 BL/6 mice were randomized into control group(equivalent volume of ultrapure water, ig), model group(equivalent volume of ultrapure water, ig), low-, medium-, and high-dose SJP groups(SJP, ig), and positive control group(prednisone acetate, ig), 12 each group. EAE was induced in mice except the control group. Administration began from the first day after immunization. The general conditions, symptom score, and body weight of the mice were recorded. On the 21 st day, mouse brain tissues were separrated. Then hematoxylin-eosin(HE) staining and Luxol Fast Blue(LFB) staining were used to detect the pathological changes of brain tissues. Immunohistochemistry(IHC) was employed to determine the myelin basic protein(MBP) level, and Western blot the expression of occludin and claudin-5, as well as the levels of interleukin-6(IL-6) and proteins in the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) pathway and their phosphorylation levels. The mRNA expression of IL-6, JAK2, and STAT3 was detected by real-time quantitative polymerase chain reaction(qPCR). Finally, molecular docking of six main active components in SJP, including emodin and curcumin, with IL-6, JAK2 and STAT3 was performed, and the binding affinity was evaluated. The results showed that the established HPLC method demonstrated high precision, reproducibility, stability, and high recovery of samples. Compared with the model group, SJP reduced the clinical symptom score and alleviate the inflammatory infiltration of brain white matter and demyelination of EAE mice. At the same time, SJP increased the expression of occludin and claudin-5, down-regulated the mRNA expression of IL-6, JAK2, and STAT3, as well as the levels of IL-6/JAK/STAT3 proteins and the phosphorylation levels, with significant difference. Molecular docking suggested that the six active components in SJP had high binding energy with IL-6, JAK2, and STAT3 proteins. The established HPLC method is simple, accurate, and highly sensitive, which can simultaneously determine the content of emodin and curcumin in SJP. SJP may alleviate the clinical symptoms of EAE by inhibiting IL-6/JAK2/STAT3 signaling pathway, protecting the blood-brain barrier, and relieving the inflammatory response and demyelinization of brain tissue.

Gut microbiome alterations in colitis rats after moxibustion at bilateral Tianshu acupoints.

BACKGROUND: The pathogenesis of ulcerative colitis (UC) is closely related to the gut microbiota. Moxibustion has been used to improve the inflammation and gastrointestinal dysfunctions in gastrointestinal disorders such as UC. In this study, we investigated whether moxibustion could improve the gut microbial dysbiosis induced by dextran sulphate sodium. METHODS: Twenty-five male rats were randomly assigned into five groups. The UC rat model was established by administering DSS solution. The rats in the moxibustion and normal rats with moxibustion groups were treated with moxibustion at Tianshu (bilateral, ST25) points, and the mesalazine group rats were treated with mesalazine once daily for 7 consecutive days. Disease activity index (DAI) and haematoxylin and eosin staining were used to evaluate the effect of moxibustion. Gut microbiota profiling was conducted by metagenomic high throughput sequencing technology. The gut microbiota composition, diversity and function were analyzed and compared using metagenomics methodologies. RESULTS: The DAI scores and histopathology scores in the moxibustion and mesalazine groups were significantly decreased compared with the UC group (P < 0.01). Moxibustion treatment increased abundance levels of Bacteroidetes, Actinobacteria, Ascomycota, Synergistetes and decreased abundance of Firmicutes, Proteobacteria. At the genus level, the abundance of Bacteroides, Bacteroides_bacterium_M7, Prevotella, Bacteroidales_bacterium_H2, were increased and Bacteroides_bacterium_H3, Parabacteroides, Porphyromonas, Alistipes, Parasutterella were decreased in the UC group in comparsion with those in the NG group. Moxibustion increased the abundance of Bacteroides and Bacteroides_bacterium_H3 and decreased Bacteroides_bacterium_M7, Prevotella, Bacteroidales_bacterium_H2. In UC group, the specie Bacteroides_massiliensis was negatively (P < 0.05) correlated with IL-23, Bacteroides_eggerthii_CAG109 and Bacteroides_eggerthii were negatively (P < 0.05) correlated with TGF-ß. And the species Prevotella_sp_CAG1031 and Bacteroides_bacterium_H2 were significant positively (P < 0.05) correlated with IL-23. In addition, compare with the normal group, genes involved in certain metabolic pathways, such as energy production and conversion, amino acid transport and metabolism, carbohydrate transport and metabolism, were under-represented in the UC group, and these changes in the metabolic pathways could be reversed by moxibustion treatment and mesalazine treatment. CONCLUSIONS: Our findings suggest that moxibustion treatment may protect the host from mucosal inflammation by modulating the intestinal microbiota community.

Herb-partitioned moxibustion regulated the miRNA expression profile in the thyroid tissues of rats with experimental autoimmune thyroiditi.

OBJECTIVE: To observe the effect of herb-partitioned moxibustion (HPM) on the miRNA expression profile of thyroid tissue in experimental autoimmune thyroiditis (EAT) rats. METHODS: Rats were randomly divided into normal control (NC) group, EAT model (EAT) group, HPM group and western medicine (Med) group. EAT model rats were prepared by a combined immunization with complete and incomplete Freund's adjuvant emulsified with porcine thyroglobulin and iodine. Rats in the HPM group were treated with HPM, while rats in the Med group were treated with levothyrocine (1 µg/2 mL) by gavage. HE staining was used to observe the pathological morphological changes of thyroid tissue, ELISAs was uaed to detect the serum concentrations of TGAb, TPOAb, FT3, FT4, TSH. We then performed high-throughput miRNA sequencing to analyse the miRNA expression profiles in the thyroid tissues, followed by a bioinformatics analysis. RT-qPCR was used to verify the identified differentially expressed miRNAs. RESULTS: HPM improved the thyroid tissue morphology and reduced serum TPOAb, TGAb, TSH concentration in EAT rats (P < 0.05), but with no obvious effect on FT3 and FT4 concentration. While the TSH, FT3 and FT4 concentration was significantly changed in the Med group (P < 0.01 or P < 0.05) compared with that of EAT group. Sequencing results showed that a total of 17 miRNAs were upregulated, and 4 were downregulated in the EAT rats, in which the expression levels of miR-346 and miR-331-5p were reversed by HPM. The target genes of the miRNAs that regulated by HPM were associated with a variety of immune factors and immune signals. RT-qPCR verification showed that the expression of miRNA-346 and miRNA-331-5p was consistent with the sequencing results. CONCLUSIONS: HPM could regulate the the expression of miRNA-346 and miRNA-331-5p, then act on their target genes to immune and inflammation-related pathways, which may be one of the mechanisms of HPM on EAT rats.

Influence of herb-partitioned moxibustion at Qihai (CV6) and bilateral Tianshu (ST25) and Shangjuxu (ST37) acupoints on toll-like receptors 4 signaling pathways in patients with ulcerative coliti.

OBJECTIVE: To evaluate the efficacy of herb-partitioned moxibustion (HPM) at Qihai (CV6), Tianshu (ST25) and Shangjuxu (ST37) acupoints in relieving symptoms and the immune regulation of HPM on the toll-like receptors 4 (TLR4) signaling pathway in ulcerative colitis (UC) patients. METHODS: A randomized, single-blind study was conducted 63 patients to receive HPM or sham HPM treatment. The efficacy outcomes included scores of the Mayo, Baron, inflammatory bowel disease questionnaire (IBDQ), self-rating depression scale (SDS), self-rating anxiety scale (SAS). HE staining was used to observe the histopathological changes of the colon. The expression of inflammatory cytokines and TLR4 signaling pathway related molecules were determined by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Baron, SDS, SAS scores were significantly decreased in moxibustion group (P < 0.05), IBDQ score was significantly greater in the moxibustion group than in the sham moxibustion group (P < 0.05). Histopathology of mucosal biopsies showed that both two groups improved in mucosa after treatment. The expression levels of tumor necrosis factor-α, interleukin-2, interleukin-12, interferon-γ, and TLR4, lipopolysaccharide, myeloid differentiation factor 88, interleukin receptor associated kinase, tumor necrosis factor receptor associated factor 6 and nuclear factor kappa-B p65 were significantly lower in the moxibustion group than in the sham moxibustion group (P < 0.05). CONCLUSION: This study showed that HPM at Qihai?(CV6),?Tianshu?(ST25) and?Shangjuxu (ST37) acupoints is effective to relieve symptoms, anxiety, depression and improving life quality in UC patients, which may be related to the immune regulation of HPM on TLR4 signaling pathway.

Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn's disease via signaling involving distinct classes of phosphatidylinositol 3-kinases.

BACKGROUND: Autophagy is an evolutionarily conserved biological process in eukaryotic cells that involves lysosomal-mediated degradation and recycling of related cellular components. Recent studies have shown that autophagy plays an important role in the pathogenesis of Crohn's disease (CD). Herbal cake-partitioned moxibustion (HM) has been historically practiced to treat CD. However, the mechanism by which HM regulates colonic autophagy in CD remains unclear. AIM: To observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism. METHODS: Rats were randomly divided into a normal control (NC) group, a CD group, an HM group, an insulin + CD (I + CD) group, an insulin + HM (I + HM) group, a rapamycin + CD (RA + CD) group, and a rapamycin + HM (RA + HM) group. 2,4,6-trinitrobenzenesulfonic acid was administered to establish a CD model. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and the formation of autophagosomes was observed by electron microscopy. The expression of autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Insulin and rapamycin were used to inhibit and activate colonic autophagy, respectively. The mRNA expression levels of phosphatidylinositol 3-kinase class I (PI3KC1), Akt1, LC3B, sequestosome 1 (p62), and mammalian target of rapamycin (mTOR) were evaluated by RT-qPCR. The protein expression levels of interleukin 18 (IL-18), tumor necrosis factor-α (TNF-α), nuclear factor κB/p65 (NF-κB p65), LC3B, p62, coiled-coil myosin-like BCL2-interacting protein (Beclin-1), p-mTOR, PI3KC1, class III phosphatidylinositol 3-kinase (PI3KC3/Vps34), and p-Akt were evaluated by Western blot analysis. RESULTS: Compared with the NC group, the CD group showed severe damage to colon tissues and higher expression levels of IL-18 and NF-κB p65 in colon tissues (P < 0.01 for both). Compared with the CD group, the HM group showed significantly lower levels of these proteins (P IL-18 < 0.01 and P p65 < 0.05). There were no significant differences in the expression of TNF-α protein in colon tissue among the rat groups. Typical autophagic vesicles were found in both the CD and HM groups. The expression of the autophagy proteins LC3B and Beclin-1 was upregulated (P < 0.01 for both) in the colon tissues of rats in the CD group compared with the NC group, while the protein expression of p62 and p-mTOR was downregulated (P < 0.01 for both). However, these expression trends were significantly reversed in the HM group compared with the CD group (P LC3B < 0.01, P Beclin-1 < 0.05, P p62 < 0.05, and P m-TOR < 0.05). Compared with those in the RA + CD group, the mRNA expression levels of PI3KC1, Akt1, mTOR, and p62 in the RA + HM group were significantly higher (P PI3KC1 < 0.01 and P Akt1, mTOR, and p62 < 0.05), while those of LC3B were significantly lower (P < 0.05). Compared with the RA + CD group, the RA + HM group exhibited significantly higher PI3KC1, p-Akt1, and p-mTOR protein levels (P PI3KC1 < 0.01, P p-Akt1 < 0.05, and P p-mTOR < 0.01), a higher p62 protein level (P = 0.057), and significantly lower LC3B and Vps34 protein levels (P < 0.01 for both) in colon tissue. CONCLUSION: HM can activate PI3KC1/Akt1/mTOR signaling while inhibiting the PI3KC3 (Vps34)-Beclin-1 protein complex in the colon tissues of CD rats, thereby inhibiting overactivated autophagy and thus exerting a therapeutic effect.

[Investigating effect of Faeces Bombycis on intestinal microflora in rats with syndrome of damp retention in middle-jiao by high-throughput sequencing].

The aim of this paper was to investigate the effect of Faeces Bombycis(FB) on the intestinal microflora in rats with syndrome of damp retention in middle-jiao, and to explore its mechanism in regulating intestinal microflora from the perspective of microorganisms contained in FB. The contents of antidiuretic hormone(ADH) and C-reactive protein(CRP) in serum and aquaporin 3(AQP3) in jejunum were determined by enzyme-linked immunosorbent assay(ELISA). Illumina Miseq platform was used for high-throughput sequencing of the rat feces and FB. The ELISA results showed that as compared with the normal control group, the contents of ADH and CRP in the model group were significantly increased(P<0.05), and the content of AQP3 was significantly decreased(P<0.05). After drug administration, the ADH, CRP and AQP3 contents were recovered. Sequencing of rat feces showed that the ACE, Chao1 and Shannon indexes of the intestinal microflora were the lowest in the model group. As compared with the normal control group, the levels from phylum to genus were all significantly changed in model group, and Proteobacteria, Acinetobacter, Anaerobacter, Pseudomonas, and Parabacteroides levels were significantly increased(P<0.05), while Marvinbryantia level was significantly decreased(P<0.05). As compared with the model group, Proteobacteria was significantly decreased in the FB low and high dose groups(P<0.05), and Acinetobacter, Anaerobacter, Pseudomonas, Parabacteroides levels were significantly decreased in the low, medium and high dose groups(P<0.05), while Lachnoanaerobaculum, Intestinimonas and Marvinbryantia were increased significantly in the high dose group(P<0.05). Sequencing analysis of FB showed that the relative abundance of Leclercia, Pantoea, Brachybacterium, Shimwellia, Hartmannibacter, Klebsiella, Serratia, Aurantimonas, Paenibacillus and Bacillus was high in the FB, but they were basically not present or little in the rat feces. In conclusion, FB may play a role in the treatment of "syndrome of damp retention in middle-jiao" by balancing the intestinal microflora, and this effect may be related to the metabolites of microorganisms in the FB.

Adjuvant 131I-metuximab for hepatocellular carcinoma after liver resection: a randomised, controlled, multicentre, open-label, phase 2 trial.

BACKGROUND: Effective adjuvant treatment after hepatectomy for hepatocellular carcinoma (HCC) is an important area of research. Radioactive iodine (131I)-labelled metuximab is a radiolabelled monoclonal antibody against the CD147 (also known as basigin or HAb18G) antigen that is expressed in HCC. We aimed to examine the role of 131I-metuximab as an adjuvant therapy after HCC resection. METHODS: This randomised, controlled, multicentre, open-label, phase 2 trial was done at five medical centres in China. Patients aged 18-75 years who underwent curative-intent resection of histologically confirmed HCC expressing CD147 were randomly assigned (1:1) by a computer-generated random sequence, stratified by centre, to receive either adjuvant transarterial injection of one dose of 27·75 MBq/kg 131I-metuximab 4-6 weeks after the hepatectomy (treatment group) or no adjuvant treatment (control group). Patients and physicians were not masked to the study groups. The primary outcome was 5-year recurrence-free survival (RFS) in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, NCT00819650. FINDINGS: Between April 1, 2009, and Nov 30, 2012, 485 patients were screened for eligibility. 329 (68%) of these patients were excluded and 156 (32%) were randomly assigned to receive either 131I-metuximab (n=78) or no adjuvant treatment (n=78). The median follow-up was 55·9 months (IQR 18·6-79·4). In the intention-to-treat population, the 5-year RFS was 43·4% (95% CI 33·6-55·9) in the 131I-metuximab group and 21·7% (14·2-33·1) in the control group (hazard ratio 0·49 [95% CI 0·34-0·72]; Z=2·96, p=0·0031). 131I-metuximab-associated adverse events occurred within the first 4 weeks in 34 (45%) of 76 patients, seven (21%) of whom had grade 3 or 4 adverse events. These adverse events were all resolved with appropriate treatment within 2 weeks of being identified. INTERPRETATION: Adjuvant 131I-metuximab treatment significantly improved the 5-year RFS of patients after hepatectomy for HCC tumours expressing CD147. This treatment was well tolerated by patients. FUNDING: State Key Project on Infectious Diseases of China.

Maillard Reaction Products of Stir Fried Hordei Fructus Germinatus Are Important for Its Efficacy in Treating Functional Dyspepsia.

Hordei Fructus Germinatus (HFG) has been used as a traditional medicine to treat functional dyspepsia (FD) in China. Stir fried HFG (F-HFG) containing Maillard reaction products (MRPs) is used more widely than the raw HFG (R-HFG). However, the exact mechanisms in its functionality remain unclear. This article investigated the effect of R-HFG, F-HFG, and MRPs on brain-gut peptides, gut microbiota, and digestive enzymes using an FD animal mode. After administration of R-HFG, F-HFG, and MRPs, higher mRNA expression level of gastrin (GAS) and lower mRNA expression level of vasoactive intestinal peptide (VIP) were exhibited in F-HFG and MRPs rats than R-HFG rats (P < .05). Furthermore, compared with the R-HFG group, the contents of motilin (MTL) and GAS showed an upward tendency, whereas the contents of VIP and chokcystokinin (CCK) showed a downward tendency in the F-HFG group. In addition, bacterial communities in the control, F-HFG, and MRPs groups clustered closely to one another, and bacterial communities in the model and recovery groups clustered together, whereas the bacterial communities in the R-HFG group were clustered into a category. Moreover, there were no apparent differences in brain-gut peptides and gut microbiota between the F-HFG and MRPs groups. However, after the oral administration of R-HFG, F-HFG, and MRPs, the level of digestive enzyme did not show a significant change as compared with the recovery group. These results indicated that the stronger effect of F-HFG could be attributed to the MRPs produced during stir frying, and MRPs possessed the effect of regulating brain-gut peptides and gut microbiota.

Combined Antiangiogenic Therapy and Immunotherapy Is Effective for Pancreatic Cancer With Mismatch Repair Proficiency but High Tumor Mutation Burden: A Case Report.

Immunotherapy has been recommended as a second-line treatment only for high microsatellite instability or DNA mismatch repair deficiency advanced pancreatic cancer in National Comprehensive Cancer Network guidelines. Here, we report a case with a good response to immunotherapy in pancreatic cancer with mismatch repair proficiency. A 55-year-old woman diagnosed with pancreatic cancer cT4N1M1 (liver, lung) who harbored ERBB2 mutations with high tumor mutation burden (TMB) underwent multiple therapies and survived 19 months. A partial response in pancreatic cancer was observed when the patient was treated with combined antiangiogenic therapy and immunotherapy after a series of ineffective treatments. Neutrophil-to-lymphocyte ratio (NLR), a predictive marker of efficacy of immunotherapy, confirmed that immunotherapy resulted in the partial response in pancreatic cancer. To our knowledge, this is the first to report advanced pancreatic cancer with mismatch repair proficiency had a good response to immunotherapy, and this is the first to report an association between high blood-based TMB or NLR and improved clinical outcomes in pancreatic cancer. Therefore, TMB may also be a biomarker for immunotherapy of pancreatic cancer, and NLR may be a prospective predictive marker for efficacy of immunotherapy in pancreatic cancer.

Effect of mild moxibustion on intestinal microbiota and NLRP6 inflammasome signaling in rats with post-inflammatory irritable bowel syndrome.

BACKGROUND: About one-third of refractory irritable bowel syndrome (IBS) cases are caused by gastrointestinal (GI) infection/inflammation, known as post-infectious/post-inflammatory IBS (PI-IBS). Although it is known that intestinal microbiota and host NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammsome signaling are closely related to PI-IBS and moxibustion has a therapeutic effect on PI-IBS, whether moxibustion regulates the intestinal flora and host NLRP6 events in PI-IBS remains unclear. AIM: To examine the regulatory effect of moxibustion on intestinal microbiota and host NLRP6 inflammatory signaling in PI-IBS. METHODS: Sprague-Dawley rats were divided into a normal control group, a model control group, a mild moxibustion group, and a sham mild moxibustion group. PI-IBS rats in the mild moxibustion group were treated with moxibusiton at bilateral Tianshu (ST 25) and Zusanli (ST36) for 7 consecutive days for 10 min each time. The sham group rats were given the same treatment as the mild moxibustion group except the moxa stick was not ignited. Abdominal withdrawal reflex (AWR) score was measured to assess the visceral sensitivity, and colon histopathology and ultrastructure, colonic myeloperoxidase (MPO) activity, and serum C-reactive protein (CRP) level were measured to evaluate low-grade colonic inflammation in rats. The relative abundance of selected intestinal bacteria in rat feces was detected by 16S rDNA PCR and the NLRP6 inflammsome signaling in the colon was detected by immunofluorescence, qRT-PCR, and Western blot. RESULTS: The AWR score was significantly decreased and the low-grade intestinal inflammation reflected by serum CRP and colonic MPO levels was inhibited in the mild moxibustion group compared with the sham group. Mild moxibustion remarkably increased the relative DNA abundances of Lactobacillus, Bifidobacterium, and Faecalibacterium prausnitzii but decreased that of Escherichia coli in the gut of PI-IBS rats. Additionally, mild moxibustion induced mRNA and protein expression of intestine lectin 1 but inhibited the expression of IL-1ß, IL-18, and resistance-like molecule ß by promoting the NLRP6 and reducing the mRNA and protein expression of apoptosis-associated speck-like protein containing CARD (ASC) and cysteinyl-aspartate-specific proteinase 1 (Caspase-1). The relative DNA abundances of Lactobacillus, Bifidobacteria, Faecalibacterium prausnitzii, and Escherichia coli in each group were correlated with the mRNA and protein expression of NLRP6, ASC, and Caspase-1 in the colon. CONCLUSION: These findings indicated that mild moxibustion can relieve low-grade GI inflammation and alleviate visceral hypersensitivity in PI-IBS by regulating intestinal microbes and controlling NLRP6 inflammasome signaling.

Herbs-partitioned moxibustion alleviates aberrant intestinal epithelial cell apoptosis by upregulating A20 expression in a mouse model of Crohn's disease.

BACKGROUND: A20 inhibits intestinal epithelial cell apoptosis in Crohn's disease, and herbs-partitioned moxibustion (HPM) has been demonstrated to be an effective treatment for Crohn's disease. However, the mechanism by which HPM reduces intestinal epithelial cell apoptosis in Crohn's disease has not been thoroughly elucidated to date. AIM: To elucidate whether HPM exerts its effects by upregulating A20 to affect intestinal epithelial cell apoptosis in a Crohn's disease mouse model. METHODS: In this study, mice with A20 deletion in intestinal epithelial cells (A20IEC-KO) were utilized to establish a Crohn's disease mouse model with 2,4,6-trinitrobenzene sulfonic acid (TNBS) administration, as well as wild-type mice. Mice were randomly divided into normal control (NC), model control (MC), mesalazine (MESA), and HPM groups. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and serum endotoxin and apoptosis of epithelial cells were evaluated by enzyme-linked immunosorbent assay and terminal dUTP nick-end labeling assay accordingly. The protein expression levels of A20 and tumor necrosis factor receptor 1 (TNFR1)-related signaling molecules were evaluated by Western blot, and co-expression of A20 and TNFR1-associated death domain (TRADD) and co-expression of A20 and receptor-interacting protein 1 (RIP1) were observed by double immunofluorescence staining. RESULTS: The intestinal epithelial barrier was noted to have an improvement in the HPM group of wild-type (WT) mice compared with that in A20IEC-KO mice. Compared with A20 IEC-KO HPM mice, serum endotoxin levels and apoptosis percentages were decreased (P < 0.01), A20 expression levels were increased (P < 0.01), and expression of TNFR1, TRADDD, and RIP1 was decreased in the HPM group of WT mice (P TNFR1 < 0.05, P TRADD < 0.01, P RIP1 < 0.01). Both of the co-expression of A20/TRADD and A20/RIP1 showed a predominantly yellow fluorescence in the HPM group of WT mice, while a predominantly red fluorescence was noted in the HPM group of A20IEC-KO mice. CONCLUSION: Our findings suggest that HPM in treating Crohn's disease functions possibly via upregulation of the A20 expression level, resulting in downregulation of TNFR1, TRADD, and RIP1 to alleviate increased cell apoptosis in the intestinal epithelial barrier in Crohn's disease.

[Preparation and evaluation of four kinds of mixed essential oil liposomes in Jieyu Anshen Formula].

In order to increase the stability and solubility of essential oil in Jieyu Anshen Formula, this study was to prepare the essential oil into liposomes. In this experiment, the method for the determination of encapsulation efficiency of liposomes was established by ultraviolet spectrophotometer and dextran gel column. The encapsulation efficiency and particle size of liposomes were used as evaluation indexes for single factor investigation and Box-Behnken design-response surface method was used to optimize the design. Then the optimal formulation of volatile oil liposome was characterized using methyleugenol, elemin, ß-asarone and α-asarone as index components. Finally, the in vitro transdermal properties of liposomes were studied by modified Franz diffusion cell. The results showed that the concentration of lecithin, the mass ratio of lecithin to volatile oil, and the stirring speed were the three most significant factors affecting the liposome preparation. The optimum formulation of volatile oil liposome was as follows: the concentration of lecithin was 7 g·L~(-1); mass ratio of lecithin to volatile oil was 5∶1; and the stirring speed was 330 r·min~(-1). Under such conditions, the prepared liposomes had blue emulsion light, good fluidity, half translucent, with particle size of(102.6±0.35) nm, Zeta potential of(-17.8±0.306) mV, permeability of(1.67±1.01)%, and stable property if liposome was stored at 4 ℃. 24 h after percutaneous administration, the cumulative osmotic capacity per unit time was(30.485 2±1.238 9),(34.794 8±0.928 3),(26.677 1±1.171 7),(3.066 2±0.175 3) µg·cm~(-2)respectively for methyleugenol, elemin, ß-asarone and α-asarone. In vitro transdermal behaviors of methyleugenol, elemin, ß-asarone and α-asarone in liposomes were all consistent with Higuchi equation. The prepared volatile oil liposomes met the relevant quality requirements, providing a reference for further research on preparation of multi-component Chinese medicine essential oil liposomes.