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Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Here, we generate multi-omic profiling of 257 primary and 176 metastatic regions from 182 HCC patients. Primary tumors rich in hypoxia signatures facilitated polyclonal dissemination. Genomic divergence between primary and metastatic HCC is high, and early dissemination is prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases is associated with decreased T cell reactivity, resulting from disruptions to neoantigen presentation. We identify somatic copy number alterations as highly selected events driving metastasis. Subclones without Wnt mutations show a stronger selective advantage for metastasis than those with Wnt mutations and are characterized by a microenvironment rich in activated fibroblasts favoring a pro-metastatic phenotype. Finally, metastases without Wnt mutations exhibit higher enrichment of immunosuppressive B cells that mediate terminal exhaustion of CD8 + T cells via HLA-E:CD94-NKG2A checkpoint axis. Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Rhesus monkeys are increasingly used in biomedical research, which makes their hematological and biochemical parameters increasingly important in preclinical research. Since age and sex can influence blood parameters, establishing reference intervals for such parameters based on age and sex becomes along with identifying the effect of age and sex on those parameters., Methods: A total of 1385 healthy Chinese rhesus monkeys (548 males and 837 females) anesthetized with ketamine were selected and segregated by age (six groups) and sex. A total of 21 hematological and 26 biochemical parameters were measured, and the effects of age and sex were analyzed., Results: We established baseline indices for hematological and biochemical parameters based on age and sex, separately, and observed significant impacts of age, sex, and age-sex interactions on blood parameters. Among different age groups, significant differences were found in WBC, NEUT%, LYM%, EO%, LYM#, EO#, MCV, RDW-CV, PLT, MPV, PDW, PCT, TP, Alb, GLB, A/G, ALT, AST, ALP, TBIL, GGT, BUN, Cre, GLU, CK, TRIG, LDL, HCY, IL-6 FOL, Vit B12, VIT D-T, PTH, and AMH. Additionally, significant differences were observed in RBC, HGB, HCT, MPV, Alb, BUN, Cre, GLU, CHOL, TRIG, HDL, LDL, HCY, and VIT D-T between the two sexes. An age-sex interaction exerted a significant effect on WBC, NEUT#, MCV, MCHC, PDW, GLB, ALP, Cre, CHOL, TRIG, HDL, LDL, HCY, IL-6, Vit B12, VIT D-T. However, neither age, sex, and age-sex interactions exerted significant effects on MO%, MOMO#, MCH, RDW-SD, CRP, and CT., Conclusion: Our study investigated the blood parameters of rhesus monkeys to provide a reference basis for rhesus monkey-related scientific experimental research., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

To investigate the potential effects and mechanism of wogonin on dextran sulfate sodium (DSS)-induced colitis, 70 male mice were administered wogonin (12.5, 25, 50 mg·kg -1 ·d -1 , i.g.) for 10 days, meanwhile, in order to induce colitis, the mice were free to drink 3% DSS for 6 days. We found that wogonin could obviously ameliorate DSS-induced colitis, including preventing colon shortening and inhibiting pathological damage. In addition, wogonin could increase the expression of PPARγ, which not only restores intestinal epithelial hypoxia but also inhibits iNOS protein to reduce intestinal nitrite levels. All these effects facilitated a reduction in the abundance of Enterobacteriaceae in DSS-induced colitis mice. Therefore, compared with the DSS group, the number of Enterobacteriaceae in the intestinal flora was significantly reduced after administration of wogonin or rosiglitazone by 16s rDNA technology. We also verified that wogonin could promote the expression of PPARγ mRNA and protein in Caco-2 cells, and this effect disappeared when PPARγ signal was inhibited. In conclusion, our study suggested that wogonin can activate the PPARγ signal of the Intestinal epithelium to ameliorate the Intestinal inflammation caused by Enterobacteriaceae bacteria expansion., (© 2022 John Wiley & Sons Ltd.)

Ethnopharmacological Relevance: Gegen Qinlian decoction (GQD) is a traditional Chinese medicine derived from Treatise on febrile diseases and is clinically used for the treatment of acute ulcerative colitis (UC). However, the potential mechanism of GQD treatment for UC remains elusive., Aim of Study: In this study, we aimed to explore the involvement of gut microbiota-related tryptophan metabolism in mediating protective effects of GQD against intestinal barrier damage., Materials and Methods: Mice with colitis were treated with 3% dextran sulfate sodium (DSS) for 6 days. The therapeutic effects of GQD in UC mice were examined based on body weight, disease activity index (DAI), organ index, length and pathological changes in the colon. The distribution of fluorescein isothiocyanate dextran (FITC-dextran) in the intestinal tract was observed using small animal imaging, while concentration of FITC-dextran in serum was detected using a fluorescein microplate analyser. Bacterial infiltration in colon tissues was observed by fluorescence in situ hybridisation (FISH), and the bacterial load in mesenteric lymph nodes (MLNs) was further examined through bacterial culture. Subsequently, colonic goblet cells were detected using Alcian blue staining. The tight junctions of the colonic epithelium were observed using transmission electron microscopy, and the expression of tight junction proteins was detected by immunofluorescence (IF) and western blot. In addition, flow cytometry was used to analyse the proportion of interleukin-22-positive (IL-22 + ) ILC3 cells in lamina propria lymphocytes, and the content of IL-22 in colon homogenates was determined using an ELISA kit. In addition, targeted tryptophan metabolomics was used to detect the concentration of indole derivatives produced by tryptophan metabolism in faeces, and 16S rDNA was used to investigate the composition and abundance of gut microbiota-related tryptophan metabolism., Results: Administration of GQD significantly alleviated the pathological symptoms, including weight loss, increased DAI score, changes in organ index, colon shortening, and colon pathological injury in UC mice. In addition, GQD reduced the diffusion of FITC-dextran in the intestinal tract, the content of FITC-dextran in serum, and bacterial infiltration in MLNs and colon tissues. Additionally, GQD significantly increased the number of colonic goblet cells, repaired the structure of epithelial tight junctions and increased the expression of tight junction proteins. Furthermore, GQD significantly increased the proportion of IL-22 + ILC3 in the lamina propria, the expression of CYP1A1 protein in colon tissue, and the level of IL-22 in colon homogenates. However, the above protective effects of GQD were inhibited by co-administration of GQD and aryl hydrocarbon receptor (AhR) antagonist. Additionally, GQD restored the content of indole derivatives generated by tryptophan metabolism, regulated the diversity of the gut microbiota, and significantly increased the abundance of genes related to tryptophan metabolism., Conclusion: Our results confirmed that GQD repaired the damaged intestinal barrier in UC mice by regulating gut microbiota-related tryptophan metabolism and restoring the generation of indole derivatives to activate AhR-mediated IL-22 production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Dahuang Mudan decoction (DMD) is a classic prescription for treating intestinal carbuncle from Zhang Zhongjing's "Essentials of the Golden Chamber" in the Han Dynasty. Recent studies also prove that DMD has a therapeutic effect on ulcerative colitis (UC), but its mechanism is still unclear., Aim of Study: In this study, we aim to assess the therapeutic effect of DMD on DSS-induced chronic colitis in mice and deeply expound its underlying regulative mechanism., Materials and Methods: The efficacy of DMD on mice with 2% DSS-induced chronic colitis was examined by changes in mouse body weight, DAI score, colon length changes, peripheral blood white blood cells (WBC) and red blood cells (RBC) counts, and hemoglobin (HGB) content, using mesalazine as a positive control. A small animal imaging system observed the FITC-Dextran fluorescence distribution in mice, and the contents of IL-22 and IL-17A in colon tissue homogenate supernatant and LPS in peripheral blood were detected by ELISA. Fluorescence in situ molecular hybridization and bacterial culture were used to investigate bacterial infiltration in intestinal mucosa and bacterial translocation in mesenteric lymph nodes and spleen. Mice immune function was further evaluated by analyzing the changes in spleen index, thymus index, and the ratio of peripheral blood granulocytes, monocytes, and lymphocytes. Meanwhile, the proportion of NCR + group 3 innate lymphoid cells (ILC3), NCR - ILC3, and IL-22 + ILC3 in colonic lamina propria lymphocytes of mice was detected by flow cytometry. The contents of effectors IL-22, IL-17A, and GM-CSF were detected by RT-PCR. We use cell scratching to determine the effect of DMD conditioned medium on the migration of Caco-2 cells by establishing an in vitro model of MNK-3 conditioned medium (CM) intervening Caco-2 cells. RT-PCR and WB detect the expression of tight junction ZO-1, Occludin, and Claudin-1., Results: DMD restored the body weight, colon length, peripheral blood RBC numbers, and HGB content of chronic colitis mice and reduced peripheral blood WBC and colon inflammatory cell infiltration. Moreover, DMD decreased LPS content in serum, bacterial infiltration of colonic mucosa, and bacterial translocation in spleen and mesenteric lymph nodes. Simultaneously, DMD intensified the expression of ZO-1, Occludin, and Claudin-1, the ratio of NCR + ILC3 and IL-22 + ILC3, and decreased the proportion of NCR - ILC3. In vitro studies also confirmed that the conditioned medium of DMD promoted the migration of Caco-2 cells and the expression of tight junction proteins., Conclusion: Our results confirm that DMD improves inflammation and restores intestinal epithelial function in mice with chronic colitis, and the mechanism may be related to regulating ILC3 function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Context: Chinese herb Huangqin decoction (HQD) can regulate intestinal flora in ulcerative colitis (UC) mice., Objective: Our study clarifies the mechanism of HQD in regulating the intestinal flora of UC mice., Materials and Methods: Male C57BL/6 mice were randomly divided into six groups: Control, Model (3% DSS), Sulfasalazine (500 mg/kg), HQD-L (250 mg/kg), HQD-M (500 mg/kg), and HQD-H (1000 mg/kg) groups. Measurement of body weight, colon length, DAI, and haematoxylin-eosin staining were conducted. FISH and 16S rDNA detected colonic bacterial infiltration and intestinal flora changes. The expression of RegIIIγ and PRRs (NOD2, TLR5, TLR4) were detected by FCM and WB, respectively. In addition, WB, qPCR, or IHC were used to detect the expression of NOD2, MyD88, RIP2, and NF-κB p65 in the colon. ELISA was used to determine cytokines., Results: Compared with the model group (DAI score, 2.38 ± 0.05; histological score, 4.08 ± 0.54), HQD treatment significantly reduced the DAI score (L, 2.16 ± 0.09; M, 1.45 ± 0.05; H, 1.18 ± 0.05) and histological score (L, 3.16 ± 0.82; M, 2.50 ± 0.81; H, 1.51 ± 0.76); restored the weight, the colonic length ( p  < 0.05). 16S rDNA identification showed HQD regulated the balance of intestinal flora. Moreover, HQD suppressed the expression of RegIIIγ ( p  < 0.05) and prevented colonic bacterial infiltration. Furthermore, WB results showed NOD2, and TLR4 were inhibited by HQD, especially NOD2 ( p  < 0.01). The data of WB, qPCR, and IHC demonstrated that the NOD2-dependent pathway was inhibited by HQD ( p  < 0.01)., Discussion and Conclusions: HQD (1000 mg/kg) regulates the intestinal flora of colitis mice, mainly characterized as inhibition of the NOD2-dependent pathway. These results indicate that HQD has potential.

Background: Emodin is an active ingredient of traditional Chinese medicine Rheum palmatum L. and Polygonum cuspidatum, which possesses anti-inflammatory and intestinal mucosal protection effects. Our previous study found that emodin significantly alleviated ulcerative colitis induced by sodium dextran sulfate (DSS). In this study, we found the underlying mechanism of emodin on ulcerative colitis (UC)., Purpose: We aimed to further explore the mechanism of emodin in the treatment of ulcerative colitis from the perspective of metabolism and intestinal flora., Methods: Ulcerative colitis was induced by 3% sodium dextran sulfate (DSS) on mice, and the mice were respectively treated with mesalazine, rosiglitazone, emodin, and emodin combined with GW9662 (PPARγ inhibitor) simultaneously. Weight changes, the disease activity index (DAI), colonic length, and pathologic changes in colon were used to evaluate the efficacy of emodin. LC-MS/MS was performed for metabolomics analysis of colon. In addition, intestinal flora was assessed using 16S rDNA sequencing. A vector-based short hairpin RNA (shRNA) method was used to silence PPARγ gene expression in Caco-2 cells., Results: Emodin binds to the active site of PPARγ protein and forms hydrogen bond interaction with ARG288 and CYS285 amino acids. Furthermore, Emodin significantly promotes the protein expression of PPARγ, while inhibiting iNOS and NF-kB p65 in UC mice, however, this effect is hardly shown when it is combined with GW9662 (the inhibitor of PPARγ). Meanwhile, emodin suppresses the expression of iNOS in Caco-2 cells induced with IFNγ and IL-22, but has no effect on its expression in shPPARγ-Caco-2 cells. In addition, through activating PPARγ signal pathway, emodin is capable of regulating colonic metabolism including oxidative phosphorylation and citrulline metabolism and effecting luminal availability of oxygen and nitrate. This promotes the recovery of anoxic environment of colon epithelial cells, which strains the growth and expansion of Enterobacteriaceae., Conclusion: The mechanism of Emodin in the treatment of ulcerative colitis relies on its regulation of PPARγ signal pathway, which could modulate colonic metabolism and restore intestinal homeostasis., (Copyright © 2022. Published by Elsevier GmbH.)

As one of the ligands of aryl hydrocarbon receptor (AhR), baicalein, isolated from Scutellaria baicalensis Georgi , has been proved to exert potential therapeutic effects on ulcerative colitis (UC), but its therapeutic mechanism remains obscure. Authentically, ulcerative colitis can be alleviated by regulating the differentiation of naïve CD4 + T cells via AhR activation. So, our study planned to prove the hypothesis that baicalein protected mice against UC by regulating the balance of Th17/Treg cells via AhR activation. Immunofluorescence and western blot results showed that baicalein could promote AhR activation and induce it to transfer to the nucleus. We further determined the effect of baicalein on naïve CD4 + T cell differentiation in vitro by magnetic cell separation and drug intervention. The results showed that baicalein could promote Treg cell differentiation by activating AhR. In vivo study, UC mice were established by free drinking of dextran sulfate sodium (DSS) for 7 days and then were orally administrated by baicalein (10, 20, and 40 mg/kg), TCDD (AhR agonist), and CH223191 (antagonist). The results demonstrated that baicalein improved the symptoms of UC mice, regulated the balance of Th17/Treg cells, and restored the balance of proinflammatory cytokines such as IL-17, IL-6, and TNF- α ; anti-inflammatory cytokines such as IL-10 and TGF- β ; and epithelial protective cytokine IL-22 in UC mice, and these effects were related to AhR. Taken together, our research found that baicalein might be a potential drug for UC via regulating Treg cell differentiation and maintaining immune homeostasis and attempted to shed a light on the pivotal role of AhR in these effects., Competing Interests: All authors do not have conflicts of interest to declare., (Copyright © 2020 Chang Liu et al.)

Currently, the potential role of the alterations occurring in the liver immune system and intestinal flora in liver injury remains unknown. Our study aimed to explore the impacts of intestinal microbial barrier damage induced by ceftriaxone on liver immunity. We developed the BALB/c mice model by administering ceftriaxone. The intestinal microbial barrier damage was observed by 16S rRNA, and the pathological changes of intestines and livers were detected by H&E or transmission electron microscope. The activation of immunocytes were tested by Flow Cytometry; the expression of LPS, ALT, AST, IL-6 and TNF-α were detected by Limulus Test or ELISA. Compared to the control, the intestinal microbes significantly decreased in ceftriaxone group. Additionally, the weight of cecum contents increased, the intestinal wall became thinner and the villus in the small intestine and cecum were damaged. The expression of LPS and the ratio of liver lymphocytes were significantly increased. H&E results indicated the structures of liver arose the pathologic changes. Meanwhile, the content of serum ALT, AST, IL-6 and TNF-α increased. Collectively, our study indicates that the damages of gut microbial barrier induced by ceftriaxone prompted activation of immunocytes and release of inflammatory cytokines, which may lead to chronic inflammation in liver., (© FEMS 2020.)