Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC 50 13 nM against mouse TRPC6, t 1/2 8.5-13.5 hours) with 85-and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis. TRPC6 | ion channels | calcium | nuclear factor of activated T cells | fibrosis

The emerging evidence supports the use of prebiotics like herb-derived polysaccharides for treating nonalcoholic fatty liver disease (NAFLD) by modulating gut microbiome. The present study was initiated on the microbiota-dependent anti-NAFLD effect of Astragalus polysaccharides (APS) extracted from Astragalus mongholicus Bunge in high-fat diet (HFD)-fed mice. However, the exact mechanisms underlying the beneficial effects of APS on NAFLD formation remain poorly understood.Co-housing experiment was used to assess the microbiota dependent anti-NAFLD effect of APS. Then, targeted metabolomics and metagenomics were adopted for determining short-chain fatty acids (SCFAs) and bacteria that were specifically enriched by APS. Further in vitro experiment was carried out to test the capacity of SCFAs-producing of identified bacterium. Finally, the anti-NAFLD efficacy of identified bacterium was tested in HFD-fed mice.Our results first demonstrated the anti-NAFLD effect of APS in HFD-fed mice and the contribution of gut microbiota. Moreover, our results indicated that SCFAs, predominantly acetic acid were elevated in APS-supplemented mice and ex vivo experiment. Metagenomics revealed that D. vulgaris from Desulfovibrio genus was not only enriched by APS, but also a potent generator of acetic acid, which showed significant anti-NAFLD effects in HFD-fed mice. In addition, D. vulgaris modulated the hepatic gene expression pattern of lipids metabolism, particularly suppressed hepatic fatty acid synthase (FASN) and CD36 protein expression.Our results demonstrate that APS enriched D. vulgaris is effective on attenuating hepatic steatosis possibly through producing acetic acid, and modulation on hepatic lipids metabolism in mice. Further studies are warranted to explore the long-term impacts of D. vulgaris on host metabolism and the underlying mechanism.

Aging is usually characterized with inflammation and disordered bile acids (BAs) homeostasis, as well as gut dysbiosis. The pathophysiological changes during aging are also sexual specific. However, it remains unclear about the modulating process among gut microbiota, BA metabolism, and inflammation during aging. In this study, we established a direct link between gut microbiota and BA profile changes in the liver, serum, and four intestinal segments of both sexes during aging and gut microbiota remodeling by co-housing old mice with young ones. We found aging reduced Actinobacteria in male mice but increased Firmicutes in female mice. Among the top 10 altered genera with aging, 4 genera changed oppositely between male and female mice, and most of the changes were reversed by co-housing in both sexes. Gut microbiota remodeling by co-housing partly rescued the systemically dysregulated BA homeostasis induced by aging in a sex-and tissuespecific manner. Aging had greater impacts on hepatic BA profile in females, but intestinal BA profile in males. In addition, aging increased hepatic and colonic deoxycholic acid in male mice, but reduced them in females. Moreover, muricholic acids shifted markedly in the intestine, especially in old male mice, and partially reversed by co-housing. Notably, the ratios of primary to secondary BAs in the liver, serum, and all four intestinal segments were increased in old mice and reduced by cohousing in both sexes. Together, the presented data revealed that sex divergent changes of gut microbiota and BA profile in multiple body compartments during aging and gut microbiota remodeling, highlighting the sex-specific prevention and treatment of aging-related disorders by targeting gut microbiota-regulated BA metabolism should particularly be given more attention.

Most herbal polysaccharides possess multiple benefits against metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD) and obesity. However, the underlying mechanisms are largely unknown. Here, male C57BL/6J mice were fed with chow or high-fat diet (HFD) with or without Astragalus polysaccharides (APS) supplementation, and gut microbial profile and metabolite profile were studied by metagenomic sequencing and untargeted metabolomics, respectively. APS was effective in alleviating HFD-induced metabolic disorders, with the alteration of gut microbiota composition and function. A total of 188 species, which mainly from Bacteroidetes, Actinobacteria, Firmicutes, and Proteobacteria phyla, and 36 metabolites were markedly changed by HFD and revered by APS. Additionally, the altered glutathione metabolism and purine metabolism pathways were identified by both metagenomic function analysis and metabolite pathway enrichment analysis. Furthermore, the gut microbial alteration was associated with the changes of key intestinal metabolites. We found 31 and 20 species were correlated with purine metabolism and glutathione metabolism, respectively. Together, our results showed significant metagenomic and metabolomic changes after HFD feeding and APS intervention, revealed the potential correlation between gut microbial species and metabolites, and highlighted mechanisms of herb-derived polysaccharides by modulating gut microbiome and host metabolism underlying their benefits on metabolic disorders.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.