Eggshell membrane modulates gut microbiota to prevent murine pre‐cachexia through suppression of T helper cell differentiation

Abstract

Background Cachexia is a life-threatening condition observed in several pathologies, such as cancer or chronic diseases. Interleukin 10 (Il10) gene transfer is known to improve cachexia by downregulating Il6. Here, we used an IL10-knockout mouse model to simulate cachexia and investigate the effects of eggshell membrane (ESM), a resistant protein, on general pre-cachexia symptoms, which is particularly important for the development of cachexia therapeutics. Methods Five-week-old male C57BL6/J mice were fed an AIN-93G powdered diet (WT), and 5-week-old male B6.129P2-Il10 < tm1Cgn>/J (IL10 À/À ) mice were fed either the AIN-93G diet (KO) or an 8% ESM-containing diet (KOE) for 28 weeks. The tissue weight and levels of anaemia-, blood glucose-, lipid metabolism-, and muscular and colonic inflammation-related biochemical markers were measured. Transcriptomic analysis on liver and colon mucus and proteomic analysis on skeletal muscle were performed. Ingenuity Pathway Analysis was used to identify molecular pathways and networks. Caecal short-chain fatty acids (SCFAs) were identified using HPLC, and caecal bacteria DNA were subjected to metagenomic analysis. Flow cytometry analysis was performed to measure the CD4 + IL17 + T cells in mesenteric lymph nodes. ResultsThe body weight, weight of gastrocnemius muscle and fat tissues, colon weight/length ratio, plasma HDL and NEFA, muscular PECAM-1 levels (P < 0.01), plasma glucose and colonic mucosal myeloperoxidase activity (P < 0.05) and T helper (Th) 17 cell abundance (P = 0.071) were improved in KOE mice over KO mice. Proteomic analysis indicated the protective role of ESM in muscle weakness and maintenance of muscle formation (>1.5-fold). Transcriptomic analysis revealed that ESM supplementation suppressed the LPS/IL1-mediated inhibition of RXR function pathway in the liver and downregulated the colonic mucosal expression of chemokines and Th cell differentiation-related markers (P < 0.01) by suppressing the upstream BATF pathway. Analysis of the intestinal microenvironment revealed that ESM supplementation ameliorated the microbial alpha diversity and the abundance of microbiota associated with the degree of inflammation (P < 0.05) and increased the level of total organic acids, particularly of SCFAs such as butyrate (2.3fold), which could inhibit Th1 and Th17 production. Conclusions ESM supplementation ameliorated the chief symptoms of cachexia, including anorexia, lean fat tissue mass, skeletal muscle wasting and reduced physical function. ESM also improved colon and skeletal muscle inflammation, lipid metabolism and microbial dysbiosis. These results along with the suppressed differentiation of Th cells could be associated with the beneficial intestinal microenvironment and, subsequently, attenuation of pre-cachexia. Our findings provide insights into the potential of ESM in complementary interventions for pre-cachexia prevention.

The Loss of YTHDC1 in Gut Macrophages Exacerbates Inflammatory Bowel Disease

“…In contrast to wild‐type mice, the compositions of microbiotas in Il10 −/− mice are also changed. [ 24 ] In agreement with the notion that Il10 −/− mice would not develop colitis until 2 months, [ 15a ] we observed evident rectal prolapse in 8‐week‐old Il10 −/− mice but not in 4‐week‐old ones (Figure S9c, Supporting Information). Consistently, colitis development and microbiota dysbiosis were also observed in 8‐week‐old Il10 −/− mice (Figure S9d–f, Supporting Information).…”

Dietary intake of micronized avian eggshell membrane in aged mice reduces circulating inflammatory markers, increases microbiota diversity, and attenuates skeletal muscle aging

“…Since in vitro digestion demonstrated poor digestibility of ESM, particularly under the elderly gastrointestinal condition, the observed effects of ESM on skeletal muscle might be due to undigested ESM reaching the gut and interacting with the gut microbiota. Previous experiments in mice suffering from inflammatory bowel disease and pre-cachexia showed that ESM improved the microbiota dysbiosis, with significant change in Bacterioidetes, Firmicutes and Proteobacteria ( 13 , 25 ). The cecum microbiota was analyzed in all mice groups by 16S rRNA amplicon sequencing, showing that a diet containing 8% ESM had an impact on microbiota diversity and composition ( Figure 7 ).…”

“…Eggshell membrane (ESM) is an interesting biological material in this perspective due to its high content of bioactive compounds. Clinical trials and in vitro experiments have shown that ESM has promising benefits for joint health, wound healing, and skin health [reviewed by ( 12 , 13 )]. It is a low-cost by-product readily available as a remnant of the chicken egg processing (breaking) industry.…”

“…However, no further characterization at the molecular level of skeletal muscle was performed in their study. A recent study has shown that ESM prevents murine pre-cachexia in young mice ( 13 ). To the best of our knowledge, no studies have investigated the potential of ESM on skeletal muscle ageing.…”

Abnormal lipid metabolism in cancer-associated cachexia and potential therapy strategy

“…Another study implicates adipocytes as predominantly negative regulators of the surrounding myeloid cells ( 128 ). At the same time, studies have shown that the imbalance of the immune system affects the gut microbiota ( 129 ), which also plays an important role in CAC. One clinical study has shown that compared with non-cachexic people, Proteobacteria , an unknown genus from the Enterobacteriaceae family, and Veillonella were more abundant among CAC patients ( 130 ).…”