ASKA technology-based pull-down method reveals a suppressive effect of ASK1 on the inflammatory NOD-RIPK2 pathway in brown adipocytes.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

GeGen QinLian decoction alleviate influenza virus infectious pneumonia through intestinal flora.

Influenza in humans is often accompanied by gastroenteritis-like symptoms. GeGen QinLian decoction (GQD), a Chinese herb formula, has been widely used to treat infectious diarrhea for centuries and has the effect of restoring intestinal flora. Studies have also reported that GQD were used to treat patients with influenza. However, whether regulating the intestinal flora is one of the ways GQD treats influenza has not been confirmed. In present research, we conducted a systemic pharmacological study, and the results showed that GQD may acts through multiple targets and pathways. In influenza-infected mice, GQD treatment reduced mortality and lung inflammation. Most importantly, the mortality and lung inflammation were also reduced in influenza-infected mice that have undergone fecal microbiota transplantation (FMT) from GQD (FMT-GQD) treated mice. GQD treatment or FMT-GQD treatment restores the intestinal flora, resulting in an increase in Akkermansia_muciniphila, Desulfovibrio_C21_c20 and Lactobacillus_salivarius, and a decrease in Escherichia_coli. FMT-GQD treatment inhibited the NOD/RIP2/NF-κB signaling pathway in the intestine and affected the expression of downstream related inflammatory cytokines in mesenteric lymph nodes (mLNs) and serum. In addition, FMT-GQD treatment showed systemic protection by restraining the inflammatory differentiation of CD4+ T cells. In conclusion, our study shows that GQD can affect systemic immunity, at least in part, through the intestinal flora, thereby protect the mice against influenza virus infectious pneumonia.

Regulation of temozolomide resistance in glioma cells via the RIP2/NF-κB/MGMT pathway.

BACKGROUND: Temozolomide (TMZ) is a first-line chemotherapy drug for the treatment of malignant glioma and resistance to it poses a major challenge. Receptor-interacting protein 2 (RIP2) is associated with the malignant character of cancer cells. However, it remains unclear whether RIP2 is involved in TMZ resistance in glioma. METHODS: RIP2 expression was inhibited in TMZ-resistant glioma cells and normal glioma cells by using small interfering RNA (siRNA) against RIP2. Plasmid transfection method was used to overexpress RIP2. Cell counting kit-8 assays were performed to evaluate cell viability. Western blotting or immunofluorescence was performed to determine RIP2, NF-κB, and MGMT expression in cells. Flow cytometry was used to investigate cell apoptosis. TMZ-resistant glioma xenograft models were established to evaluate the role of the RIP2/NF-κB/MGMT signaling pathway in drug resistance. RESULTS: We observed that RIP2 expression was upregulated in TMZ-resistant glioma cells, whereas silencing of RIP2 expression enhanced cellular sensitivity to TMZ. Similarly, upon the induction of RIP2 overexpression, glioma cells developed resistance to TMZ. The molecular mechanism underlying the process indicated that RIP2 can activate the NF-κB signaling pathway and upregulate the expression of O6-methylguanine-DNA methyltransferase (MGMT), following which the glioma cells develop drug resistance. In the TMZ-resistant glioma xenograft model, treatment with JSH-23 (an NF-κB inhibitor) and lomeguatrib (an MGMT inhibitor) could enhance the sensitivity of the transplanted tumor to TMZ. CONCLUSION: We report that the RIP2/NF-κB/MGMT signaling pathway is involved in the regulation of TMZ resistance. Interference with NF-κB or MGMT activity could constitute a novel strategy for the treatment of RIP2-positive TMZ-resistant glioma.