RESUMEN

OBJECTIVE: Acupuncture is effective for irritable bowel syndrome (IBS); however, the mechanisms of action are not fully understood. We aim to explore the mechanism of electroacupuncture (EA) in the dual regulation of disorders of gut-brain interaction. METHODS: A rat model of IBS was generated by chronic unpredictable mild stress (CUMS). Eight of 32 rats were assigned to the blank control group. The remaining 24 rats received CUMS for 14 days. Then, the rats surviving and successfully modelled were randomly divided into the CUMS group, the CUMS+EA group, and the CUMS+PB (pinaverium bromide) group. In the next 14 days of treatment, rats in the CUMS+EA group were acupunctured at ST25 (Tianshu), ST36 (Zusanli), SP6 (Sanyinjiao), and LR3 (Taichong) for 15 min every day. Rats in the CUMS+PB group were treated by the administration of gavage with 2.7 mg/mL pinaverium every day. Visceral pain threshold, the percentage of time spent in open arms (OT%) in the elevated plus maze test (EPMT), and the sucrose preference (SP%) in the sucrose preference test (SPT) were measured at baseline, day 15, and day 30. The expression of zonula occludens-1 (ZO-1), the morphology of the connective structure of intestinal epithelium, the CRF and CRF-R1 mRNA expression in the hypothalamus, and the double staining of intestinal mucosal mast cells (IMMC) and CRF-R1 were measured at the end of the experiment. RESULTS: Compared with the blank control group, visceral pain threshold pressure, the expression of ZO-1, OT%, SP%, CRF, and CRF-R1 mRNA expression in the hypothalamus, and double staining of IMMC and CRF-R1 were decreased significantly in the CUMS group. Meanwhile, the morphology of the connective structure in the CUMS group was indistinct. Compared with the CUMS group, SP% was significantly increased in the CUMS+EA group, but there was no significant difference for it in the CUMS+PB group. The morphology of the connective structure in the two treatment groups was clear and seeable. And the expression of other parameters mentioned above was apparently increased in the two treatment groups. Compared with the CUMS+PB group, the expression of ZO-1 in the CUMS+EA group was significantly enhanced. And no obvious difference for other parameters was found between the two treatment groups. CONCLUSIONS: EA treatment can decrease the expression of hypothalamic CRF and CRF-R1, relieve anxiety and depression, meanwhile reduce the expression of CRF-R1 in the gastrointestinal mucosa, increase ZO-1 expression, and adjust tight junctions (TJs) to repair the intestinal mucosal barrier. The above roles suggest that EA may play a dual role in alleviating the gastrointestinal and psychological symptoms of IBS, suggesting a potentially dual therapeutic role for EA in regulating disorders of gut-brain interaction in IBS rats.

RESUMEN

Tyrosine phosphorylation, as a hallmark in cellular signal transduction, is important for a diverse array of cellular processes, such as proliferation, metabolism, motility, and survival. Aberrant tyrosine phosphorylation plays a causal role in many diseases, especially the cancer. Detecting protein phosphorylation status in the cancer cells or tissues is vital for assessing the pathological phase, discovering the cancer biomarkers, and identifying the drug targets. However, the common biochemical detection methods remain through anti-pTyr antibodies, which are known to have limited sensitivity, poor reproducibility and high cost. Recent studies have proved that superbinder SH2 domain is a good replacement of anti-pTyr antibodies for the specific enrichment of pTyr peptides in phosphoproteomics analysis. In this work, we exploited a series of affinity reagents based on superbinder SH2 derived from Src protein for detecting the pTyr-containing proteins to replace anti-pY antibodies in immunoblotting and immunofluorescence techniques. The excellent performance of HRP-sSH2 and EGFP-sSH2 was verified by the analysis of several different tumor cell samples and was compared with most commonly used commercial antibodies. EGFP-sSH2-(Arg)9 might be applied as the probe for direct fluorescence imaging in live cells via efficiently penetrating cell membranes and specifically binding with pTyr proteins. In summary, we have developed three novel, convenient, sensitive, and cost-effective affinity reagents that would have wide applications in protein tyrosine phosphorylation analysis for the tumor research and clinical diagnosis.

Asunto(s)

RESUMEN

BACKGROUND: Melanoma is a malignant tumor with high misdiagnosis rate and poor prognosis. The bio-targeted therapy is a prevailing method in the treatment of melanoma; however, the accompanying drug resistance is inevitable. SH2 superbinder, a triple-mutant of the Src Homology 2 (SH2) domain, shows potent antitumor ability by replacing natural SH2-containing proteins and blocking multiple pY-based signaling pathways. Polyarginine (Arg)9, a powerful vector for intracellular delivery of large molecules, could transport therapeutic agents across cell membrane. The purpose of this study is to construct (Arg)9-SH2 superbinder and investigate its effects on melanoma cells, expecting to provide potential new approaches for anti-cancer therapy and overcoming the unavoidable drug resistance of single-targeted antitumor agents. METHODS: (Arg)9 and SH2 superbinder were fused to form (Arg)9-SH2 superbinder via genetic engineering. Pull down assay was performed to identify that (Arg)9-SH2 superbinder could capture a wide variety of pY proteins. Immunofluorescence was used to detect the efficiency of (Arg)9-SH2 superbinder entering cells. The proliferation ability was assessed by MTT and colony formation assay. In addition, wound healing and transwell assay were performed to evaluate migration of B16F10, A375 and A375/DDP cells. Moreover, apoptosis caused by (Arg)9-SH2 superbinder was analyzed by flow cytometry-based Annexin V/PI. Furthermore, western blot revealed that (Arg)9-SH2 superbinder influenced some pY-related signaling pathways. Finally, B16F10 xenograft model was established to confirm whether (Arg)9-SH2 superbinder could restrain the growth of tumor. RESULTS: Our data showed that (Arg)9-SH2 superbinder had the ability to enter melanoma cells effectively and displayed strong affinities for various pY proteins. Furthermore, (Arg)9-SH2 superbinder could repress proliferation, migration and induce apoptosis of melanoma cells by regulating PI3K/AKT, MAPK/ERK and JAK/STAT signaling pathways. Importantly, (Arg)9-SH2 superbinder could significantly inhibit the growth of tumor in mice. CONCLUSIONS: (Arg)9-SH2 superbinder exhibited high affinities for pY proteins, which showed effective anticancer ability by replacing SH2-containing proteins and blocking diverse pY-based pathways. The remarkable ability of (Arg)9-SH2 superbinder to inhibit cancer cell proliferation and tumor growth might open the door to explore the SH2 superbinder as a therapeutic agent for cancer treatment.

Asunto(s)