The Role Of Non-depolarizing Muscular Relaxants On Enhancing The Responses Of Inflammation And The Underlying Mechanism

We have known that there is cholinergic anti-inflammatory pathway (CAP), which efferent vagus nerve signals suppress the release of pro-inflammatory cytokine and relieve the inflammatory responses. There were proof that prove NMBA can bind to a7-nAChR and inhibit its function. So we suppose that NDMA can bind to a7-nAChR on the membrane of immune cells, and exacerbate the inflammatory responses. ObjectsTo confirm non depolarizing muscle relaxant (NDMA) can aggravate the inflammatory response from the following aspects, and to explore its mechanism:To discuss the excitation of different concentration of nicotine to a7-nAChR expressed on the surface of HEK 293 cells, to survey the inhibition of different concentration of NDMA to a7-nAChR expressed on the surface of HEK 293 cells.To observe different concentration of NDMA markedly reverse that 1 μM nicotine attenuate the release of TNF-a and IL-10 on LPS-stimulated human macrophage culture.To investigate that systemic, hepatic and splenic TNF-a amounts of endotoxemia in the rat which receive continually intravenous infusion of NDMA.MethodsWe creat α7 nAChR and RIC-3 plasmid, and transfect a7 nAChR alone or co-transefect α7 nAChR and RIC-3 to HEK293 cells. RT-PCR, western blot and IF were used to get the proof of α7 nAChR expression on the surface of HEK 293 cells. To observe the magnitude of excitation of different concentration of nicotine to a7 nAChR expression on HEK 293 cells.To observe different concentration of NDMA markedly reverse that 1 μM nicotine attenuate the release of TNF-α and IL-10 on LPS-stimulated human macrophage cultures. ELISA was used to determine the amounts of TNF-α and IL-10 in the supernatants.Animal model of endotoxemia were used for experiments to observe that different type of NDMA affect on the amounts of systemic, hepatic and splenic TNF-a, which were determined by ELISA.ResultsImmunofluorescence showed that detected α7-nAChR protein were expressed on HEK293 cells, calcium imaging showed that a7-nAChR alone expressed on HEK 293 cells was not functional.RT-PCR and western blot were used to detect the expression of RIC-3 and a7-nAChR from the level of mRN A and protein. And calcium imaging proved that RIC-3 can enhance the functional expression of a7-nAChR on HEK 293 cells.Calcium imaging showed that NDMA (10-9-10-7M) could inhibit the excitation of 30 μM nicotine to a7-nAChR functional expressed on HEK 293 cells.Immunofluorescence showed that a7-nAChR expressed on human macrophage naturally. ELISA proved that MLA(antagonist to a7-nAChR),NDMA all can reverse that 1 μM nicotine attenuate the release of TNF-α on LPS-stimulated human macrophage culture (P<0.05), but not the release of IL-10 (P>0.05)On animal model of endotoxemia, the systemic, hepatic and splenic TNF-a amounts of group rocuronium and cisatracurium were higher than group control which just received intravenous infusion of saline (P<0..05).ConclusionRIC-3 can enhance functional expression of α7-nAChR on HEK293 cells, and we express functional a7-nAChR on HEK293 cells.NDMA can inhibit the excitation of 30 μM nicotine to a7-nAChR which expressed functionally on HEK 293 cells.Non-depolarizing muscular relaxants (NDMA) can reverse that nicotine attenuate the release of TNF-a on LPS-stimulated human macrophage culture, but not the release of IL-10.NDMA which were continually infused through the veins can exacerbate the inflammatory responses of endotoxemic rat.In the conclusion, NDMA can exacerbate the inflammatory responses through blocking a7-nAChR.