The Effects Of α₇-ACh Receptors In Mouse Kupffer Cells On The Cholinergic Anti-inflammatory Pathway

This study aimed to determine whether there were alpha 7 nicotinic acetylcholine receptors(α7 nAChR) expressed in mouse Kupffer cells and to understand their effects on inflammation caused by LPS. Firstly, Kupffer cells were isolated from male BLAB/c mice by collagenase digestion and differential centrifugation using Percoll. The purity and viability of Kupffer cells isolated were about 80% to 90% as assessed by trypan blue exclusion and flow cytometry. Secondly, Kupffer cells labeled with FITC-αBGT were observed by Laser scanning confocal microscope, which proved the expression ofα7 acetylcholine receptors. We got the same result via western blotting test. Thirdly, TNF-α,IL-10 and HMGB-1 in vivo and in vitro were detected with Elisa after LPS being administered. Finally, 100 mice were randomly divided into four groups:Group I(only lethal dose of LPS was given),Group II(nicotine beside LPS was given), Group III(LPS,nicotine and GdCl3 were given). Group IV(LPS and nicotine were given and the left cevical vagus nerve was cut off). The mortality of mice was observed for 72 hours.Our study showed that Kupffer cells isolated from male BALB/c mice did posessα7 acetylcholine receptors, which played a critical role in regulating inflammation caused by lethal dose of LPS administration.Objective: Isolation,culture and identify for Kupffer cells ; observe the expression ofα7 acetylcholine receptors in Kupffer cells by Laser scanning confocal microscope and Western blotting; test the changes of TNF-α,IL-10 and HMGB-1 both in vivo and in vitro after LPS being administered; observe the Changes of Mortality of Mice after Lethal Dose of LPS was given.Methods: (1)Kupffer cells were isolated by collagenase digestion and differential centrifugation from male BLAB/c mice(aged 6 to 8 weeks), while purity and viability were assessed by trypan blue exclusion and flow cytometry;(2) Isolated Kupffer cells labeled with FITC-αBGT were observed under Laser scanning confocal microscope to test the The Expression of alpha 7 Acetylcholine Receptors. The western blotting test was also adopted. RAW264.7 was used as positive control(3) 100nM LPS was given to isolated Kupffer cells, with or without nicotine of 1 mM . Changes of TNF-α,IL-10 and HMGB-1 were determined at 4h , 12h or 24h, respectively. The same treatment as the cells were performed on mice groups;(4) 100 BALB/c male mice were randomly divided into four group:Group I (only lethal dose of LPS was given), Group II ( nicotine and LPS were given),Group III( LPS,nicotine and GdCl3 were given),and Group IV (LPS and nicotine were given and the left cevical vagus nerve was cut off).The mortality of mice was observed for 72 hours.Results: (1)The yields of isolated Kupffer cells could reach about 1- 2×107, with purity and viability reaching as high as about 80% - 90%,which could meet our needs;(2) Expression ofα7 acetylcholine receptors in Kupffer cells was confirmed by Laser scanning confocal microscope and Western blotting;(3) After nicotine was administered, the level of TNF-αand HMGB-1 increased and the level of IL-10 decreased. Given left cervical vagus nerve cut off orα-BGT ,the effect of nicotine was weakened;(4) Group I had the highest mortality rate,while in group II nicotine did reduce the mortality rate dramatically . After left cervical vagus nerve was cut off orα-BGT was given, the effects of nicotine was weakened. Difference for mortality rate between Group III and Group IV was not significant.Conclusions: Kupffer cells with high purity and viability could be harvested by the method of type IV collagenase digestion and differential centrifugation using Percoll as described elsewhere after our slight modifications . Its purity and viability could meet our needs and and the successful isolation of Kupffer cells was the basis for our further study. Kupffer cells played a crucial rule in modulating inflammation acts of bodies and the effect of nicotine was weakened after left cervical vagus nerve was cut off orα-BGT was given.In other words, the effect of nicotine could be partially blocked after left cervical vagus nerve was cut off orα-BGT was given. It was verified that the anti-inflammatory effect of nicotine was partly by left cervical vagus nerve and alpha 7 acetylcholine receptors might play a critical role.