The Study On The Effects Of Carbachol On Cytokine Releases From Rat Peritoneal Macrophages Stimulated By LPS And Its Receptor

Object: The present study was performed to: (1) observe the effect of carbachol on the releases of inflammatory cytokines from rat peritoneal macrophages stimulated by lipopolysaccharide; (2) investigate the receptor of the inhibitive effects of carbachol on the releases of proinflammatory cytokines from macrophages stimulated by LPS.Methods: Rat peritoneal macrophages were stimulated with lipopolysaccharide (LPS, 100ng/mL) to establish an inflammatory cell model. The study was carried out by methods of ELISA test and immunofluorescence experiment and divided into two parts. Part one: To observe the effect of carbachol on the releases of inflammatory cytokines from macrophages stimulated by lipopolysaccharide and comparison of the effects of different cholinergic agonists. Male Wistar rat peritoneal macrophages were added to 24-well plates (10⁶ cells per well) and treated with carbachol ( K1 2mmol/L, K2 0.2mmol/L, K3 0.02mmol/L, K4 2μmol/L, K5 0.2μmol/L, K6 0.02μmol/L) , nicotinic cholinergic receptor agonist nicotine or muscarinic cholinergic receptor agonist muscarine for 15min. Then cells were exposed to LPS and incubated in 37°C, 5%CO₂ incubator. Supernatants were collected 4h after the addition of LPS and assayed for the levels of proinflammatory cytokine TNF-a. IL-6 and anti-inflammatory cytokine IL-10. Cells were divided into 5 groups: â‘  normal control group(C); â‘¡LPS group(L); â‘¢carbachol group(K): being further divided into 6 subgroups (K1～K6) with different concentrations of carbachol to observe the difference between groups and determinate the concentration of cholinergic agonists of subsequent experiments; â‘£nicotine group(N); ⑤muscarine group(M). Part two: To investigate the receptor of the anti-inflammatory effect ofcarbachol on macrophages. Macrophages were added to 24-well plates and firstly treated with muscarinic cholinergic receptor antagonist atropine or special antagonist of a7 subunit of nicotinic cholinergic receptor(a7nAChR) a-bungarotoxin for 15 min. The subsequent steps were done the same as part one. Supernatants were collected and assayed for the levels of TNF-a and IL-6.Cells were divided into 6 groups: â‘  carbachol group(K); â‘¡ nicotine group(N); â‘¢ atropine+carbachol group(AK); â‘£ atropine+nicotine group(AN); ⑤ a-Bgt+carbachol group(BK); â‘¥ a-Bgt+nicotine group(BN). For immunofluorescence experiment, macrophages were added to 6-well plates with a glass slice in each well. Cells were treated with carbachol or nicotine (5mmol/L) and 15min later incubated with FITC-labeled a-Bgt. After being washed with PBS and fixed, cells were mounted for viewing and photographing by fluorescent microscopy and fluorescent confocal microscopy. Cells were divided into 4 groups: â‘ negative control group; â‘¡ positive control group; â‘¢ nicotine control group; â‘£carbachol group.Results: 1. Inflammatory cytokines including TNF-a, IL-6 and IL-10 could be detected with low values in normal control group and increased significantly after LPS stimulation. 2. Levels of TNF-a of all six subgroups of carbachol were lower than that of L group, among which K1, K2 and K3 group were of statistic significance. Then K3 was chosen as the cholinergic agonist concentration in subsequent experiments. 3.Comparing to that of L group, Levels of TNF- a and IL-6 of K group were significantly lowered, while level of IL-10 had no significant change. 4. Levels of TNF-a and IL-6 of K group, N group and M group were significant lower than that of L group. Comparing to that of M group, Levels of TNF-a and IL-6 of K group and N group were much lower; No significant difference could be observed between K group and N group; Level of IL-10 didn't alter significantly between L, K and N groups. 5. Comparing to that of K group, levels of TNF-a and IL-6 of AK group had no significant changes, which was the same between N group and AN group. While comparing to that ofK group, levels of TNF-a and IL-6 of BK group increased significantly and presented the same between N group and BN group. 6. Result of immunofluorescence experiments. Stained with FITC-a-Bgt, intensive fluorescence could be observed in the location of macrophage membranes. Pretreatment of nicotine and carbachol of high concentration markedly reduced the fluorescence intensity.Conclusions: l.Carbachol significantly reduced the levels of TNF-a and IL-6 released from rat peritoneal macrophages stimulated by LPS, which might be dose-dependent, while had no significant effect on the release of IL-10. 2. This inhibitive effect of carbachol was similar to that of nicotine but significantly stronger than that of muscarine. 3.a-Bgt markedly reduced the binding of carbachol and nicotine to macrophages and thus inhibited their anti-inflammatory effects, while atropine did not, indicating that carbachol, just like nicotine, play its role by activating a7nAChR.