| Inflammation is a common and important basic pathological process in lifecycle. Defensive reactions, stimulated by outside or inner injury factors, in living tissues with vascular system are collectively known as inflammatory reaction. In certain cases, inflammation can induce some diseases, such as inflammation of the brain can oppress the central nerve system. Myocarditis, in the case of serious influence, will affect the function of the heart, too severe allergic reactions, even can be life threatening. A variety of clinical symptoms for chronic diseases show circadian rhythms. For example, Myocardial infarction patients have a higher incidence rate in the morning; Patients with Rheumatoid arthritis(RA) show joint stiffness in the morning. Chronopharmacology, as a cross subject of time biology and pharmacology, although have long been known to clinical doctors that can help improve curative effect of conventional treatment, reduce side effects and develop into Chronotherapy. But so far fewer has been recognized. The main reason is that the existing C hronopharmacology can only partially improve the therapeutic effect and not materially cure disease and that is why few research resources are input. Therefore, for Chronotherapy for inflammatory disease has very big research space.neurotransmitter acetylcholine by membrane nicotine receptor alpha 7 mediated, which can effectively restrain the macrophages release of inflammatory factor, and then adjust the degree of inflammation. It is a neural system to make systematic inflammation regulation, thus a nerve immune regulating pathway. The theory was first published by Borovikova in Nature, then our lab also did a lot of research work by using the pathways on the mechanism of atherosclerosis(atherosclerosis, AS) and myocardial infarction(myocardial infarction, MI) in the later time, proved that the pathway plays a role in related diseases, and proposed many new point of views. O ur laboratory has the automatic time-rhythm animal-breeding rooms, so we connected the relevant knowledge of cholinergic anti- inflammatory pathway and circadian rhythm. Through research, we found the topic on whether the anti- inflammatory function of the cholinergic anti- inflammatory pathway have a circadian rhythm has never been reported. Then we thought whether the pathway of anti- inflammatory function has circadian rhythm? If proved yes, couldn’t this hypothesis provide new ideas for clinical treatment? This topic revolves around alpha 7- n AC h R, andtries to verify whether the cholinergic anti- inflammatory pathway have anti- inflammatory function; prove if there is a circadian rhythm; determine its relation between the pathway and GCs anti- inflammatory rhythms. Based on these assumptions, we design the corresponding experiment. Detailed experimental results are as follows:Experime nt 1: the cholinergic anti-inflammatory pathway has significant anti-inflammatory effectWe set the mice model by injecting specific blocker Mifepristone and MLA to block GR and alpha 7- n ACh R respectively before injecting low concentration of LPS. We found that the expression of inflammation factors(IL-1bβ, TNF-α and IL-6) in blocked groups, compared with what in the control groups, increased significantly, while the result in the Mifepristone group increased more obviously.Experime nt 2: the expression of GCs and VAChT have circadian rhythms, anti-inflammatory rhythm of cholinergic anti-inflammatory pathway may have complementary relationship with anti-inflammatory rhythm of GCs.The normal mice were divided into eight groups. Blood samples were taken from one of the groups 3 hours after another group, and then GCs concentration was tested in serum. We found that the concentration of the GCs bear a difference between daytime and nighttime. 0 o’clock at night was the highest point, and the value was 382.9 ng/ml(n = 7, p < 0.05), while 15 o’clock had the lowest point, which had a value of 69.5 ng/ml(n = 7, p < 0.05). The expression of VAC h T in liver and kidney was relatively less in active phase than in dormant phase. The rhythm was contrary to GCs’.Experime nt 3: the expression of inflammatory factors shows circadian rhythm changesLPS-induced mice were divided into eight groups. Blood samples were taken and tested from one of the groups 3 hours after another group. We found that the expression rhythm of three kinds of inflammatory factors(IL-1bβ, TNF-α and IL-6) had similar rhythm among groups, which showed significant inverse relationship with GCs secretion. The expression of inflammatory factors in dormant phase was more than that in active phase, and the highest point happened at 15 o’clock, the lowest point was at six o’clock.Experime nt 4: Mice with endoxemia has circadian rhythm changes on survival rateMice with endoxemia were divided into eight groups, which were injected every 3 hours and observed for 72 hours. We fo und that the average survival rate of mice in activephase(21:00, 0:00, 3:00, 6:00) was significantly higher than that in the dormant phase(9:00, 12:00, 15:00, 18:00). The result at 15:00 compared with that at 3:00 had significant differences(χ2 = 7.428, P = 0.006).Experime nt 5: The anti-inflammatory effects of cholinergic anti-inflammatory pathway was not able to change the rhythm of inflammatory cytokines’ expressionLPS-induced mice were divided into 8 groups randomly, injected with propylene glycol, Mifepristone(40 mg/kg), MLA(10 mg/kg) and combination of Mifepristone and MLA at 3 o’clock and 15 o’clock, respectively. We found that the expression of inflammatory factors in the group given the same dr ug were higher in dormant phase than that in active phase. For MLA group, the expression of IL-6 was elevated both in active and dormant phase, the result had significant difference(P < 0.05); compared with control groups, Mifepriston group also had a significantly increase in expression of inflammatory factors, then the differences of TNF-α and IL-6 between daytime and nighttime disappeared. For group of MLA plus Mifepriston, the inflammatory factors expression were further improved, and the results about TNF-α and IL-6 have no obvious difference between daytime and nighttime, then the expression of IL-1β had a higher concentration in dormant phase, the result had significant differences.Experime nt 6: the function of the choline rgic anti-inflammatory pathway is insufficient to alter the survival rate difference between day and night for mice model with poison pyemiaMice with endoxemia were divided into eight groups, injected with propylene glycol, Mifepristone(40 mg/kg), MLA(10 mg/kg) and combination of Mifepristone and MLA at 3 o’clock and 15 o’clock, respectively. There was a significant difference about the Survival Rate of mice between the active phase and the dormant phase in the control group, and the mice injected at 3AM showed higher Survival Rate.(χ2=4.041, P=0.044); for the MLA group, the Survival Rate dropped significantly and had the same rhythm to co ntrol group(χ2=5.520, P=0.019); the Survival Rate dropped sharply in the Mifestone group(15PM: χ2=29.121, P<0.01,3AM: χ2=37.299, P<0.01) and the MLA plus Mifestone group(15PM: χ2=29.237,P<0.01,3AM: χ2=38.199, P<0.01) compared to control group, and there was no significant difference between the active phase and the dormant phase; there was also different Survival Rate between the MLA group a nd Mifestone group(15PM: χ2=23.381, P<0.01,3AM: χ2=33.670, P<0.01)Conclusion:1. GCs, and cholinergic anti- inflammatory pathway play a very important role in body anti-inflammatory mechanism.2. The anti- inflammatory function of the cholinergic anti- inflammatory pathway is weaker than that of GCs. When anti- inflammatory effects are suppressed in GCs, anti- inflammatory activity of cholinergic anti- inflammatory pathway will not be able to change the anti- inflammatory rhythm of the body as a whole, and there were no obvious circadian rhythm.3. The body contains other anti- inflammatory mechanisms besides GCs and cholinergic anti- inflammatory pathway, whose circadian rhythm is the same as GCs’ anti-inflammatory rhythm. |
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