| A number of chronic progressive diseases, such as arthritis, sepsis, ulcerative colitis, andatherosclerosis, severely endanger health and life. With the world’s population aging, theprevalence of these debilitating inflammatory diseases will increase year by year, which hasbecome one of the most concerning public health problems. In clinical settings, successfullytreating these diseases is still highly challenging for physicians, mainly owing to the complexityof inflammatory response cascade which involves in numerous pro-inflammatory cytokines andmediators. According to the definite analgesic and anti-inflammatory effects, non-steroidalanti-inflammatory drugs (NSAIDs) have always been recommended as the first-line drugs forthe treatment of arthritis in many countries. Despite effective in relieving pain and improvinginflammatory symptoms, NSAIDs fail to prevent the progression of inflammation-baseddiseases, and some dose-related side effects largely restrict their clinical applications.Furthermore, to our knowledge, the clinically available anti-inflammatory drugs are alldescribed to be ineffective in the therapy of sepsis characterized by an uncontrolled systemicinflammatory response syndrome. Therefore, the limitations of current anti-inflammatory drugsagainst such intractable diseases are well recognized, and looking for new anti-inflammatorytargets or developing the more effective anti-inflammatory pharmacological interventons areconsiderable interest and urgently needed.Activation of alpha7nicotinic acetylcholine receptor (α7nAChR) has been recentlydescribed to be an extremely prospective strategy for the prevention and treatment of chronicdebilitating inflammatory diseases, since abundant investigations have provided strongpreclinically relevent evidence that stimulating α7nAChR by pharmacological interventionscan remarkably inhibit the synthesis and release of pro-inflammatory cytokines from immunecells and/or endothelial cells, including TNF-alpha, interleukins, and high mobility groupprotein1(HMGB1). A promising candidate in this area is choline, a natural specific α7nAChRagonist that has been shown to have analgesic effects on various inflammatory pain models andexert protective effects against experimental sepsis in mice. However, it is unclear whether choline affords anti-inflammatory effect on non-infectious acute inflammation. More associatedevidences are also required to confirm the efficacy of choline on the prevention and treatmentof sepsis.The simultaneous use of clinically effective anti-inflammatory drugs with differentmechanisms of action is one of the most feasible ways to develop a more effectivepharmacological approach for the treatment of inflammatory diseases. Aspirin, one of thetraditional NSAIDs targeting cyclooxygenase, is the most frequently prescribed drugs forrelieving pain and attenuating inflammatory symptoms. Although aspirin medication in ananti-inflammatory dosage is known to increase the risk of gastrointestinal bleeding andperforation, it has been reported to be much safe for the long-term use of aspirin at relativelysmall dosages when treating patients with a procoagulant and inflammatory state. Moreinterestingly, aspirin was recently revealed to have several therapeutic advantages over the otherNSAIDs. On one hand, through cyclooxygenase-dependent or-independent mechanisms,aspirin not only promotes the formation of novel lipid mediators (aspirin-triggered lipoxins) toplay a potent anti-inflammatory action, but also displays some intriguing traits to modulate theimmune response during autoimmune-based inflammatory diseases. On the other hand, it hasbeen argued that the selective cyclooxygenase-2inhibitors are not superiorer to aspirin withrespect to the anti-inflammatory effects, but potentially increase the risk of catastrophiccardiovascular events. Moreover, clinical evidence indicated that the use of aspirin wasassociated with reduced medial tibial cartilage loss in patients with arthritis and lowerhospitalization rate in patients with sepsis, indicating that aspirin will be more widelyprescribed to treat inflammation-based diseases in the future. Thus, it is of great value to studyhow to make aspirin retain its potent anti-inflammatory efficacy with lower doses, therebyavoiding the adverse effects usually acquired through high dose aspirin treatment.Because the concurrent use of drugs with different mechanisms of action is generally auseful approach for achieving effective control of inflammation while minimizing dose-relatedside effects, the key question is therefore to find an anti-inflammatory candidate well suited tocombine with aspirin.Since choline inhibits the release of a variety of pro-inflammatory cytokines by activatingα7nAChR whereas aspirin attenuates inflammatory response by multiple pathways,co-treatment with choline and aspirin is theoretically postulated to display a more potentanti-inflammatory effect. Notably, our previous observations showed that choline couldpotentiate the antinociceptive action of aspirin at small doses in several inflammatory painmodels. Therefore, we hypothesized that choline and aspirin may have synergisticanti-inflammatory effects, which may be a more effective anti-inflammatory approach with afavorable safety profile. At present, there is no reports regarding this theme worldwide. The first chapter of this thesis was to evaluate the anti-inflammatory effects of choline and theinteraction between choline and aspirin on carrageenan-induced paw edema in mice byisobolographic analysis, as well as the relationship between the anti-inflammatory action and α7nAChR. The second chapter was to evaluate the effects of single application of choline oraspirin for the prevention and treatment of LPS-induced sepsis, and also to investigate theinteraction between choline and aspirin against LPS-induced sepsis and mortality in mice.Furthermore, the pharmacological mechanisms responsible for the combined effects of cholineand aspirin on LPS-stimulated inflammatory response were also observed, especially for theirroles in the release of pro-inflammatory cytokines and mediators.The key findings listed below:1. Pharmacological characteristics of inhibitory effects of choline or co-treatment withcholine and aspirin on λ-carrageenan-induced paw edema in micePaw edema was induced by intraplantarly injecting30μl of1%carrageenan into the lefthind paw. The paw swelling rates (%, mean±S.E.M.) measured at1,2,3,4,5, and6h aftercarrageenan administration were12.7±1.3%,17.5±1.5%,22.1±2.1%,19.8±2.0%,17.2±1.8%, and16.0±2.2%, respectively. The peak value of edema formation occurred3h aftercarrageenan injection, whereas edema was waning from4to6h. Therefore, the effects of drugson edema within3h after carrageenan injection were principally observed, and their effects atthe time point of3-h were used to calculate the efficacy of the drugs against inflammatoryswelling.Prophylactic treatment with aspirin in doses ranging from11.25to200mg/kgdose-dependently inhibited the λ-carrageenan-induced paw edema. When the average degree ofinflammatory swelling of the control group was set as100%at3h after the carrageenaninjection, the normalized swelling rates after aspirin administration in doses of11.25,22.5,45,100, and200mg/kg were94.6%,93.8%,72.0%,54.8%and43.7%, respectively; the calculated50%effective dose (ED50) value was136.73mg/kg (95%confidence interval:87.41to213.87mg/kg).Pretreatment with choline in doses ranging from12.5to80mg/kg also dose-dependentlysuppressed the λ-carrageenan-induced paw edema, and the normalized swelling rates aftercholine administrations in doses of12.5,25,50, and80mg/kg were:91.1%,73.9%,70.5%, and66.9%, respectively; the calculated ED50value was over200.31mg/kg (95%confidenceinterval:75.07to534.48mg/kg).Combined choline and aspirin treatment at doses in a constant ratio produced significantanti-inflammatory effects in a dose-dependent manner. After pretreatment with drugs(choline+aspirin) in combination at doses of (3.125+2.8),(6.25+5.625),(12.5+11.25),(25+22.5), and (50+45) mg/kg, the normalized swelling rates at3-h were83.2%,71.5%,59.7%, 46.3%, and34.7%, respectively. Isobolographic analysis was further performed to assess theanti-inflammatory interaction between choline and aspirin by using CalcuSyn2.0software.There was a significant leftward shift in the dose-response curve of co-administration withcholine and aspirin when compared to those of drug administration alone. The determined ED50values for drugs in combination were21.31mg/kg (19.73to23.04) for choline and19.19mg/kg (17.76to20.73) for aspirin. The estimated combination index (CI) values at ED50, ED75,and ED90were0.25,0.32, and0.44. As CI values in the range0.9and1.1indicate an additiveeffect, whereas CI values of <0.9indicate synergism, CI values of <0.5indicate strongsynergism, and CI values of>1.1indicate antagonism, these results demonstrated thatcombined choline and aspirin therapy had a strong synergistic anti-inflammatory effects incarrageenan-induced paw edema in mice.Pretreatment with MLA (5mg/kg), a selective α7nAChR antagonist, failed to affectcarrageenan-induced paw edema, but completely antagonized the inhibitory effects of choline50mg/kg on carrageenan-induced paw swelling. Furthermore, MLA2mg/kg also partlyreversed the amelioration on paw edema produced by co-administration with choline (12.5mg/kg) and aspirin (11.25mg/kg), suggesting that anti-inflammatory effects of choline isthrough acting on α7nAChR, and the activation of α7nAChR participates in the synergisticinteraction between choline and aspirin.2. Pharmacological characteristics of protective effects of choline or co-treatment withcholine and aspirin against LPS-induced sepsis in miceThe mouse model of uncontrolled systemic inflammatory response was successfullyestablished after intravenously injected with LPS in saline in doses of10,20,40and50mg/kg,showing an approximate100%mortality within24hours which occurred at the dose of50mg/kg of LPS examined.Pretreatment with choline (10to50mg/kg)1h prior to LPS (50mg/kg) challenge had noeffects on survival time and mortality in LPS-treated mice when compared to that of the salinegroup, whereas choline in doses of60,70and80mg/kg significantly prolonged the mediansurvival time and improved the survival rates to30%,20%, and20%, respectively; the maximalimprovement of choline was observed when the dose was used at60mg/kg, indicating arelatively low efficacy against LPS-induced mortality for choline application alone.Pretreatment with aspirin at10and20mg/kg did not affect the survival curves, whereasaspirin at40and60mg/kg administrations both significantly prolonged the median survivaltime, although they did not increase the final survival rates. However,100mg/kg aspirinafforded no amelioration on the median survival time. These results suggested that aspirin had aslight protection against LPS-induced sepsis.Although either pretreatment with20mg/kg aspirin or40mg/kg choline alone had no effects on survival time and mortality in LPS-treated mice, the combined choline (40mg/kg)and aspirin (20mg/kg) treatment remarkably ameliorated survival curve, and significantlyincreased the survival rate to60%. Furthermore, in the presence of aspirin at the fixed dose of20mg/kg, choline (10to40mg/kg) dose-dependently improved the final survival rates, in spiteof reversed effects occurring when choline was given at60mg/kg. Using Cox ProportionalHazards Regression Model for statistical analysis showed that there was a significantinteraction between two drugs, implying that co-administration of choline and aspirin exhibiteda synergistic protection against LPS-induced uncontrolled systemic inflammatory response andmortality in mice, since aspirin (20mg/kg) or choline (below40mg/kg) alone completely failedto improve the survival curve.Orthogonal design method was performed to investigate the optimal remedy of choline forthe therapeutic treatment of sepsis in LPS-treated mice. We found that therapeutic treatmentwith choline at dose of40mg/kg for three times at2-h intervals showed the optimalimprovement on the survival curve of septic mice, but the survival rate only reached up to30%.Although therapeutic co-administration with the optimal remedy of choline (40mg/kg/3times/2-h intervals) and aspirin (15and30mg/kg) did not display a significant synergisticprotection, the survival rates of septic mice still reached up to40%and40%, respectively,indicated that combined therapeutic treatment is superior to drug use alone.Pretreatment with2mg/kg MLA failed to affect the survival time and survival rate inLPS-treated mice, but fully blocked the protective effect of60mg/kg choline on septic mice,demonstrating that the improvement of choline on septic shock mice is through acting on α7nAChR. MLA (2mg/kg) also abrogated the synergistic anti-inflammatory effects by choline (40mg/kg) and aspirin (20mg/kg) in combination, implying that activation of α7nAChR isinvolved in the synergy of this two drugs.It has been well documented that the pathophysiologically increased pro-inflammatorycytokines and mediators contribute to LPS-induced sepsis as well as carrageenan-mediatedswelling. To further understand the biochemical mechanisms responsible for theanti-inflammatory effects of choline and choline-aspirin, mice were treated with LPS post drugtreatments and the cytokines and arachidonic acid metabolites in the serum were detected. Inaddition to inhibiting the production of TNF-α and HMGB1, choline at40mg/kg significantlydecreased the LPS-evoked production of IL-1β, IL-6, PGE2, PGI2, TXA2, and LTB4, withoutaffecting the release of IFN-γ and IL-10. Aspirin at20mg/kg significantly decreased the LPS-induced production of IL-1β, IL-6, IL-10, and arachidonic acidmetabolites; but did not affectthe release of IFN-γ, TNF-α, and HMGB1. Interestingly, the combination of choline and aspirinappeared to be more sufficient in preventing the release of a variety of pro-inflammatorymediators containing IFN-γ, IL-1β, IL-6, HMGB1, PGI2, and PGE2; which at least in part, contributed to their synergistic interaction against inflammatory injury.In cultured murine macrophage-like cell line RAW264.7cells, we focused our attentions onfollowing two questions:(1) whether aspirin directly enhances the inhibition of choline on therelease of TNF-α after LPS stimulation.(2) whether choline directly enhances the inhibition ofaspirin on LPS-induced cyclooxygenase and the arachidonic acid metabolism.A sharp increase of TNF-α in supernatant was observed from cultured RAW264.7cells afterLPS (100ng/ml) challenge. Choline in concentrations ranging from1to50mmol/Lconcentration-dependently inhibited the levels of TNF-α, which was consistent with previousreports. Aspirin at100μmol/L also significantly inhibited the levels of TNF-α, although thisinhibition is modestly effective. However, when co-pretreatment with choline (50mmol/L)and aspirin (100μmol/L) did not afforded an additive or supra-additive effects on TNF-αproduction. These in vitro results, together with the data in serum TNF-α, suggested that themechanism responsible for the in vivo synergism of choline and aspirin may be not throughtheir direct synergistic inhibition on TNF-α release.A sharp increase of PGI2and TXA2in supernatant was respectively observed from culturedRAW264.7cells after LPS treatment. Aspirin in concentrations ranging from0.1to30μmol/Lsignificantly inhibited the levels of PGI2and TXA2in a concentration-dependent manner.However, choline in concentrations ranging from0.1to10mmol/L failed to affectLPS-induced production of PGI2and TXA2, suggested that choline has no direct effects oncyclooxygenase activity as well as the production of PGI2and TXA2. Notably, choline1mmol/L also failed to affect the effects of aspirin on PGI2and TXA2, suggested that cholinehad no effects on the inhibition of cyclooxygenase by aspirin.It is well known that aspirin inhibits platelets aggregative activity through its inhibition onTXA2, and aspirin-induced gastrointestinal side effects is due to its inhibition on PGI2. Our invitro results showed that choline not only failed to affect the arachidonic acid metabolism, butalso did not enhances the inhibition of aspirin on LPS-induced cyclooxygenase, implying thatcholine is unlikely to increase the risk of aspirin-induced gastrointestinal ulcers or bleedingtendency when they are used in combination.Taken together, our results demonstrate that choline is able to potentiate theanti-inflammatory effects of aspirin at relatively low dose and possibly to reduce highdose-related side effects of aspirin, which appears to be a more effective anti-inflammatoryregimen with a favorable safety profile.Based upon aboved observations, we concluded that:(1) Choline exerts a significant anti-inflammatory effect on the local and systemic inflammatoryresponse through activating α7nAChR.(2) Co-treatment with choline and aspirin represents a significant synergistic anti-inflammatory effect, and activation of α7nAChR participates in this synergism.(3) Pretreatment with choline and aspirin in combination displays a synergistic protectionagainst LPS-induced sepsis in mice, possiblely via more effectively preventing the excessiverelease of pro-inflammatory cytokines and mediators triggered by LPS. |
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