| Objectives:As the first choice for the treatment of cancer pain,morphine is irreplaceable.However,the tolerance caused by multiple doses seriously hinders its clinical application,and a method to delay morphine tolerance is urgently needed.At the same time,it has been shown that the formation and regulation mechanism of pain has gender dimorphism.Gender dimorphism is becoming a general interest in the field of pain,which provides a new theoretical basis for precise treatment and has strong practical significance.This thesis aims to explore the gender differences and its mechanisms of morphine tolerance in mice.Firstly,we established a mouse model of morphine tolerance and then used behavioral methods to test whether Tlr2 knockout and NPD1 intervention could delay the formation of morphine tolerance in mice and whether there are gender differences.Secondly,we preliminarily explored the relationship between macrophage extracellular traps and the formation of morphine tolerance in male mice and cell models by using molecular biology methods,which provided a new idea for explaining gender differences in pain and opioid analgesia.Methods:1.Select both male and female C57BL/6 mice,and morphine 10 mg/kg was subcutaneously injected into the back once a day for 10 consecutive days to establish a mouse morphine tolerance model.Behavioral tests were performed by hot plate and tail-flick tests to explore whether there were gender differences in the analgesic effect of morphine.2.Select both male and female Tlr2-/-mice,and morphine 10 mg/kg was subcutaneously injected into the back for 10 consecutive days to explore whether Tlr2 knockout could delay the formation of morphine tolerance in mice and whether there are gender differences.3.Select both male and female C57BL/6 mice,and saline,NPD1(2 ng,10 ng and 50 ng)was intraperitoneally injected 30 mins before morphine injection every day to explore whether NPD1 could delay the formation of morphine tolerance in mice and whether there are gender differences.4.The expression levels of macrophages marker F4/80 and ETs marker CitH3 and PAD4 in lumbar DRG in male wild-type morphine tolerance mouse model were detected by q-PCR,WB and IF to explore whether METs was involved in development of morphine tolerance in mice.In addition,macrophages were depleted before the establishment of a morphine tolerance mouse model to explore whether the formation of ETs during morphine tolerance was inseparable from macrophages.5.RAW 264.7 was treated with 0,3,10,30,100μM morphine for 24 hours to simulate the tolerance state and observe whether morphine could induce its release of ETs.6.Select male C57BL/6 mice,and the PAD4 inhibitor Cl-amidine 10 mg/kg was intraperitoneally injected 30 min before morphine injection every day to explore whether inhibiting PAD4 could delay the formation of morphine tolerance in mice and to detect the expression changes of ETs-related molecules.7.The expression levels of macrophages marker F4/80 and ETs marker CitH3 and PAD4 in lumbar DRG in morphine tolerance mouse model were detected by q-PCR,WB and IF to explore whether Tlr2 knockout and NPD1 intervention could delay the formation of morphine tolerance was also related to METs.Results:1.Morphine could significantly increase the latency to heat pain of C57BL/6 mice in a short period of time.However,as the number of doses increased,tolerance developed,which occurred in both male and female mice.At the same time,the basic thresholds of male and female mice were significantly different in the hot plate and tail-flick tests,and the analgesic effect of morphine also had sex differences,which was more significant in male mice.2.Compared with wild-type mice,the acute analgesic effect of morphine in Tlr2-/mice was more pronounced and the development of tolerance was also slower,which was similar in both male and female mice.However,compared with Tlr2-/-female mice,Tlr2-/male mice had a higher basic threshold,and there were significant differences in the acute analgesic effect and tolerance formation.The above results indicated that the effect of morphine on Tlr2-/-mice had sexual dimorphism.3.NPD1 2 ng,10 ng and 50 ng could significantly improve the analgesic effect of morphine 0-4 hours after the initial administration and delay the formation of tolerance,and the NPD1 2 ng group has the best analgesic effect.However,unlike that in male mice,NPD1 2 ng intervention did not achieve the expected effect in female mice,neither improving the acute analgesic effect nor delaying the development of morphine tolerance was statistically significant.In addition,compared with male mice,the latency of female mice giving NPD1 2 ng was significantly lower in the tail-flick test,indicating that the effect of NPD1 on development of morphine tolerance also had sex differences.4.Q-PCR results showed that the expression level of Padi4 in DRG in male wild-type mice was gradually up-regulated with the formation of morphine tolerance.Similarly,the co-labeling of macrophage marker F4/80 and ETs marker CitH3 in DRG was also observed in immunofluorescence experiments.The expression level of PAD4 in morphine tolerance mice was significantly up-regulated compared with the control group,which was consistent with the q-PCR result,and after depletion of macrophages,the expression level of PAD4 was significantly decreased compared with the morphine group.The above evidence suggested that macrophages and METs were involved in the development of morphine tolerance in mice.5.RAW 264.7 were respectively treated with 0,3,10,30,and 100 μM Morphine for 24 hours.Western blotting showed that the two markers of ETs,PAD4 and CitH3,were increased at the protein level,and q-PCR also confirmed an increase in Padi4 mRNA,reaching a peak at the 30 μM dose with a statistically significant difference.Then,the 30μM dose was selected for immunofluorescence,which further proved that long-term morphine treatment could induce the release of ETs from macrophages.6.The behavioral results showed that a single dose of Cl-amidine did not have much effect on the heat pain latency in mice,but multiple dose could significantly delay the development of morphine tolerance.Western blotting and q-PCR also demonstrated that the expression level of PAD4,Padi4 and F4/80 increased significantly in mice treated with only morphine compared with the control group,but decreased in mice treated with Cl-amidine before morphine compared with mice treated with only morphine.It suggested that Cl-amidine could inhibit PAD4 expression,thereby reducing macrophage recruitment and ETs release.In addition,similar results were observed in the immunofluorescence of DRG tissues and corresponding cell experiments.7.Both Tlr2 knockout and NPD1 intervention can significantly reduce the mRNA expression levels of Padi4 and F4/80 and the protein expression level of PAD4 in morphine tolerance mice.Therefore,the reason Tlr2 knockout and NPD1 intervention could delay the development of morphine tolerance was also associated with inhibition of macrophage recruitment and release of ETs.Conclusions:1.There are sex differences in the acute analgesic effect and tolerance of morphine in mice.Tlr2 knockout and NPD1 intervention can delay the development of morphine tolerance with gender differences.2.It was demonstrated in male mice and cell model that macrophages and METs are involved in the formation of morphine tolerance.The reason Cl-amidine,Tlr2 knockout and NPD1 can delay the formation of morphine tolerance are also associated with inhibition of macrophage recruitment and release of ETs. |
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