Objective To investigate the effect and mechanism of ellagic acid(E A)on morphine analgesic tolerance in rats.Methods In vivo experiment,40 healthy male SD rats were randomly divided into the normal saline(NS)group,morphine(MT)group,ellagic acid(EA)group,and ellagic acid+morphine(EA+MT)group.The rat model of morphine analgesic tolerance was established by intrathecal injection of morphine.The latter three groups were administered with morphine,while all the 4 groups were infused with saline or the corresponding drug.Tail flick latency(TFL)was measured by warm water bath method and mechanical withdrawal threshold(MWT)was measured by von Frey method to observe the change trend of analgesic threshold before and after administration on the 1st-7th days.After modeling,the subject rats were anesthetized and killed,and the periaqueductal gray(PAG)tissue samples were taken.The expression of PINK1,LC3 and P62 proteins was detected by Western blot.Malondialdehyde(MDA)was determined by thiobarbituric acid method,and mitochondrial integrity was observed by transmission electron microscopy(TEM).In vitro experiments,human neuroblastoma cells(SH-SY5Y cells)were treated with EA and morphine,and divided into the control(SHAM)group,morphine(Mo)group,EA group,and EA+morphine(Ea+Mo)group.The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The content of cAMP was determined by enzyme-linked immunosorbent assay(ELISA),the level of ROS was detected by ROS fluorescent probe(DCFH-DA)and the expressions of PINK1,LC3 and P62 protein were determined by Western blot.Results Compared with MT group,TFL and MWT decreased more slowly in EA+MT group,and the difference was statistically significant(P＜0.05).Compared with NS group,the levels of PINK1 protein,the ratio of LC3Ⅱ/LC3Ⅰ,P62 protein and MDA in MT group increased,and the difference was statistically significant(P＜0.05),and mitochondrial swelling was obviously with more autophagosomes.Compared with MT group,PINK1 protein and the ratio of LC3Ⅱ/LC3I in EA+MT group significantly increased,while P62 protein and MDA levels significantly decreased(P＜0.05),and mild swelling of mitochondria with a part of autophagosomes.Compared with SHAM group,the levels of cAMP,ROS,the ratio of LC3 Ⅱ/LC3 1 and P62 protein increased,while the levels of cell viability and PINK1 protein decreased in Mo group,and the difference was statistically significant(P＜0.05).Compared with Mo group,the levels of cell viability,PINK1 protein and the ratio of LC3 Ⅱ/LC3 Ⅰ increased,and the levels of cAMP,ROS and P62 protein decreased in Ea+Mo group,the difference was statistically significant(P＜0.05).Conclusion EA has analgesic effect and can alleviate the formation of morphine analgesic tolerance,and its mechanism may be related to mitophagy.
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