| ObjectiveNeonatal hypoxic-ischemic brain damage(HIBD)is a brain injury or damage caused by neonatal cerebral hypoxia.It is also the most important cause of death of full-term infants,which can seriously affect the normal neural development of newborns.Acupuncture therapy has achieved considerable curative effect in the rehabilitative treatment of neonatal HIBD.Accordingly,this study was conducted to study the effect of acupuncture on improving the social cognitive impairment in a rat model of intrauterine distress-induced HIBD,evaluate the effect of acupuncture on the pathological changes of hippocampal tissue in the modeled HIBD rats,and discuss the effect of ubiquitination modification of NLRP3 inflammasome on inflammatory response and autophagic flux intensity.Its purpose was to clarify the mechanism of acupuncture improving the outcome of inflammatory necrosis of hippocampal neurons in the modeled HIBD rats by regulating NLRP3 ubiquitination by promoting autophagy and proteasomal degradation.It is expected to provide theoretical basis for clinical treatment of neonatal HIBD by using acupuncture.MethodsThe intrauterine distress-induced HIBD rat model was established by 10-minutedelayed cesarean section.Modeled rats were then randomly divided into four groups on the 14th day after birth,including the normal group,model group,acupuncture group and shamacupuncture group.The rats in the acupuncture group were given acupuncture intervention(once a day,needle retention for 10 minutes,14 days in total)since the 15th day after birth.Rats in the sham-acupuncture group received sham acupuncture intervention;and those in the normal group and the model group were provided with fixation for 10 minutes at the same time.This study consisted of four parts as follows:(1)Part Ⅰ:The effect of acupuncture on the growth and development of modeled HIBD rats was evaluated by comparing the survival rate,eye opening time and body weight of rats in normal group and model group on the 3rd,5th,7th,14th and 28th day after birth.(2)Part Ⅱ:The effects of acupuncture on exploratory,social and cognitive abilities of modeled HIBD rats were evaluated by open-field test,three chamber free exploring test and Morris water maze test.Hematoxylin-eosin(HE)staining and Nissl staining were performed to detect the effect of acupuncture on the pathological structure of hippocampus in modeled HIBD rats.TUNEL staining was used to observe the effect of acupuncture on the apoptosis rate of hippocampal neurons in modeled HIBD rats.(3)Part Ⅲ:The expression of serum inflammatory factors IL-18,IL-1β and Caspase-1 in rats of each group on the 14th,28th and 42nd day was detected by enzyme-linked immunosorbent assay(ELISA).Meanwhile,Western blot was used to detect the relative expressions of inflammation related protein NLRP3,ASC,Caspase-1,pro-Caspase-1,IL-18,IL-1β,pro-IL-1β and NF-kb as well as autophagy related proteins P62,LC3Ⅰ and LC3Ⅱ in rats of each group on the 14th,28th and 42nd day,with the aim to evaluate the effects of acupuncture on autophagy and inflammatory response in hippocampus of modeled HIBD rats.The ubiquitination level of NLRP3 was detected by co-immunoprecipitation(co-IP)assay in rats of each group on the 14th,28th and 42nd day after birth,with the purpose to evaluate the effect of intrauterine distress-induced ischemia and hypoxia on the level of NLRP3 ubiquitination in hippocampus of rats and the effect of acupuncture on the level of NLRP3 ubiquitination in hippocampus of modeled HIBD rats.Furthermore,real-time fluorescence quantitative PCR(qPCR)was adopted to detect the relative gene expressions of NLRP3 related ubiquitin ligases TRIM31,MARCH7,Pellion2,ARIH2 and FBXL2 on the 28th day after birth,so as to further evaluate the effect of acupuncture on the ubiquitination level of NLRP3.Furthermore,immunofluorescence staining was utilized to observe the distribution and expressions of NLRP3,ASC and Caspase-1 in the hippocampus of modeled HIBD rats and the co-labeling rate of NLRP3/ASC and NLRP3/Caspase-1.These experiments were performed to evaluate primarily the effect of acupuncture on the inflammatory response of hippocampal tissue in modeled HIBD rats,and to clarify the mechanism of acupuncture on the pathological structure of hippocampal tissue in modeled HIBD rats.Part Ⅳ:Deubiquitinase inhibitor ML323 and ubiquitin-activating enzyme inhibitor PYR41 were added for intervention.The effect of acupuncture on social ability of modeled HIBD rats was detected by three chamber free exploring test.Western blot was further carried out to measure the relative expressions of inflammation related protein NLRP3,ASC,Caspase-1,pro-Caspase-1 and IL-1β as well as autophagy related proteins P62 and LC3Ⅱ in rats of each group on the 14th and 28th day,so as to further clarify the mechanism of acupuncture protecting neurons by regulating NLRP3 ubiquitination,promoting autophagic and proteasomal degradation to inhibit inflammatory response.ResultsResults of Part Ⅰ:Within 14 days after birth,the survival rate of rats in the normal group was significantly higher than that in the model group,with statistically significant difference(P<0.01).There was statistically significant difference that the eye opening time of rats in the normal group was significantly shorter than that of rats in the model group(P<0.01).On the 3rd day after birth,the weight of rats in the normal group was much higher than that in the model group,and the difference was statistically significant(P<0.001).On the 5th,7th and 14th days after birth,the weight of rats increased significantly in both the normal group and the model group than those before(P<0.001、P<0.01、P<0.001),but the weight of rats in the normal group was still higher than that in the model group,showing statistically significant difference(P<0.05),suggesting that intrauterine distress can cause weight loss in rats.On the 28th day after birth,the weight of rats in each group increased compared with that before;while the weight of rats in the model group was significantly lower compared with the normal group,and the difference was statistically significant(P<0.001).Rats in the acupuncture group tended to weigh less than that in the normal group,but significantly heavier than that in the model group(P<0.05),suggesting that acupuncture can improve the growth and development of modeled HIBD rats.Results of Part II:1.Behavioral test results(1)Open-field test results:After 14 days of intervention,there was no significant difference in total traveling distance,vertical standing and modification times among normal group,model group,acupuncture group and sham-acupuncture group(P>0.05).(2)Three chamber free exploring test results:In the third stage of the three-box social experiment,the social time between the model group and the stranger rat No.1 was significantly lower than that of the normal group and the acupuncture group,and the difference was statistically significant(P<0.05,P<0.05),while the same as the normal group and the acupuncture group.Compared with the sham acupuncture group,there was no significant difference(P>0.05);compared with the normal group,the social time between the acupuncture group and the stranger mouse No.1 was slightly lower,but the difference was not statistically significant(P>0.05),suggesting that acupuncture can effectively improve the social ability of HIBD model rats.In the fourth stage of the three-box social experiment,the social time between the normal group and the acupuncture rats and the stranger mouse No.2 was significantly higher than that of the model group and the sham acupuncture group,and the difference was statistically significant(P<0.01,P<0.05,P<0.001,P<0.01),suggesting that acupuncture can effectively improve the social tendency of HIBD model rats and new social objects,and improve the social dysfunction of HIBD.(3)Morris water maze test results:There was significant decrease in the escape latency of rats in normal group,model group,acupuncture group and sham-acupuncture group,with significant difference in escape latency via inter-group comparison.However,in the space exploration stage,there was no significant difference between groups in the number of crossing the original platform position as well as the ratio of distance and time of rats in the quadrant of original platform position to the overall data.1.Pathology results(1)HE staining results:In the normal group,2-3 layers of pyramidal cells were visible in the hippocampus,with the observation that cells were full in shape,arranged neatly and closely,and the nucleoli were deeply stained,clear and complete.In the model group,the number of layers of pyramidal cells in hippocampal tissue decreased,the cells were distributed sparsely and arranged disorderly,with partial cell loss and nuclear shrinkage.Compared with the normal group,the acupuncture group showed slightly sparse and disordered distribution of pyramidal cells in the hippocampal tissue,and slightly reduced number of layers of pyramidal cells,but the degree was lighter than that of the model group,with the observation of nuclear shrinkage occasionally.(2)Nissl staining results:In the normal group,there was normal morphology of pyramidal cells in hippocampus,with neat and close arrangement,uniform coloring,round appearance of cells,clear nucleoli and abundant Nissl bodies in cytoplasm.In the model group,there was decrease in the number of layers of pyramidal cells in the hippocampus,accompanied by disordered arrangement,partial cell loss,nucleolar shrinkage and obviously reduced intracellular Nissl bodies.The number of layers of pyramidal cells in the hippocampus of the acupuncture group was slightly less than that of the normal group,and cells were arranged neatly,with round cell boundary,and the degree of damage was milder than that of the model group.(3)TUNEL staining results:The apoptosis rate of CA1 cells in the hippocampus of the rats in the model group was significantly higher than that in the normal group,and the difference was statistically significant(P<0.001).In addition,compared with the model group,the apoptosis rate of the rats in the acupuncture group was significantly decreased,the difference was statistically significant(P<0.001),suggesting that acupuncture can effectively reduce the apoptosis rate of hippocampal cells in HIE rats.Results of Part III:(1)The results of serum inflammatory factors in each group of rats:on the 14th day after birth,compared with the normal group,the serum inflammatory factors IL-1β,IL-18 and Caspase-1 in the model group were significantly increased(P<0.001,P<0.05,P<0.05),indicating that hypoxia-ischemia induced by HIBD modeling can increase the level of inflammation in the hippocampus of HIBD rats;on the 28th day after birth,compared with the normal group,the model The serum inflammatory factors IL-1β,IL-18 and Caspase-1 of the rats in the group were significantly increased and the differences were statistically significant(P<0.01,P<0.01,P<0.01);The levels of serum inflammatory factors IL-1β,IL18 and Caspase-1 in mice were significantly decreased(P<0.001,P<0.05,P<0.01),suggesting that acupuncture can effectively inhibit the inflammatory response induced by ischemia and hypoxia in HIBD model rats.On the 42nd day after birth,compared with the normal group,the serum inflammatory factors IL-1β,IL-18,and Caspase-1 were significantly increased in the model group and the difference was statistically significant(P<0.01,P<0.001,P<0.01).,compared with the model group,the acupuncture group significantly decreased serum inflammatory factors IL-1β,IL-18,and Caspase-1,and the difference was statistically significant(P<0.05,P<0.05,P<0.05),suggesting that acupuncture can reduce inflammation.Inhibition of the response persisted after the intervention was discontinued.(2)The expression results of inflammation and autophagy-related proteins in each group of rats:on the 14th day after birth,compared with the normal group,the hippocampal inflammation-related proteins NF-κb,NLRP3,ASC,Caspase-1,pro-The expression levels of-Caspase-1,IL-18,IL-1β,and pro-IL-1β were increased,and the differences were statistically significant(P<0.001,P<0.01,P<0.01,P<0.01,P<0.001,P<0.05,P<0.001,P<0.01),suggesting that HIBD can lead to an increase in the level of inflammation in rat hippocampus,and the inflammatory response is related to the activation of NLRP3.On the 28th day after birth,compared with the model group,the expression of autophagy-related protein LC3II and P62 in the hippocampus of the acupuncture group were significantly increased(P<0.001),and the expression of P62 was significantly increased(P<0.001).The expression levels of NF-κb,NLRP3,ASC,Caspase-1,pro-Caspase-1,IL-18,IL-1β,and proIL-1β were decreased(P<0.001,P<0.001,P<0.001,P<0.001,P<0.001,P<0.001,P<0.001,P<0.001),suggesting that acupuncture can promote autophagic flow in the hippocampus of HIBD model rats,and effectively relieve the inflammatory response caused by ischemia and hypoxia,thereby protecting the injured damage neurons.On the 42nd day after birth,the expression levels of inflammation-related proteins NF-κb,NLRP3,ASC,Caspase-1,pro-Caspase-1,IL-18,IL-1β and pro-IL-1β in the hippocampus of rats in the acupuncture group remained unchanged.lower than the model group(P<0.05,P<0.05,P<0.05.P<0.05,P<0.01,P<0.001,P<0.01.P<0.05),and the expression levels of autophagy-related proteins were compared with those in the model group,LC3Ⅱ still increased(P<0.01),and P62 decreased(P<0.01),indicating that acupuncture’s promotion of autophagy flow and inhibition of inflammatory response still existed after the intervention was stopped.(3)Detection of the ubiquitination level of NLRP3 protein in each group of rats:The ubiquitination level of NLRP3 in the hippocampus of the rats in the normal group was significantly higher than that in the model group,and the difference was statistically significant(P<0.05).The ubiquitination level of NLRP3 was higher than that in the model group(P<0.05),but there was no significant difference compared with the normal group,suggesting that acupuncture could promote the ubiquitination of NLRP3.(4)Results of real-time fluorescence quantitative PCR of rats in each group:Compared with the normal group,the relative expression levels of March7,ARIH2,and FBXL2 genes in the model group were significantly decreased(P<0.05,P<0.05,P<0.01).The relative expression levels of March7,ARIH2,and FBXL2 genes in the acupuncture group were significantly increased(P<0.01,P<0.01,P<0.01),indicating that acupuncture could significantly increase the expression levels of NLRP3-related ubiquitinases March7,ARIH2,and FBXL2.(5)NLRP3/ASC and NLRP3/Caspase-1 immunofluorescence co-labeling staining results of hippocampal tissue of rats in each group:The co-labeled pixel area of NLRP3/ASC in the model group was significantly higher than that in the normal group,and the difference was statistically significant.(P<0.01),in addition,compared with the model group,the colabeled pixel area of NLRP3/ASC in the acupuncture group was significantly reduced,suggesting that acupuncture could reduce the binding level of NLRP3 and ASC.The colabeled pixel area of NLRP3/Caspase-1 in the model group was significantly higher than that in the normal group,and the difference was statistically significant(P<0.001).Compared with the model group,the co-labeled pixel area of NLRP3/Caspase-1 in the acupuncture group was significantly reduced,suggesting that acupuncture could reduce the binding level of NLRP3 and Caspase-1.Results of Part Ⅳ:(1)The results of the three-box social behavioral experiment of rats in each group:in the third stage of the three-box social experiment,the social time between the model group and the stranger rat No.1 was significantly shorter than that of the normal group,and the difference was statistically significant(P<0.01);Compared with the model group,the social time of the acupuncture group,the ML323 group and the stranger rat No.1 was significantly different(P<0.05,P<0.05),and the difference of the social time between the PYR41 group and the stranger rat No.1 was not indicated.There was statistical significance(P>0.05),indicating that hypoxia-ischemia caused by HIBD model can lead to the decline of social ability in HIBD model rats,and increasing the level of ubiquitination in vivo can effectively improve this situation.Compared with the ML323 group,there was no significant difference in the social time between the ML323+acupuncture group and the stranger mouse No.1(P>0.05).It is considered that the level of ubiquitination in the HIBD model rats after the ML323 intervention was greatly increased,resulting in the inability of acupuncture.Further increase the level of ubiquitination.In addition,compared with the P YR31 group,the social time between the PYR41+acupuncture group and the stranger mouse No.1 showed an upward trend,but the difference was not statistically significant(P>0.05),indicating that acupuncture can increase the ubiquitination level of HIBD model mice to a certain extent.Improve the trend of social function decline.In the fourth stage of the three-box social experiment,the social time between the model group and the stranger rat No.2 was significantly shorter than that of the normal group(P<0.01);There was a statistically significant difference in the longer social time of the rat No.2(P<0.05,P<0.05),and there was no statistically significant difference in the social time between the rats in the PYR41 group and the No.2 stranger(P>0.05),indicating HIBD Hypoxia-ischemia induced by modeling can reduce the social tendency of HIBD model rats with new social objects,and increasing the level of ubiquitination in vivo can effectively improve this situation.Compared with the ML323 group,the social time between the ML323+acupuncture group and the stranger mouse No.2 showed an upward trend,but the difference was not statistically significant(P>0.05).The social time of HIBD model showed an upward trend,but the difference was not statistically significant(P>0.05),suggesting that the level of ubiquitination in HIBD model rats after acupuncture was increased,which was helpful to enhance the social tendency of HIBD rats to new social objects.(2)The expression results of autophagy and inflammation-related proteins in the rats in each group:Compared with the rats in the model group,the expression of autophagy-related protein LC3Ⅱ in the hippocampus of the rats in the acupuncture group was significantly increased(P<0.001),and the expression of P62 was significantly decreased(P<0.001).0.001),while the expression levels of inflammation-related proteins NLRP3,ASC,Caspase1,pro-Caspase-1,and IL-1β decreased and the differences were statistically significant(P<0.05,P<0.001,P<0.001,P<0.01,P<0.001).It is suggested that acupuncture can effectively inhibit the activation of NLRP3 inflammasome in the hippocampus of HIBD model mice.Compared with the model group,the expression of LC3Ⅱ in the ML323 group was significantly increased(P<0.001),the expression of P62 was significantly decreased(P<0.001),and the inflammation-related proteins NLRP3,ASC,Caspase-1,pro-Caspase-1,IL-The expression level of 1β decreased,and the difference was statistically significant(P<0.001,P<0.001,P<0.001,P<0.001,P<0.001),indicating that the increase of ubiquitination level was helpful to promote the autophagy flow and inhibit the Activation of the NLRP3 inflammasome,thereby reducing the level of inflammation.Compared with the model group,there was no significant difference in the expression of autophagy-related protein LC3Ⅱ in the PYR41 group(P>0.05),but the expression of autophagy substrate P62 was increased(P<0.05),and the inflammation-related proteins Caspase-1,pro-The expression levels of Caspase-1 and IL-1β increased and the differences were statistically significant(P<0.05.P<0.001,P<0.001),suggesting that the reduction of ubiquitin levels could inhibit autophagic flow and lead to the increase of tissue inflammation levels.The comparison between the acupuncture+PYR41 group and the PYR41 group indicated that the expression levels of NLRP3,ASC,Caspase-1 and IL-1β in the hippocampus of the rats in the acupuncture+PYR41 group were decreased and the difference was statistically significant(P<0.05,P<0.001,P<0.01,P<0.001),suggesting that acupuncture can alleviate the ubiquitinase inhibitory effect of PYR41 to a certain extent,thereby inhibiting the inflammatory response and protecting damaged neurons.Conclusion1.HIBD model rats have social dysfunction and learning and cognitive impairment.Acupuncture can improve the social dysfunction and learning and cognitive impairment of HIBD model rats,and improve the pathological structure of hippocampus in HIBD model rats.2.Acupuncture can increase the ubiquitination level of NLRP3,promote the degradation of NLRP3 autophagy and ubiquitin-proteasome pathway,inhibit the activation of NLRP3 inflammasome,and reduce the level of serum inflammatory factors,thereby promoting the damage caused by ischemia and hypoxia.brain tissue protection.3.The effect of acupuncture on the ubiquitination level of NLRP3 protein may be related to the regulation of acupuncture on NLRP3-related ubiquitinases MARCH7,ARIH2 and FBLX2. |
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