The Mechanism Of "The 3-points For Intelligence" To Improve Cognitive Impairment In HIBD Rats Involving Autophagy And Inflammasome

ObjectiveThe purpose of this study is to investigate the internal mechanism of "The 3-Points for Intelligencec" in improving cognitive impairment in hypoxic-ischemic brain damage(HIBD)model rats and to explore the effect of acupuncture on the improvement of damaged neurons in the hippocampus and the pathological changes of the brain.From the activation mechanism of NLRP3 inflammasomes,it is clarified that "The 3-Points for Intelligence" can inhibit the activation of NLRP3 inflammasomes by regulating the intensity of autophagy flux in neurons of HIBD rats and improve the pathways of neuronal damage.This study provided a theoretical basis for the mechanism of acupuncture for perinatal hypoxic-ischemic encephalopathy.MethodsA 10-minute delayed cesarean section method was used to reproduce a HIBD neonatal rat model.Acupuncture was given on the 15th day after birth of newborn rats("The 3-Points for Intelligence" includes Shenting acupoint and both Benshen acupoints).Intervention was performed once a day for 10 minutes lasting 14 days.This study consists of 4 parts:(1)The first part of the experiment evaluated the effects of acupuncture on the growth of HIBD rats,including weight measurement and mNSS neurological deficit score;(2)The second part of the experiment used open field test,new objects recognition experiment and objects position recognition experiment to evaluate the effect of acupuncture on behavioral cognitive ability of HIBD newborn rats;brain wet weights,diameter of the coronal,sagittal,and horizontal planes were measured to evaluate the changes and the effect of acupuncture on brain edema and atrophy in HIBD rats;effects of acupuncture on hippocampal morphology and neuron structure in HIBD rats were observed with HE staining and Nissl staining;effects of acupuncture on hippocampal neuron apoptosis in HIBD rats were observed with TUNEL staining;immunohistochemistry was used to observe the effect of acupuncture on the mean optical density of neuron markers NeuN and NF200 in HIBD rats to clarify the pathological mechanism of acupuncture in improving behavioral cognitive ability of HIBD rats.(3)The third part of the experiment used ELISA technology to detect the expression of oxidative stress-related indicators ROS,MDA,SOD,GSH-px and inflammatory related factors TNF-α,IL-1β,IL-18 to investigate the effects of acupuncture on serum oxidative stress and inflammatory in HIBD rats.Applying Western Blotting to detect autophagy-related proteins LC3Ⅱ,P62,Lamp1,Cathepsin D,Belinl expression,and pyroptosis related proteins NLRP3,ASC,Caspase-1,IL-1β,GSDMD expression at four points at PND1,PND3,PND7,PND14(before acupuncture)and PND28(after acupuncture)to investigate the changes and the effect of acupuncture on brain autophagy and pyroptosis in HIBD rats after ischemia and reperfusion.(4)In the fourth part of the experiment,the effects of acupuncture on behavioral cognition of HIBD rats were observed by adding autophagy activator RAPA(rapamycin)and inhibitor CQ(chloroquine)with applying sequential memory test and episodic memory test.Western blotting was used to detect the effects of acupuncture on the expression of hippocampal autophagy-related proteins LC3Ⅱ,P62,Lamp1,mTOR,p-mTOR and pyroptosis related indicators NLRP3,ASC,Caspase-1,IL-1β,GSDMD in HIBD rats.Real-time PCR to detect the effects of acupuncture on the relative mRNA expression of Map11c3a,Lampl,Khdrbs1,Nlrp3,Pycard,and Gsdmd in HIBD rats which helped to study the function of acupuncture in regulating the intensity of autophagic flux to inhibit the activation of NLRP3 inflammasomes at the level of molecular genes to reduce cell death and protect damaged neurons.Results1.Part 1 experimental resultsA 10-minute delayed cesarean section method was used to reproduce a HIBD neonatal rat model.Compared body weights of rats in each group at PND3,PND7,PND14,PND21 and PND28.The results showed that ischemia-hypoxia caused by HIBD model could reduce the weight of newborn rats at PND3.With the increase of growth time,the growth rate of HIBD rats was significantly weaker than normal rats at PND7 and PND14.HIBD caused slower weight gain in newborn rats and there was no significant change after 7 days of acupuncture.As the growth time increased,the weight of the HIBD model rats increased slowly and their growth was restricted at PND28.At the same time,acupuncture for 14 days was beneficial to HIBD rats’ weight gain.HIBD rats had significantly impaired neurological function,and the model was successful.Acupuncture could effectively improve the injured nerve function of HIBD model rats.2.Part 2 experimental results(1)Results of Open fieldHIBD did not affect the open field exploration ability of rats,and acupuncture for 7 days did not significantly affect this at PND21.After 14 days of acupuncture treatment,acupuncture could significantly improve the open field level of HIBD model rats to improve ability of HIBD rats to explore surrounding areas,but could not significantly improve the open field vertical exploration ability of HIBD rats.The rats in the acupuncture group did not show a significant increase or decrease in the modification activity,which indicated that the effect of acupuncture on the HIBD rats was not obvious.(2)Results of new object recognition experiment and object position recognition experimentThe rats in each group could recognize new and old objects and positions.Acupuncture could significantly improve the new object discrimination ability and the position recognition of HIBD rats.(3)Brain wet weight and sectional diameter of rats in each groupHIBD did not cause visible edema or atrophy in rat brain.Acupuncture had no significant effect on the brain size and weight of HIBD rats.(4)HE staining results in the hippocampus of each group of ratsHippocampal pyramidal cells of normal group rats had a clear structure and were polygonal in shape,arranged tightly and neatly,with full nuclei and deep nucleoli staining.Compared with the normal group,the pyramidal cells in the hippocampus of the model group were scattered;the gaps were widened;the cell swelling caused morphological changes;the border was not clear and most neurons were necrotic to form vacuoles,with the nucleoli contracted.Compared with the model group,the hippocampal pyramidal cells in the acupuncture group were arranged neatly and densely;a few neurons were swollen and necrotic;some nucleoli were clearly visible and the boundaries were clearer.It was suggested that acupuncture could improve the morphology and structure of HIBD damaged neurons.(5)Nissl staining results in the hippocampus of each group of ratsThe pyramidal cells in the hippocampus of the normal group were arranged neatly;some of the cytoplasm was incompletely pigmented;a small amount of nissl bodies were on both sides of the pyramidal layer and some nucleoli were condensed.Compared with the normal group,the hippocampal pyramidal cells of the model group were scattered;the gaps were significantly widened;the cell bodies were swollen to form vacuoles:the coloring was not deep and the nucleoli was significantly condensed.Compared with the model group,the hippocampal pyramidal cells in the acupuncture group were arranged neatly and densely;some nucleoli were clearly visible and the boundaries were clearer.It was suggested that HIBD caused swelling and necrosis of neurons in hippocampus of newborn rats.Acupuncture could effectively alleviate the pathological morphology of hippocampus in HIBD rats.(6)Tunnel staining results of hippocampus in each group of ratsIntrauterine distress and hypoxia-ischemia could cause neuronal apoptosis and brain damage in the hippocampus of neonatal rats.Acupuncture could significantly improve hippocampal neuron apoptosis in HIBD rats.(7)Immunohistochemical results of rats in each groupHIBD led to decrease in the expression of NeuN in the hippocampus and damage in neurons.The cells in the model group and the sham acupuncture group were lighter in color than the normal group and the cell arrangement was significantly scattered.Acupuncture could effectively improve the expression of NeuN in injured hippocampus.Hypoxia-ischemia led to damage or necrosis of hippocampal neurons in rats,and the expression of NF200 was significantly reduced.Acupuncture could effectively improve the expression of NF200 in damaged hippocampus of rats.3.Part 3 experimental results(1)ELISA results of rats serum in each groupHIBD caused the rat body to release a large amount of reactive oxygen and oxidative stress.Acupuncture coould effectively reduce ROS and MDA concentrations and oxidative stress response.HIBD caused the SOD concentration in the rats to decrease.Acupuncture could effectively increase the SOD and GSH-Px concentrations and promote clearance oxygen produced by oxidative stress to reduce cell damage.HIBD caused the rat bodies releases a large number of cellular inflammatory factors and damages the cells.Acupuncture for 14 days could effectively reduce the serum inflammatory factor concentrations of TNF-α,IL-1β,and IL-18 to reduce the degree of cell damage.(2)Changes of autophagy flux and inflammasome in hippocampus of HIBD ratsAt PND 1,HIBD caused body ischemic hypoxia and autophagy were activated to clearly damaged organelles.HIBD activated the hippocampus of the synthesis of NLRP3 inflammasome.At PND3,HIBD-induced autophagy degradation continued to increase.NLRP3 inflammasome-mediated pyroptosis continued to increase in hippocampus of HIBD rats.At PND7,HIBD-induced autophagy degradation continued to increase.Pyroptosis in HIBD rats continued to increase.At PND14,induction of autophagy in the hippocampus of HIBD rats continued to increase,but the degradation was not significantly changed.Pyroptosis in HIBD rats continued to increase and brain damage was getting worse.(3)Effect of acupuncture on autophagy and inflammasome of hippocampal neurons in rats of each groupAfter acupuncture treatment,compared with the normal group,the expression of LC3Ⅱ and LAMP1 in the hippocampus of the model group was significantly increased;the expression of P62 was significantly reduced and the differences were statistically significant.Compared with the model group,the expressions of LC3Ⅱ,P62,and LAMP 1 in hippocampus of the acupuncture group were significantly increased and the differences were statistically significant,while the sham acupuncture group had no significant changes.It was suggested that acupuncture could inhibit excessive autophagy flux in hippocampus of HIBD rats.Compared with the normal group,the expressions of NLRP3,pro-caspase-1,caspase-1,ASC,IL-1β,and GSDMD in the hippocampus of the model group were significantly increased and the differences were statistically significant.Compared with the model group,the expressions of NLRP3,pro-caspase-1,caspase-1,ASC,IL-1β and GSDMD in the hippocampus of the acupuncture group were significantly reduced and the differences were statistically significant.It is suggested that acupuncture could effectively inhibit the hippocampal neuron pyroptosis in HIBD rats and protect the damaged cells.4.Part 4 experimental results(1)Results of behavioral testThe rats in each group could recognize the order of appearance of objects and whether they match the background.The results of time memory test showed that:HIBD severely reduced the rat’s ability to recognize the appearance order of objects,and inhibiting autophagy flux could significantly improve this phenomenon.The HIBD model rats produced excessive autophagy flux after RAPA intervention.At the same time,acupuncture failed to reduce it,and brain damage was continuously aggravating,resulting in the rats’ memory ability at a low level,The inhibition of excessive autophagy flux had a tendency to alleviate the memory decline in rats.The results of episodic memory test showed that HIBD seriously reduced the ability of rats to recognize whether the object matches the environment,and inhibiting autophagy flux could significantly improve this phenomenon.HIBD model rats produced excessive autophagy flux after RAPA intervention,and autophagy flux was suppressed after acupuncture,with brain damage reducing and the memory ability of rats was improved.Inhibition of excessive autophagy flux could effectively improve the object recognition and memory ability of rats.(2)Autophagy-related protein expression results in each group of ratsThe expression of LC3Ⅱ and LAMP1 in the hippocampus of the model group were significantly increased and the expression of P62 and P-mTOR/total mTOR were reduced which indicated acupuncture inhibited degradation of autophagy flux.The expression of LC3Ⅱ in the RAPA group was significantly increased and the expression of P62 and P-mTOR/total mTOR were reduced.LAMP 1 showed an upward trend which suggested that autophagy flux was promoted by continuous overdose.The LC3Ⅱ,P62,LAMP1,and P-mTOR/total mTOR in group CQ rats were all increased,suggesting that autophagy flux degradation was inhibited.The acupuncture + RAPA group had higher expressions of LC3Ⅱ and P62 with statistical significance.Compared with the CQ group,the acupuncture+CQ group showed lower expressions of LC3Ⅱ and P62.It was suggested that acupuncture could inhibit excessive autophagy flux in HIBD rats.(3)Expression results of pyroptosis-related proteins in each group of ratsThe expressions of NLRP3,ASC,pro-caspase-1,caspase-1,IL-1β,and GSDMD in the hippocampus of the model group were increased,which suggested that hypoxic-ischemia activated NLRP3 inflammasome in rat neurons and aggravated the pyroptosis.The expressions of NLRP3,ASC,pro-caspase-1,caspase-1,IL-1β,GSDMD in the hippocampus of the acupuncture group were reduced,which indicated acupuncture inhibited neuronal NLRP3 inflammasome activation in HIBD rats.The expressions of ASC,pro-caspase-1,and IL-1β in hippocampus of RAPA group were increased,which indicated increase of autophagy flux promoted exacerbation of pyroptosis.The expressions of NLRP3,ASC,pro-caspase-1 and GSDMD in hippocampus of CQ group rats were decreased which indicated reducing excess autophagy flux could suppress neuronal pyroptosis.Acupuncture+RAPA group compared with RAPA group,the expression of ASC,pro-caspase-1,IL-1β,GSDMD in hippocampus of rats were reduced which suggested that acupuncture could moderately reduce excessive autophagy flux in HIBD rats and inhibit cell pyroptosis.(4)Real-time PCR results of each group of ratsThe relative expression levels of LC3,P62 and LAMP1 mRNA in the hippocampus of the model group were increased.The relative expression levels of LC3 and LAMP1 mRNA in the acupuncture group were increased;P62 was decreased which indicated acupuncture inhibited excessive autophagy flux.The relative expression levels of LC3,P62,LAMP1 mRNA in RAPA group were increased,which indicated RAPA enhances autophagy flux in HIBD rats.The relative expression of P62 mRNA in CQ group rats decreased,suggesting that CQ inhibited autophagy by inhibiting the production of autophagy substrate.The relative expression of P62 mRNA in the acupuncture+RAPA group was reduced,suggesting that acupuncture inhibited excessive autophagy flux by inhibiting the production of autophagy substrates.The relative expression levels of NLRP3,ASC and GSDMD mRNA in the hippocampus of the model group increased.The relative expression levels of NLRP3,ASC and GSDMD mRNA in hippocampus of the acupuncture group were reduced,suggesting that acupuncture inhibited HIBD pyroptosis.The relative expression of NLRP3 and GSDMD mRNA in hippocampus of RAPA group were increased,suggesting that autophagy enhancement aggravated pyroptosis in HIBD rats.The relative expression levels of NLRP3,ASC,GSDMD mRNA in hippocampus of CQ group rats were reduced,suggesting that blocking autophagy degradation could effectively reduce the pyroptosis of HIBD rats.The relative expression of NLRP3 mRNA decreased of the acupuncture+RAPA group.Compared with the acupuncture+CQ group,the relative expression of NLRP3 mRNA increased of the acupuncture+CQ group,which suggested that acupuncture inhibited the production and activity of NLRP3 inflammasomes to attenuate neuronal pyroptosis in HIBD rats.ConclusionThe mechanism of acupuncture effectively improving the behavioral memory impairment and brain pathological changes in HIBD rats maybe relate to the activation of neuronal NLRP3 inflammasome.This study confirmed that a new mechanism for acupuncture to improve hypoxic-ischemic brain damage in HIBD neonatal rats with is regulating the autophagic flux and the activation of NLRP3 inflammasomes to decrease pyroptosis caused by hypoxia-ischemia in the hippocampus of rats to protect damaged neurons.