Effects Of Acupuncture On Microglia Polarization And TLR4/TRIF/MyD88 Signaling Pathway In TBI Rats

Objective: Flow cytometry,co-immunoprecipitation and other techniques were used to detect the effects of acupuncture on M1 and M2 polarization of microglia and TLR4/TRIF/My D88 signaling pathway in rats with traumatic cranial injury(TBI).The effect and mechanism of acupuncture on the microglia polarization after cranial injury were investigated via the TLR4/TRIF/My D88 signaling pathway.Methods:Experiment 1: 70 SPF male Sprague-Dawley rats were randomly divided into normal group(10 rats),model group(30 rats),and acupuncture group(30 rats).According to the treatment time,the model and acupuncture group were both divided into 3subgroups(1d,3d,5d),10 rats in each subgroup.The model and acupuncture group were established moderate TBI model,referencing to the Feeney's free-falling epidural percussion model method.The acupuncture group received treatment within12 hours after modeling,according to the selected acupoints GV20,GV26,GV16 through GV15,bilateral LI4.Applying twisting and purging method,each acupoints was hand-manipulate for 1min,once every 5min,retained for 15 min,performed once a day.The model group was fixed for 15 minutes without treatment,no treatment was given to the normal group.,The injured brain tissue and peripheral cortex were sampled 4 hours after the treatment.The m NSS method was used to evaluate neurological deficits after modeling and before sampling;Nissl's and HE staining were to observe the morphological changes of damaged brain tissue;flow cytometry was to test the M1 and M2 polarized microglia of injured region on the 1st,3rd and 5th days;co-immunoprecipitation was to detect the total expression of TLR4,TRIF,My D88 and the combination of TLR4 with TRIF and My D88 of each group on the 1st,3rd and 5th days.Experiment 2: 48 SPF male SD rats were modeled according to the above method before randomly divided into blocking group and blocking plus acupuncture group,24 rats in each group.And according to the treatment time,each group was randomly divided into 3 time subgroups of 1d,3d and 5d,8 rats in each subgroup.The blocking group and blocking plus acupuncture group received injection of the TLR4 inhibitor TAK-242 via tail vein 1 time/day.The acupuncture treatment performed 1 hour after injection,with the same acupuncture method mentioned above.Flow cytometry was used to detect M1 type polarization of microglia at day 1,3 and 5.Results:1.Results of m NSS neurobehavioral score: no difference of neurobehavioral scores in each group showed before modeling.The scores of the model group and acupuncture group were higher than normal group(P<0.01)on the first day after modeling;no statistical significance appeared between the model and acupuncture group,(P>0.05).The nerve function score of the model group increased from day 3 to5,while the score of acupuncture group gradually decreased from day 3 to 5,which was statistically significant compared with the model group(P<0.05).2.Pathological staining results:2.1 HE staining results: in the model group,from the 1st to 3rd day,the damaged area and its surrounding brain tissues were loose in morphology and structure,interstitial edema,reduced and disordered cell number,hypertrophy and swelling of nerve cells,which were vacuol-like,with cell nucleus migration and contraction,and inflammatory cell infiltration.On the 5th day,the edema of the cells in the model group was slightly improved,and a large number of nuclei were seen to be fragmented,dissolved or wrinkled,some tissues were necrotic,with unclear boundary.Compared with the model group,cell edema was significantly improved in the acupuncture group on the 5th day,with a small amount of nuclear consolidation and a large number of glial cells and fibers filling the lesion area.2.2 Nissl's staining: rats of the model group showed damaged cortical tissues,inflammatory infiltration,loose neuronal arrangement,irregular morphology,vacuol-like degeneration,nuclear consolidation,unclear nucleoli,greatly reduced of Nissl's corpuscles and colour shallow.With the extension of injury time,local inflammation and neuronal injury were further aggravated.The situation of the acupuncture group was better than model group.On the 3rd to 5th day,the inflammatory infiltration of the lesion and the neuronal injury were gradually reduced, the neurons were gradually increased and arranged in a relatively regular manner,the vacuol-like degeneration and nuclear reconsolidation were gradually reduced,and the number of Nissl's corpuscles was increased and became fuller.The number,morphology and integrity of neurons were improved.3.Results of microglia M1 and M2 type polarization detected by flow cytometry:(microglia were labeled with CD11 b,CD86 in CD11b(CD11b/CD86)manifest the M1 polarized microglia,while CD206 in CD11b(CD11b/CD206)labeled the M2 polarized microglia.):(1)CD11b/CD86 : low expression in normal group.The expression of the model group rose on the 1st day,peaked on the 3rd day,lasting to the5 th day,P<0.01.Compared with model group,the acupuncture group showed no significant difference in expression on the 1st day(P>0.05),while a significant difference appeared on the 3rd to 5th days(P<0.01).(2)CD11b/CD206: low expression in normal group.The expression of model group was very little on the 1st and 3rd day,and there was no significant difference compared with normal group(P>0.05);on the 5th day,there was a significant difference compared with normal group(P<0.01).There was no significant difference between acupuncture group and model group on the 1st day(P>0.05),and the expression continuously increased on the 3rd to 5th day,all of which were higher than those of the model group(P<0.01).4.The co-immunoprecipitation results of TLR4/TRIF/My D88:4.1 The total expression of TLR4?TRIF?My D88 in microglia(Input TLR4?Input TRIF?Input My D88):Input TLR4:low expression of TLR4 in normal group.The was a gradually increase of expression of model group from the 3rd to 5th day,compared with normal group(P<0.05);compared to model group,the data of acupuncture group increased on the 1st day(P<0.01),while the Input TLR4 decreased obviously from the3 rd to 5th day,P<0.01.(2)Input TRIF?Input My D88:no significant difference showed among the normal group,model group and acupuncture group on the 1st,3rd and 5th day(P>0.05).4.2 TRIF,My D88 binding to TLR4 in microglia(IP TRIF?IP My D88): In the normal group,the expression of IP TRIF and IP My D88 in microglia were all at low levels.Compared with normal group,the expression of IP TRIF and IP MyD88 gradually increased from the 1st to 5th day(P<0.01);the expressions of the acupuncture group were lower than that of model group on the 1st to 5th day(P<0.01).5.The M1 polarization results of microglia cells after blocking TLR4:The expression of CD11b/CD86 in the blocking group was inhibited,and the overall expression was stable.Although the expression of CD11b/CD86 in the blocking group showed an upward trend on day 1 to 5,the difference at each time point was not statistically significant(P>0.05).There was a decreasing trend of the expression in the blocking plus acupuncture group at day 1~5,but there was no statistically significant difference at each time point(P>0.05).Compared with the blocking group,the expression of CD11b/CD86 in the blocking plus acupuncture group was not statistically significant at each time point(P>0.05).Conclusion:1.Acupuncture can improve the general condition of rats with TBI,reducing the m NSS nerve score,promoting the recovery of nerve function,improving the pathological injury of brain tissue of TBI rats.2.On the one hand,acupuncture can inhibit the M1 polarization in microglia,which is neurotoxic and constantly increasing,after TBI,alleviating the secondary injury caused by excessive neuroinflammation;on the other hand,it can accelerate the promotion of neuroprotective M2 polarization,restore the dynamic balance of M1 and M2 polarization,which is beneficial to nerve repair.Acupuncture has a bidirectional benign regulation effect on the M1 and M2 polarization in microglia after TBI.3.Acupuncture can down-regulate the total amount of TLR4 in microglia of TBI rats,inhibit the binding of TRIF,My D88 and TLR4,and inhibit the intracellular pathways(TRIF and My D88)of TLR4.And this might be an important mechanism of acupuncture inhibit the M1 polarization of microglia after TBI,indirectly promoting M2 polarization(negative regulation).4.After blocking the TLR4/TRIF/My D88 pathway with TAK-242,the M1 polarization of microglia and the effect of acupuncture on this polarization were inhibited.This proves that TLR4/TRIF/My D88 pathway is the main regulatory pathway for M1 polarization of microglia,and the inhibition of TLR4/TRIF/My D88 pathway by acupuncture is the main pathway for inhibition of M1 polarization of microglia.