The Mechanism Of Acupuncture Regulating Photoreceptor Cell Apoptosis Model JAK2/STAT3 Pathway To Inhibit Retinal Microglia

Objective: Retinal Degenerative(RD)is based on photoreceptor cell apoptosis.The activation of microglia can aggravate photoreceptor cell apoptosis.JAK2/STAT3 pathway is related to glial cell differentiation and neuronal apoptosis.It was proved that acupuncture may inhibit the activation of retinal microglia,but the mechanism needs to be explored.The purpose of this study is to explore the mechanism of acupuncture regulating the JAK2/STAT3 pathway to inhibit the activation of microglia and slow down the apoptosis of photoreceptor cells.Methods:1.The activation of microglia in the photoreceptor cell apoptosis model:(1)The migration and activation of retinal microglia in the photoreceptor apoptosis rat model: SD rats were divided into blank group,model 1 day group,model 7 day group,model 14 day group,and model 28 day group.The rats in the model group were all established by intraperitoneal injection of MNU.The retinal morphology and photoreceptor cell apoptosis were observed by HE staining and TUNEL method.Iba1 was labeled with immunofluorescence to observe the migration status of retinal microglia.(2)Establishment and evaluation of in vitro glucose deprivation photoreceptor cell apoptosis model: 661 W cells in vitro were cultured with energy depletion and glucose deprivation for 4h,8h,12 h,and 24 h using the glucose deprivation method,and the 661 W cell apoptosis model was established through LDH.And flow cytometry to detect the expression of cell necrosis and apoptosis.(3)Activation of microglia in the photoreceptor cell apoptosis model in vitro:Establish co-culture system of 661 W cell apoptosis model and BV2 microglia at 4time points of 4h,8h,12 h,and 24 h,and use flow cytometry to detect each At the time point 661 W cells expressed apoptosis,and the levels of IL-1? and TNF-?inflammatory factors in microglia were detected by ELISA.2.Study on the mechanism of inhibiting the activation of microglia by the JAK2/STAT3 pathway:(1)The effect of inhibiting the JAK2/STAT3 pathway on the activation and migration of retinal microglia: establish photoreceptor cell apoptosis SD rat model,rats divided into blank group,model group,DMSO control group,AG490 low,medium and high dose groups(5mg/kg,10mg/kg,15mg/kg),retinal morphological changes and photoreceptor cell apoptosis were detected by HE staining and TUNEL method,and observe the activation and migration status of retinal microglia by immunofluorescence labeling,Western blot and RT-PCR were used to detect the expression of JAK2,STAT3 protein and m RNA in the retina.(2)Inhibition of the effect of JAK2/STAT3 pathway on microglia activity in vitro:Establish an in vitro 661 W cell apoptosis model and BV2 microglia co-culture system,and use JAK2/STAT3 pathway antagonist-AG490 at low,medium and high concentrations(10umol,50 umol,100umol)in co-culture system.Flow cytometry to observe the apoptosis of 661 W cells,ELISA method to detect the expression levels of IL-1? and TNF-?,Western blot and RT-PCR detect the protein expression and m RNA level of JAK2 and STAT3.3.Study on the effect and mechanism of acupuncture regulating photoreceptor cell apoptosis model JAK2/STAT3 pathway to inhibit the activation of retinal microglia:(1)Study on the effect of acupuncture on inhibiting the activation of retinal microglia: establish a photoreceptor apoptosis model in SD rats and divide them into blank group,model group,vitamin A group,sham acupuncture group and acupuncture group.After 28 days of intervention in each group,HE staining and TUNEL method were used to observe the changes of retinal tissue morphology and photoreceptor cell apoptosis in each group;immunofluorescence method was used to observe the migration of retinal microglia marker Iba1 in each group;ELISA was used to detect the expression of IL-1? and TNF-?.(2)Study on the mechanism of acupuncture regulating JAK2/STAT3 pathway to inhibit retinal microglia: establish photoreceptor cell apoptosis rat model,rats divided into blank group,model group,vitamin A group,sham acupuncture group and acupuncture group.The protein and m RNA expression of key factors JAK2,STAT3,IL-6,MCP1 and SOCS3 in the rat retina were detected by Western blot and RT-PCR.Results:1.The activation of microglia in the photoreceptor cell apoptosis model:(1)The MNU photoreceptor cell apoptosis model rats were successfully established.The retina of the model group were slightly loosely arranged in 1 day group,and the layers of the retina of the model group were disordered;the photoreceptor cells in the model group 14 days group were significantly pyknotic;the28 days model group was disordered,and the number of cells in the inner and outer nuclear layers was significantly reduced;the thickness of the outer nuclear layer of the retina was positively correlated with the modeling time;TUNEL showed that except for the blank group,the retinal apoptosis rate of the other groups increased with time,The apoptotic rate of 28 days was the highest in the group;immunofluorescence showed that the expression of Iba1 in the retina of the blank group was mainly located in the inner layer of the retina and was in a resting state;Iba1(+)showed a small amount of migration in the model 1 day group,and Iba1(+)in the model 7 day group migrating,and the cell synapses can be seen in a slender appearance;a large amount of Iba1(+)in the outer layer of the retina can be seen in the 14 day model group,and the appearance of Iba1(+)can be seen to be amoebic.In the 28 day model group,Iba1(+)is obviously amoebic,showing its stable state.(2)Successfully established the vitro photoreceptor cell apoptosis model under glucose deprivation conditions.No obvious changes were seen after 4h glucose deprivation.In the 8h group,there were some cell membrane destruction and cell body shrinkage changes;the 12 h group showed that the synapses became longer,the cell morphology changed from flat to irregular shape,and floating cells increased;In the 24 h group,the cell membrane of 661 W was damaged,the cell body was retracted obviously,and cell disintegration was seen.The necrosis rate and apoptosis rate of661 W cells in the 8h,12 h,and 24 h groups of glucose deprivation were significantly higher than those of the normal group(all P<0.01),and the necrosis rate(P>0.05)and apoptosis rate(P<0.05)of the 4h glucose deprivation group)Slightly higher than 4h normal group;661W cell necrosis rate and apoptosis rate are positively correlated with glucose deprivation time,24 h apoptosis rate reached 86.30±3.43%,indicating that the in vitro photoreceptor cell apoptosis model was successfully established.(3)After successfully establishing glucose deprivation photoreceptor cell apoptosis model,661 W cell apoptosis model was established co-cultured with BV2 microglia,the apoptotic rate of 661 W cells increased with the increase of co-cultivation time.After 24 hours of co-cultivation,the apoptotic rate of BV2 microglia with glucose deprived 661 W cell group(81.24±3.32%)was significantly higher in the control group(P<0.01),the levels of IL-1? and TNF-? inflammatory factors in microglia co-cultured with glucose-deprived 661 W cells increased with time.After 24 hours,IL-1? and TNF-? level reaches the highest point.2.Study on the mechanism of inhibiting the activation of microglia by the JAK2/STAT3 pathway:(1)MNU model rats were intraperitoneally injected with the antagonist AG490 to block the JAK2/STAT3 pathway.The results showed that the number of photoreceptor cells in the retina of each AG490 intervention group was lower than that of the model group and the DMSO control group,and the AG490 high-dose group had significant apoptosis rate lower than the model group(P<0.01),the model group can see large number of Iba1(+)expression in the subretinal cavity and outer nuclear layer.As the dose of AG490 increases,the expression of Iba1(+)in each group shows a decreasing trend,and the high dose of AG490,the number of Iba1(+)cells in the outer nuclear layer of the retina in the group was significantly less than that in the model group;Western blot and RT-PCR results showed that the JAK2 and STAT3 protein levels in the AG490 low,medium and high dose group were lower than those in the model group(all P<0.01).(2)After treatment with AG490 antagonist at various concentrations,the expression of IL-1? and TNF-? inflammatory factors and the apoptosis rate of 661 W cells in the co-culture system were lower than those in the model group.JAK2,STAT3 protein and m RNA levels increased with the dose of antagonist.The expression showed a decreasing trend,and the levels of high-dose were significantly lower than those of other groups(P<0.01).3.Study on the effect and mechanism of acupuncture regulating photoreceptor cell apoptosis model JAK2/STAT3 pathway to inhibit the activation of retinal microglia:(1)In each group of rats,HE staining showed that the retinal structure of the blank group was not significantly changed.The retina of the model group was arranged loosely and irregularly.The retinal changes of the sham acupuncture group were similar to those of the model group,but lighter than the model group.The vitamin A group and the acupuncture group were improved compared with the model group;TUNEL method detected photoreceptor cell apoptosis.The acupuncture group showed that the number of apoptosis in the acupuncture group was less than that of the model group(P<0.05);the microglia marker of the model group Iba1(+)showed that the expression was mainly located in the ONL and subretinal space;compared with the model group,the expression of Iba1 in the sham acupuncture group is reduced,and the expression of Iba1 in the vitamin A group and the acupuncture group is significantly weaker than that in the model group;The expression of IL-1? and TNF-? in the model group were significantly higher than the blank group.The levels of IL-1? and TNF-? in the acupuncture group were significantly lower than the model group(all P<0.01).(2)Western blot and RT-PCR results showed that the retina JAK2,STAT3,IL-6and MCP1 protein and m RNA expression in the acupuncture group were significantly lower than those in the model group,and the SOCS3 protein expression and m RNA were significantly higher than those in the model group(all P< 0.01);Conclusion:1.JAK2/STAT3 pathway plays an important role in the activation of microglia and antagonizes JAK2/STAT3.The pathway can reduce the inflammatory level of microglia,inhibit their activation and migration state,and slow down the process of photoreceptor cell apoptosis;2.Acupuncture can inhibit the activation and migration of retinal microglia,and reduce the release of IL-1? and TNF-? inflammatory factors,protect the pathological degeneration of RD.The mechanism is related to the JAK2/STAT3 pathway,which up-regulate JAK2,STAT3,IL-6 and MCP1,and down-regulate the SOCS3 factor.