罌粟堿抑制視網(wǎng)膜小膠質(zhì)細胞活化、保護神經(jīng)節(jié)細胞的實驗研究
發(fā)布時間：2018-09-04 12:35
【摘要】：青光眼是當今世界上第一大不可逆的致盲性眼病,典型特征是視網(wǎng)膜神經(jīng)節(jié)細胞的進行性死亡和視野缺損。盡管多種原因與青光眼性神經(jīng)節(jié)細胞損傷有關(guān),但視網(wǎng)膜小膠質(zhì)細胞的過度活化扮演著重要角色:(1)青光眼早期,視網(wǎng)膜小膠質(zhì)細胞即出現(xiàn)增殖并遷徙至視網(wǎng)膜內(nèi)層,由分支狀為阿米巴樣吞噬細胞;(2)活化的小膠質(zhì)細胞釋放的TNF-α、IL-1β等炎癥介質(zhì)與視神經(jīng)損傷程度一致,抑制炎癥介質(zhì)釋放可以減輕神經(jīng)節(jié)細胞損傷;(3)活化后小膠質(zhì)細胞異常的吞噬功能是造成視網(wǎng)膜神經(jīng)節(jié)細胞二次損傷的重要原因。大量研究表明,抑制小膠質(zhì)細胞過度活化能夠有效減輕視網(wǎng)膜神經(jīng)節(jié)細胞損傷,促進視覺功能恢復(fù)。c AMP與小膠質(zhì)細胞活化和中樞神經(jīng)元存活密切相關(guān)。(1)活化c AMP/PKA信號通路能夠通過抑制NF-κB活性減輕小膠質(zhì)細胞的炎癥因子釋放和吞噬功能,促進抗炎因子表達。(2)上調(diào)胞漿c AMP含量還能通過激活c AMP/PKA/CREB信號通路提高中樞神經(jīng)元對抗應(yīng)激損傷的能力,抑制神經(jīng)元凋亡。罌粟堿是一種磷酸二酯酶抑制劑,能夠抑制c AMP降解,提高胞漿內(nèi)c AMP含量。因此,我們推測罌粟堿能夠抑制小膠質(zhì)細胞過度活化,促進視網(wǎng)膜神經(jīng)節(jié)細胞存活。本研究中,我們擬通過(1)使用BV2小膠質(zhì)細胞系和原代視網(wǎng)膜小膠質(zhì)細胞,研究罌粟堿在體外培養(yǎng)條件下對LPS活化的小膠質(zhì)細胞形態(tài)學(xué)改變、炎癥因子表達、抗炎因子表達、表型轉(zhuǎn)換的影響及分子機制;(2)使用大鼠視神經(jīng)橫斷傷模型,研究活體條件下罌粟堿對視網(wǎng)膜小膠質(zhì)細胞形態(tài)、增殖、遷徙、炎癥因子表達及視網(wǎng)膜神經(jīng)節(jié)細胞生存的作用。第一部分:罌粟堿通過NF-κB通路抑制LPS誘導(dǎo)的BV2細胞炎癥活化1材料與方法我們使用LPS誘導(dǎo)的BV2小膠質(zhì)細胞活化模型,觀察不同濃度的罌粟堿作用下,BV2小膠質(zhì)細胞(1)形態(tài)學(xué)變化;(2)炎癥因子的轉(zhuǎn)錄和釋放;(3)表型轉(zhuǎn)換。然后定量分析NF-κB通路的活化水平,探討罌粟堿抑制小膠質(zhì)細胞活化的分子機制。分組:PBS對照組、LPS組、不同濃度的罌粟堿組。2結(jié)果2.1罌粟堿劑量依賴性的抑制LPS誘導(dǎo)的小膠質(zhì)細胞形態(tài)學(xué)變化。正常培養(yǎng)下,大約(5.6±1.2)%的BV2細胞呈阿米巴樣。LPS刺激活化后,阿米巴樣細胞比例顯著升高(89.4±4.5)%,罌粟堿可以劑量依賴性的抑制小膠質(zhì)細胞形態(tài)學(xué)變化(P0.01)。2.2罌粟堿劑量依賴性的抑制LPS誘導(dǎo)的小膠質(zhì)細胞炎癥因子轉(zhuǎn)錄和釋放。正常培養(yǎng)下,小膠質(zhì)細胞釋放極微量的TNF-α(24.54±5.23)pg/ml和IL-1β(20.04±2.13)pg/ml等炎癥因子,LPS上調(diào)了上述炎癥因子的表達數(shù)倍[TNF-α:(687.83±19.78)pg/ml,IL-1β:(153.88±4.69)pg/ml],而罌粟堿能夠劑量依賴性的抑制這種效果[10μg/ml罌粟堿作用下:TNF-α:(315.42±14.58)pg/ml,IL-1β:(35.12±5.26)pg/ml]。2.3罌粟堿促進BV2細胞表型轉(zhuǎn)換。正常培養(yǎng)下BV2細胞表達極低水平的M1標記(COX-2和i NOS)和M2b標記(IL-1ra和SOCS3),LPS能夠顯著上調(diào)M1型標記和M2b型標記的表達(P0.001),而2μg/ml和10μg/ml罌粟堿能劑量依賴性的逆轉(zhuǎn)上述效果并促進M2a型標記(Arg 1和CD206)的表達(P0.05)。2.4罌粟堿通過NF-κB通路抑制LPS誘導(dǎo)的小膠質(zhì)細胞活化。正常情況下,BV2小膠質(zhì)細胞IKK磷酸化水平較低,P65主要分布于胞漿。LPS作用后,小膠質(zhì)細胞IKK的磷酸化水平明顯上調(diào),P65出現(xiàn)顯著的核轉(zhuǎn)移。免疫印跡顯示:罌粟堿能夠劑量依賴性的抑制上述效果(P0.05)。第二部分:罌粟堿通過c AMP/PKA/NF-κB通路抑制LPS誘導(dǎo)的大鼠原代小膠質(zhì)細胞活化1材料與方法分離、純化大鼠視網(wǎng)膜小膠質(zhì)細胞,建立LPS誘導(dǎo)的小膠質(zhì)細胞活化模型。觀察不同濃度的罌粟堿作用下原代視網(wǎng)膜小膠質(zhì)細胞的形態(tài)學(xué)變化、炎癥因子/抗炎因子的表達以及表型轉(zhuǎn)換,觀察(1)不同劑量的罌粟堿作用下,c AMP/PKA、NF-κB通路的活化水平;(2)抑制c AMP/PKA的關(guān)鍵酶后NF-κB的活化狀態(tài)變化。進一步研究c AMP/PKA、NF-κB在罌粟堿抑制LPS誘導(dǎo)的小膠質(zhì)細胞炎癥中的關(guān)系。分組:PBS對照組、LPS組、罌粟堿組(0.4μg/ml組,2μg/ml組和10μg/ml組)。2結(jié)果2.1震搖法聯(lián)合差速貼壁法獲得純度為97.99%的原代大鼠視網(wǎng)膜小膠質(zhì)細胞。2.2罌粟堿抑制原代小膠質(zhì)細胞炎癥因子轉(zhuǎn)錄和釋放:與陰性對照組相比,LPS刺激上調(diào)了(8.00±1.41)倍TNF-α轉(zhuǎn)錄和(14.48±2.08)倍IL-1β轉(zhuǎn)錄,罌粟堿劑量依賴性的抑制了這種效果(P0.01)。與轉(zhuǎn)錄水平類似,預(yù)置罌粟堿能夠劑量依賴性的減少TNF-α和IL-1β的釋放(P0.01)。2.3罌粟堿不逆轉(zhuǎn)LPS引起的原代小膠質(zhì)細胞形態(tài)學(xué)變化。靜息狀態(tài)下,(10.17±2.01)%的原代小膠質(zhì)細胞呈阿米巴樣,LPS顯著增加了阿米巴樣小膠質(zhì)細胞的比例(92.03±5.64)%。與BV2細胞明顯不同,不同劑量的罌粟堿罌粟堿組(0.4μg/ml組,2μg/ml組和10μg/ml組)并不能逆轉(zhuǎn)小膠質(zhì)細胞的形態(tài)改變(與LPS組比較,P=0.737,P=0.290,P=0.290)。2.4罌粟堿促進原代小膠質(zhì)細胞抗炎因子(IL-10)的表達。靜息狀態(tài)下,原代小膠質(zhì)細胞IL-10表達水平很低(20.09±3.30)pg/ml。LPS刺激顯著提高了小膠質(zhì)細胞IL-10的表達(77.83±9.31)pg/ml,而預(yù)置罌粟堿能夠更進一步提高IL-10的表達(0.4μg/ml組除外)[2μg/ml罌粟堿:(77.83±9.31)pg/ml,與LPS組相比,P=0.020;10μg/ml罌粟堿:(77.83±9.31)pg/ml,與LPS組相比,P0.001]。2.5罌粟堿促進LPS活化的原代小膠質(zhì)細胞表型轉(zhuǎn)換。2μg/ml和10μg/ml的罌粟堿能夠抑制原代小膠質(zhì)細胞M1型標記(COX-2和i NOS)(P0.01),上調(diào)M2a型標記Arg1(P0.05)和CD206的表達。2.6罌粟堿抑制視網(wǎng)膜小膠質(zhì)細胞活化受c AMP/PKA/CREB和NF-κB信號通路調(diào)控。2.6.1罌粟堿活化小膠質(zhì)細胞c AMP/PKA/CREB通路。靜息狀態(tài)下,小膠質(zhì)細胞c AMP的含量和CREB的磷酸化水平較低。10μg/ml的罌粟堿顯著上調(diào)了小膠質(zhì)細胞c AMP的含量(P0.001)和CREB的磷酸化水平(P0.001),但LPS部分抑制了這種效果(與LPS組相比,P0.01)。2.6.2罌粟堿抑制原代小膠質(zhì)細胞NF-κB信號通路的激活:與BV2細胞類似,LPS刺激后原代小膠質(zhì)細胞核P65顯著上調(diào)約8倍,但該作用可以被10μg/ml的罌粟堿阻斷約65%。預(yù)置10μg/ml罌粟堿也能抑制LPS誘導(dǎo)的小膠質(zhì)細胞IKK磷酸化約55.7%。2.6.3 NF-κB受c AMP/PKA通路調(diào)控:200μM的rp-isomer或5μM的H-89能夠顯著阻斷罌粟堿對視網(wǎng)膜小膠質(zhì)細胞TNF-α、IL-1β等炎癥因子釋放(P0.001)及P65核轉(zhuǎn)移(64.47%-69.30%)的抑制。第三部分:罌粟堿抑制視神經(jīng)橫斷傷模型大鼠小膠質(zhì)細胞活化和神經(jīng)節(jié)細胞死亡1材料與方法制作大鼠視神經(jīng)橫斷傷模型,玻璃體腔內(nèi)注射不同濃度的罌粟堿。7天后處死大鼠,觀察:小膠質(zhì)細胞的數(shù)量、形態(tài)及遷徙、炎癥因子的釋放;視網(wǎng)膜神經(jīng)節(jié)細胞的數(shù)量、c AMP/PKA/CREB信號通路的活化情況。分組:罌粟堿組(50μg/ml,200μg/ml,500μg/ml)和PBS對照組。2結(jié)果2.1罌粟堿抑制視網(wǎng)膜小膠質(zhì)細胞的增殖、遷徙及形態(tài)學(xué)改變。視神經(jīng)橫斷傷后,小膠質(zhì)細胞大量增殖并由內(nèi)、外從狀層遷徙至神經(jīng)節(jié)細胞層,變成阿米巴樣吞噬細胞。罌粟堿顯著減少,但不能完全阻斷視網(wǎng)膜神經(jīng)節(jié)細胞層阿米巴樣小膠質(zhì)細胞的遷徙和增殖([對照組(15.60±2.30)/切片V.S 500μg/ml罌粟堿組(4.80±1.30)/切片)。500μg/ml的罌粟堿還能減少吞噬視網(wǎng)膜神經(jīng)節(jié)細胞的小膠質(zhì)細胞數(shù)量([對照組(250.20±9.20)/mm2V.S 500μg/ml的罌粟堿組(149.60±14.33)/mm2)。2.2罌粟堿抑制視網(wǎng)膜TNF-α和IL-1β的表達。視神經(jīng)橫斷傷后1周,TNF-α的轉(zhuǎn)錄上調(diào)了(3.86±1.27)倍,玻璃體腔內(nèi)注射罌粟堿顯著抑制了TNF-α的轉(zhuǎn)錄的轉(zhuǎn)錄(P0.05)。罌粟堿對IL-1β具有類似的抑制效果(P0.05)。2.3罌粟堿抑制視網(wǎng)膜神經(jīng)節(jié)細胞的死亡2.3.1全視網(wǎng)膜鋪片RGC密度:視神經(jīng)橫斷傷后7天,視網(wǎng)膜周邊部、中間部、中央部及平均RGC密度分別為1223.77±139.10/mm2,1393.62±112.59/mm2,1607.00±76.89/mm2,1408.13±192.90/mm2。玻璃體腔注射罌粟堿能夠顯著提高全視網(wǎng)膜各個區(qū)域RGC的密度。2.3.2視網(wǎng)膜切片RGC計數(shù):與全視網(wǎng)膜鋪片類似,玻璃體腔注射罌粟堿也顯著提高了視網(wǎng)膜切片上Brn-3a陽性的神經(jīng)節(jié)細胞數(shù)量(PBS對照組:(8.02±1.07)細胞/切片V.S 500μg/ml罌粟堿組:(12.82±1.71)細胞/切片)。2.4罌粟堿能夠提高視網(wǎng)膜神經(jīng)節(jié)細胞CREB的磷酸化水平。視神經(jīng)橫斷傷后1周,PBS對照組各層視網(wǎng)膜幾乎無p-CREB陽性的細胞。玻璃體腔內(nèi)注射不同濃度(50μg/ml-500μg/ml)的罌粟堿均能顯著提高視網(wǎng)膜神經(jīng)節(jié)細胞和內(nèi)核層神經(jīng)元的CREB磷酸化水平。
[Abstract]:Glaucoma is the world's largest irreversible blinding ophthalmopathy, characterized by progressive retinal ganglion cell death and visual field defect. Although many causes are associated with glaucomatous ganglion cell injury, the excessive activation of retinal microglia plays an important role: (1) early glaucoma, retinal microglia Cells proliferate and migrate to the inner layer of the retina, and are branched into amoebic phagocytes. (2) Activated microglia release TNF-a, IL-1 beta and other inflammatory mediators consistent with the degree of optic nerve injury, inhibition of the release of inflammatory mediators can reduce the damage of ganglion cells; (3) After activation, the abnormal phagocytosis of microglia is caused. A large number of studies have shown that inhibition of microglia over-activation can effectively reduce retinal ganglion cell injury and promote visual function recovery. c-AMP is closely related to microglia activation and central neuron survival. (1) activation of c-AMP/PKA signaling pathway can inhibit NF-kappa B by inhibiting NF-kappa B. Papaverine is a phosphodiesterase inhibitor that can inhibit the degradation of C AMP. We hypothesized that papaverine could inhibit the over-activation of microglia and promote the survival of retinal ganglion cells. In this study, we intend to study the morphology of LPS-activated microglia in vitro by using BV2 microglia and primary retinal microglia. The effects of papaverine on the morphology, proliferation, migration, expression of inflammatory factors and survival of retinal ganglion cells in vivo were studied using rat optic nerve transection model. Pathway Inhibits LPS-induced BV2 Cell Inflammatory Activation 1 Material and Methods We used LPS-induced BV2 microglia activation model to observe the morphological changes of BV2 microglia (1), (2) transcription and release of inflammatory factors, (3) phenotypic transformation, and then quantitatively analyze the activation level of NF-kappa B pathway to explore the effect of papaverine on BV2 microglia. Results 2.1 Papaverine inhibited LPS-induced microglial morphological changes in a dose-dependent manner. Under normal culture, about (5.6 (1.2)) percent of BV2 cells showed amoebic morphology. After LPS stimulation, the proportion of amoebic cells was significant. Papaverine inhibited LPS-induced microglial inflammatory cytokine transcription and release in a dose-dependent manner (P 0.01). Under normal culture, microglial cells released minimal amounts of TNF-alpha (24.54+5.23) pg/ml and IL-1 beta (20.04+2.13) pg/ml. Factor and LPS up-regulated the expression of these inflammatory factors several times [TNF-a: (687.83 [19.78] pg/ml, IL-1 beta: (153.88 [4.69] pg/ml], and papaverine could inhibit this effect in a dose-dependent manner [10 ug/ml papaverine: TNF-a: (315.42 [14.58] pg/ml), IL-1 beta: (35.12 [5.26] pg/ml]. LPS could significantly up-regulate the expression of M1 and M2b markers (P 0.001) in BV2 cells at very low levels (COX-2 and I NOS) and M2b markers (IL-1ra and SOCS3), while 2 ug/ml and 10 ug/ml papaverine could reverse the above effect and promote the expression of M2a markers (Arg-1 and CD206) in a dose-dependent manner (P 0.05). Pathway inhibited LPS-induced microglial activation. Normally, the IKK phosphorylation level of BV2 microglia was low, and P65 was mainly distributed in the cytoplasm. After LPS treatment, the IKK phosphorylation level of microglia was significantly up-regulated, and P65 showed significant nuclear metastasis. Immunoblotting showed that papaverine could inhibit the above effects in a dose-dependent manner (P 0.05). Part two: Papaverine inhibits LPS-induced microglia activation in rats through the c-AMP/PKA/NF-kappa B pathway. Material and method were isolated and purified. LPS-induced microglia activation model was established. Morphological changes and inflammation of primary retinal microglia were observed under different concentrations of papaverine. To observe the expression of factor/anti-inflammatory factor and phenotypic transition, we observed (1) the activation level of c-AMP/PKA and NF-kappa B pathway under different doses of papaverine; (2) the activation state of NF-kappa B after inhibiting the key enzymes of c-AMP/PKA. Results 2.1 Shaking method combined with differential adherence method was used to obtain 97.99% purity of primary rat retinal microglia. 2.2 Papaverine inhibited the transcription and release of inflammatory factors in primary microglia: Compared with negative control group, LPS stimulation increased (8.00 (+ 1.41) times TNF-alpha The effect was inhibited by papaverine in a dose-dependent manner (P 0.01). Similar to the transcriptional level, preset papaverine reduced the release of TNF-a and IL-1 beta in a dose-dependent manner (P 0.01). 2.3 papaverine did not reverse the morphological changes of primary microglia induced by LPS. In resting state, (10.17+2.01)% of the primary microglia were inhibited by papaverine. Different from BV2 cells, different doses of papaverine (0.4 ug/ml group, 2 ug/ml group and 10 ug/ml group) could not reverse the morphological changes of microglia (compared with LPS group, P = 0.737, P = 0.290, P = 0.290).2.4 poppy. LPS stimulation significantly increased the expression of IL-10 in primary microglia (77.83+9.31) pg/ml, but papaverine pretreatment could further increase the expression of IL-10 (except 0.4 ug/ml group) [2 ug/ml papaverine]. Papaverine (77.83 [9.31] pg / ml, P = 0.020; 10 UG / ml papaverine: (77.83 [9.31] pg / ml, compared with LPS group, P 0.001]. 2.5 papaverine promoted LPS-activated primary microglia phenotypic transition. 2 UG / ml and 10 UG / ml papaverine inhibited primary microglia M1-type markers (COX-2 and I NOS) (P 0.01), up-regulated M2a-type markers Arg1 (P 0.0.01). 5) and CD206 expression.2.6 Papaverine inhibits retinal microglia activation regulated by C AMP/PKA/CREB and NF-kappa B signaling pathways. MP content (P 0.001) and CREB phosphorylation (P 0.001), but LPS partially inhibited this effect (compared with LPS group, P 0.01). Inhibition of IKK phosphorylation in LPS-induced microglia was also inhibited by 10 ug/ml papaverine. 2.6.3 NF-kappa B was regulated by the c-AMP/PKA pathway. 200 UG rp-isomer or 5 UG H-89 significantly blocked papaverine release of inflammatory factors such as TNF-a, IL-1 beta (P 0.001) and P65 nuclear metastasis (64.47% -69.30%). Part 3: Papaverine inhibited the activation of microglia and the death of ganglion cells in the rat model of optic nerve transection 1 Material and Methods The rat model of optic nerve transection was made by intravitreal injection of different concentrations of papaverine. Result 2.1 Papaverine inhibited the proliferation, migration and morphological changes of retinal microglia. After optic nerve transection, microglia proliferated and migrated from the inner and outer layers. Papaverine significantly decreased, but could not completely block the migration and proliferation of amebic microglia in the retinal ganglion cell layer ([control group (15.60 [2.30) / slice V.S 500 UG / ml papaverine group (4.80 [1.30) / slice)]. 500 UG / ml papaverine also reduced the phagocytosis of retinal nerves. The number of microglia in ganglion cells ([control group (250.20 Papaverine had a similar inhibitory effect on IL-1 beta (P 0.05). 2.3 Papaverine inhibited retinal ganglion cell death 2.3.1 RGC density: 7 days after optic nerve transection, the density of RGC in peripheral, middle, central and average retinal areas was 1223.77 (+139.10/mm2), 1393.62 (+112.59/mm2), 1607.00 (+76.89/mm2), 1408.8, respectively. Intravitreal papaverine injection significantly increased the RGC density in various regions of the whole retina. 2.3.2 RGC counts in retinal slices: Similar to panretinal slices, intravitreal papaverine injection significantly increased the number of Brn-3a-positive ganglion cells in retinal slices (PBS control group: (8.02 + 1.07) cells / slices V.S 50 0 UG / ml papaverine group: (12.82 (- 1.71) cells / slices).2.4 papaverine can increase the phosphorylation of CREB in retinal ganglion cells. CREB phosphorylation level in ganglion cells and inner nuclear neurons.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R775
，
本文編號：2222117
[Abstract]:Glaucoma is the world's largest irreversible blinding ophthalmopathy, characterized by progressive retinal ganglion cell death and visual field defect. Although many causes are associated with glaucomatous ganglion cell injury, the excessive activation of retinal microglia plays an important role: (1) early glaucoma, retinal microglia Cells proliferate and migrate to the inner layer of the retina, and are branched into amoebic phagocytes. (2) Activated microglia release TNF-a, IL-1 beta and other inflammatory mediators consistent with the degree of optic nerve injury, inhibition of the release of inflammatory mediators can reduce the damage of ganglion cells; (3) After activation, the abnormal phagocytosis of microglia is caused. A large number of studies have shown that inhibition of microglia over-activation can effectively reduce retinal ganglion cell injury and promote visual function recovery. c-AMP is closely related to microglia activation and central neuron survival. (1) activation of c-AMP/PKA signaling pathway can inhibit NF-kappa B by inhibiting NF-kappa B. Papaverine is a phosphodiesterase inhibitor that can inhibit the degradation of C AMP. We hypothesized that papaverine could inhibit the over-activation of microglia and promote the survival of retinal ganglion cells. In this study, we intend to study the morphology of LPS-activated microglia in vitro by using BV2 microglia and primary retinal microglia. The effects of papaverine on the morphology, proliferation, migration, expression of inflammatory factors and survival of retinal ganglion cells in vivo were studied using rat optic nerve transection model. Pathway Inhibits LPS-induced BV2 Cell Inflammatory Activation 1 Material and Methods We used LPS-induced BV2 microglia activation model to observe the morphological changes of BV2 microglia (1), (2) transcription and release of inflammatory factors, (3) phenotypic transformation, and then quantitatively analyze the activation level of NF-kappa B pathway to explore the effect of papaverine on BV2 microglia. Results 2.1 Papaverine inhibited LPS-induced microglial morphological changes in a dose-dependent manner. Under normal culture, about (5.6 (1.2)) percent of BV2 cells showed amoebic morphology. After LPS stimulation, the proportion of amoebic cells was significant. Papaverine inhibited LPS-induced microglial inflammatory cytokine transcription and release in a dose-dependent manner (P 0.01). Under normal culture, microglial cells released minimal amounts of TNF-alpha (24.54+5.23) pg/ml and IL-1 beta (20.04+2.13) pg/ml. Factor and LPS up-regulated the expression of these inflammatory factors several times [TNF-a: (687.83 [19.78] pg/ml, IL-1 beta: (153.88 [4.69] pg/ml], and papaverine could inhibit this effect in a dose-dependent manner [10 ug/ml papaverine: TNF-a: (315.42 [14.58] pg/ml), IL-1 beta: (35.12 [5.26] pg/ml]. LPS could significantly up-regulate the expression of M1 and M2b markers (P 0.001) in BV2 cells at very low levels (COX-2 and I NOS) and M2b markers (IL-1ra and SOCS3), while 2 ug/ml and 10 ug/ml papaverine could reverse the above effect and promote the expression of M2a markers (Arg-1 and CD206) in a dose-dependent manner (P 0.05). Pathway inhibited LPS-induced microglial activation. Normally, the IKK phosphorylation level of BV2 microglia was low, and P65 was mainly distributed in the cytoplasm. After LPS treatment, the IKK phosphorylation level of microglia was significantly up-regulated, and P65 showed significant nuclear metastasis. Immunoblotting showed that papaverine could inhibit the above effects in a dose-dependent manner (P 0.05). Part two: Papaverine inhibits LPS-induced microglia activation in rats through the c-AMP/PKA/NF-kappa B pathway. Material and method were isolated and purified. LPS-induced microglia activation model was established. Morphological changes and inflammation of primary retinal microglia were observed under different concentrations of papaverine. To observe the expression of factor/anti-inflammatory factor and phenotypic transition, we observed (1) the activation level of c-AMP/PKA and NF-kappa B pathway under different doses of papaverine; (2) the activation state of NF-kappa B after inhibiting the key enzymes of c-AMP/PKA. Results 2.1 Shaking method combined with differential adherence method was used to obtain 97.99% purity of primary rat retinal microglia. 2.2 Papaverine inhibited the transcription and release of inflammatory factors in primary microglia: Compared with negative control group, LPS stimulation increased (8.00 (+ 1.41) times TNF-alpha The effect was inhibited by papaverine in a dose-dependent manner (P 0.01). Similar to the transcriptional level, preset papaverine reduced the release of TNF-a and IL-1 beta in a dose-dependent manner (P 0.01). 2.3 papaverine did not reverse the morphological changes of primary microglia induced by LPS. In resting state, (10.17+2.01)% of the primary microglia were inhibited by papaverine. Different from BV2 cells, different doses of papaverine (0.4 ug/ml group, 2 ug/ml group and 10 ug/ml group) could not reverse the morphological changes of microglia (compared with LPS group, P = 0.737, P = 0.290, P = 0.290).2.4 poppy. LPS stimulation significantly increased the expression of IL-10 in primary microglia (77.83+9.31) pg/ml, but papaverine pretreatment could further increase the expression of IL-10 (except 0.4 ug/ml group) [2 ug/ml papaverine]. Papaverine (77.83 [9.31] pg / ml, P = 0.020; 10 UG / ml papaverine: (77.83 [9.31] pg / ml, compared with LPS group, P 0.001]. 2.5 papaverine promoted LPS-activated primary microglia phenotypic transition. 2 UG / ml and 10 UG / ml papaverine inhibited primary microglia M1-type markers (COX-2 and I NOS) (P 0.01), up-regulated M2a-type markers Arg1 (P 0.0.01). 5) and CD206 expression.2.6 Papaverine inhibits retinal microglia activation regulated by C AMP/PKA/CREB and NF-kappa B signaling pathways. MP content (P 0.001) and CREB phosphorylation (P 0.001), but LPS partially inhibited this effect (compared with LPS group, P 0.01). Inhibition of IKK phosphorylation in LPS-induced microglia was also inhibited by 10 ug/ml papaverine. 2.6.3 NF-kappa B was regulated by the c-AMP/PKA pathway. 200 UG rp-isomer or 5 UG H-89 significantly blocked papaverine release of inflammatory factors such as TNF-a, IL-1 beta (P 0.001) and P65 nuclear metastasis (64.47% -69.30%). Part 3: Papaverine inhibited the activation of microglia and the death of ganglion cells in the rat model of optic nerve transection 1 Material and Methods The rat model of optic nerve transection was made by intravitreal injection of different concentrations of papaverine. Result 2.1 Papaverine inhibited the proliferation, migration and morphological changes of retinal microglia. After optic nerve transection, microglia proliferated and migrated from the inner and outer layers. Papaverine significantly decreased, but could not completely block the migration and proliferation of amebic microglia in the retinal ganglion cell layer ([control group (15.60 [2.30) / slice V.S 500 UG / ml papaverine group (4.80 [1.30) / slice)]. 500 UG / ml papaverine also reduced the phagocytosis of retinal nerves. The number of microglia in ganglion cells ([control group (250.20 Papaverine had a similar inhibitory effect on IL-1 beta (P 0.05). 2.3 Papaverine inhibited retinal ganglion cell death 2.3.1 RGC density: 7 days after optic nerve transection, the density of RGC in peripheral, middle, central and average retinal areas was 1223.77 (+139.10/mm2), 1393.62 (+112.59/mm2), 1607.00 (+76.89/mm2), 1408.8, respectively. Intravitreal papaverine injection significantly increased the RGC density in various regions of the whole retina. 2.3.2 RGC counts in retinal slices: Similar to panretinal slices, intravitreal papaverine injection significantly increased the number of Brn-3a-positive ganglion cells in retinal slices (PBS control group: (8.02 + 1.07) cells / slices V.S 50 0 UG / ml papaverine group: (12.82 (- 1.71) cells / slices).2.4 papaverine can increase the phosphorylation of CREB in retinal ganglion cells. CREB phosphorylation level in ganglion cells and inner nuclear neurons.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R775
，
本文編號：2222117
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