秦皮滴眼液抑制PI3K/Akt/mTOR信号通路缓解兔自身免疫性干眼的实验研究

Qinpi eyedrop reliving rabbit autoimmune dry eye via PI3K/Akt/mTOR pathway

目的研究秦皮滴眼液改善兔自身免疫性干眼泪液学指标及眼表慢性炎症的机制。方法健康成年雌性新西兰大白兔随机分为对照组(CG)、模型组(MG)和秦皮组(QP),各12只。MG、QP组通过耳缘静脉回输激活的自体外周血淋巴细胞(PBL)建立兔自身免疫性干眼模型。CG、MG组兔予磷酸盐缓冲液滴眼,QP组兔予秦皮滴眼液滴眼,连续给药6周。给药完成后检测兔泪液学指标,包括泪液分泌量(SⅠT)、泪膜破裂时间(BUT)、角膜荧光素染色(FL)评分;取兔右眼泪腺进行称重,苏木精-伊红(HE)染色观察泪腺、角膜结构,过碘酸雪夫(PAS)染色观察结膜杯状细胞形态及密度;实时荧光定量PCR(RT-qPCR)法和Western Blot法分别检测磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关的蛋白激酶B(Akt)、叉头翼状螺旋转录因子3(Foxp3)、维甲酸核孤儿受体-γt(ROR-γt)mRNA和蛋白的表达水平。结果(1)泪液学指标:与CG组比较,MG组给药6周后兔的SⅠT、BUT降低(t_(SⅠT)=9.493,t_(BUT)=7.889,均P=0.000),FL评分升高(t=8.751,P=0.000);与MG组比较,QP组给药6周后兔的SⅠT、BUT升高(t_(SⅠT)=2.800,P=0.009;t_(BUT)=2.976,P=0.005),FL评分降低(t=2.586,P=0.014),差异均有统计学意义。(2)泪腺重量和组织形态:与CG组比较,MG组泪腺重量降低(t=6.038,P=0.000);与MG组比较,QP组泪腺重量升高(t=2.175,P=0.037),差异均有统计学意义。MG组泪腺腺泡结构破坏、大量淋巴细胞浸润,QP组泪腺淋巴细胞聚集较MG组少。(3)角膜组织形态:与CG组比较,MG组角膜增厚、细胞形态及排列不规则,QP组较CG组无明显改变。(4)结膜组织形态:与CG组比较,MG组结膜上皮细胞破坏,杯状细胞数降低(t=4.808,P=0.000);与MG组比较,QP组结膜上皮细胞排列整齐、杯状细胞数升高(t=2.295,P=0.028),差异有统计学意义。(5)PI3K/Akt/mTOR信号通路:与CG组比较,MG组Akt、ROR-γt的mRNA和蛋白表达水平升高(mRNA:t_(Akt)=11.810,t_(ROR-γt)=10.520,均P=0.000。蛋白:t_(p-Akt)=2.494,P=0.018;t_(ROR-γt)=2.325,P=0.026),Foxp3的mRNA和蛋白表达水平降低(:t_(mRNA)=11.350,t_(蛋白)=11.210,均P=0.000);与MG组比较,QP组Akt、ROR-γt的mRNA和蛋白表达水平降低(mRNA:t_(Akt)=5.879,t_(ROR-γt)=6.698,均P=0.000。蛋白:t_(p-Akt)=5.276,t_(ROR-γt)=6.232,均P=0.000),Foxp3的mRNA和蛋白表达水平升高(t_(mRNA)=5.003,t_(蛋白)=7.474,均P=0.000),差异均有统计学意义。结论秦皮滴眼液可以通过抑制PI3K/Akt/mTOR信号通路缓解兔自身免疫性干眼。

OBJECTIVE To investigate the mechanism of Qinpi eyedrop improving the lacrimal indicators and the chronic ocular surface inflammations of rabbit with autoimmune dry eyes.METHODS Female adult New Zealand rabbits were randomly divided to control group(CG),model group(MG)and Qinpi group(QP),and 12 in each group.In MG and QP group,rabbit autoimmune dry eyes were established with activated autologous PBL via ear margin veins.Phosphate buffered saline was topically administrated to rabbits in CG and MG,Qinpi eyedrop was topically administrated to rabbits in QP group for six weeks.After administration of six weeks,lacrimal indicators including SchirmerⅠtest(SⅠT),tear film break-up times(BUT),cornea fluorescein(FL)scores were determined;Lacrimal gland weights of right eyes,hematoxylin-eosin(HE)staining of lacrimal and cornea,periodic acid-Schiff(PAS)staining of conjunctiva goblet cells densities and morphology were observed;Phosphatidylinositiol 3-kinases/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway related protein kinase B(Akt),forkhead or winged helix transcription factor 3(Foxp3),retinoic acid nuclear orphan receptorγt(ROR-γt)mRNA and protein expressions were detected by real time quantitative PCR(RT-PCR)and Western Blot.RESULTS(1)Lacrimal indicators:After administration of six weeks,compared with CG,tear secretions and BUTs in MG decreased(t_(SⅠT)=9.493,t_(BUT)=7.889,both P=0.000),FL scores increased(t=8.751,P=0.000);Compared with MG,tear secretions and BUTs in QP group increased(t_(SⅠT)=2.800,P=0.009;t_(BUT)=2.976,P=0.005),FL scores decreased(t=2.586,P=0.014),the differences were all statistically significant.(2)Lacrimal grand morphology and weights:Compared with CG,lacrimal grand weights in MG group decreased(t=6.038,P=0.000);Compared with MG,lacrimal grand weights in QP group increased(t=2.175,P=0.037),the differences were all statistically significant.The lacrimal acinar structures were destroyed and a large number of lymphocytes infiltrated in MG and the accumulation of lacrimal lymphocytes in QP group was less than that in MG.(3)Cornea morphology:Compared with CG,cornea in MG was thickened and cells were irregular and uneven distributed,while there was hardly alteration in QP group.(4)Conjunctiva morphology:Compared with CG,the destruction of conjunctiva epithelial cells was obvious in MG and goblet cells density decreased(t=4.808,P=0.000);Compared with MG,the conjunctiva epithelial cells were distributed neatly in QP group and goblet cells density increased(t=2.295,P=0.028),the differences were statistically significant.(5)PI3K/Akt/mTOR signaling pathway:Compared with CG,Akt,ROR-γt mRNA and protein expressions in MG increased(m_(RNA:tAkt)=11.810,tROR-γt=10.520,both P=0.000;Proteins:t_(p-Akt)=2.494,P=0.018;t_(ROR-γt)=2.325,P=0.026),Foxp3 mRNA and protein expressions decreased(t_(mRNA)=11.350,t_(Protein)=11.210,both P=0.000);Compared with MG,Akt,ROR-γt mRNA and protein expressions in QP group decreased(mRNA:t_(Akt)=5.879,t_(ROR-γt)=6.698,both P=0.000;Proteins:t_(p-Akt)=5.276,t_(ROR-γt)=6.232,both P=0.000),Foxp3 mRNA and protein expressions increased(t_(mRNA)=5.003,t_(Protein)=7.474,both P=0.000),the differences were all statistically significant.CONCLUSIONS Qinpi eyedrops can alleviate autoimmune dry eye in rabbits by inhibiting the PI3K/Akt/mTOR signaling pathway.