基于VIP/cAMP/PKA/AQP5信号通路探讨针刺治疗水液缺乏型干眼的作用机制  

Acupuncture promotes tear secretion by up-regulating VIP/cAMP/PKA/AQP5 signaling in guinea pigs with aqueous tear deficiency dry eye

作　　者：刘雪[1,2] 李影 沈乎醒[3] 高卫萍 赵楠 黎柳娇 刘成勇 LIU Xue;LI Ying;SHEN Hu-xing;GAO Wei-ping;ZHAO Nan;LI Liu-jiao;LIU Cheng-yong(The First Clinical Medical College,Nanjing University of Chinese Medicine,Nanjing 210023,China;Ophthalmology Department,the Affiliated Hospital of Gansu University of Chinese Medicine,Lanzhou 730000;Ophthalmology Department,the Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210024;Clinical Medical College,Gansu University of Chinese Medicine,Lanzhou 730000;Acupuncture and Rehabilitation Department,the Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210024)

机构地区：[1]南京中医药大学第一临床学院,南京210023 [2]甘肃中医药大学附属医院眼科,兰州730000 [3]南京中医药大学附属医院眼科,南京210024 [4]甘肃中医药大学中医临床学院,兰州730000 [5]南京中医药大学附属医院针灸康复科,南京210024

出　　处：《针刺研究》2023年第10期1025-1032,共8页Acupuncture Research

基　　金：国家自然科学基金项目(No.82074526);甘肃省青年科技基金项目(No.22JR5RA615)。

摘　　要：目的:观察针刺对水液缺乏型干眼(ATD)豚鼠眼表症状及泪腺组织中血管活性肠肽(VIP)/环磷酸腺苷(cAMP)/蛋白激酶A(PKA)/水通道蛋白5(AQP5)信号通路相关蛋白表达的影响,探讨针刺治疗ATD的作用机制。方法:40只豚鼠随机分为空白组、模型组、针刺组、假针组和西药组,每组8只。皮下注射氢溴酸东莨菪碱复制ATD模型。针刺组针刺双侧“睛明”“攒竹”“丝竹空”“太阳”“瞳子髎”,每次15 min,每日1次;假针组每5 min钝针点压上述穴位1次,共3次;西药组双眼滴玻璃酸钠滴眼液,3次/d,每次1滴。以上3组均治疗14 d。分别于造模前、造模后及干预后进行酚红棉线泪液分泌量(PRT)实验、测定泪膜破裂时间(BUT)和角膜荧光素钠染色评分(FLS)。干预后泪腺称重,计算泪腺指数;HE染色法观察泪腺组织病理形态变化;免疫荧光染色法检测泪腺组织AQP5的表达;ELISA法检测泪腺组织VIP和AQP5含量;Western blot法检测泪腺组织VIP、cAMP、PKA、磷酸化(p)-PKA及AQP5蛋白表达。结果:造模后,与空白组比较,其余4组PRT减少、BUT缩短(P<0.01,P<0.05),FLS升高(P<0.05,P<0.01)。干预后,与空白组比较,模型组PRT减少、BUT缩短(P<0.01),FLS升高(P<0.01),泪腺指数降低(P<0.01),泪腺组织VIP含量、AQP5免疫荧光强度及含量降低(P<0.01),VIP、cAMP、PKA、p-PKA和AQP5蛋白表达降低(P<0.01,P<0.05)。干预后,与模型组比较,针刺组和西药组PRT增多、BUT延长(P<0.01,P<0.05),FLS降低(P<0.01,P<0.05),泪腺指数升高(P<0.05),泪腺组织VIP含量、AQP5免疫荧光强度及含量升高(P<0.01);针刺组泪腺组织VIP、cAMP、PKA、p-PKA、AQP5蛋白表达升高(P<0.05,P<0.01);西药组VIP和AQP5的蛋白表达升高(P<0.05)。与假针组比较,针刺组和西药组PRT增多、BUT延长(P<0.01,P<0.05),FLS降低(P<0.01,P<0.05),泪腺组织VIP含量、AQP5免疫荧光强度及含量升高(P<0.05,P<0.01);针刺组泪腺组织VIP、cAMP、PKA、AQP5蛋白表达升高(P<0.05,P<0.0Objective To observe the effect of acupuncture on the ocular surface symptoms and the protein expression of vasoactive intestinal peptide(VIP)/cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/aquaporin 5(AQP5)signaling pathway in lacrimal gland tissue of aqueous tear deficiency(ATD)type dry eye model,so as to investigate its mechanism underlying improvement of ATD.Methods British shorthair guinea pigs were randomly divided into blank control,model,acupuncture,sham-acupuncture and medication group,with 8 guinea pigs in each group.The ATD model was established by subcutaneous injection of scopolamine hydrobromide(0.6 mg/dose,4 times/d for 10 days).For guinea pigs of the acupuncture group,filiform needles were inserted into bilateral“Jingming”(BL1),“Cuanzhu”(BL2),“Sizhukong”(TE23),“Taiyang”(EX-HN5),and“Tongziliao”(GB1)for 15 min.For guinea pigs of the sham-acupuncture group,a blunt filiform needle was used to repeatedly prick(not pierce)the skin of the same acu⁃points mentioned above.The treatment in the above two groups was conducted once daily for 14 days.The guinea pigs in the medication group received administration of sodium hyaluronate eye drops in both eyes,three times a day for 14 days.The objective tests of tear film break-up time(BUT),corneal fluorescein staining score(FLS)and phenol red thread(PRT)test were conducted before and after modeling and after the intervention.After the intervention,the lacri⁃mal index(weight of lacrimal gland/body weight)was calculated.Histopathological changes of the lacrimal gland were observed after H.E.staining.The expression of AQP5 in the lacrimal gland were detected by immunofluorescence,and the contents of VIP and AQP5 in the lacrimal gland were measured by ELISA,the protein expression levels of VIP,cAMP,PKA,p-PKA and AQP5 in the lacrimal gland were detected by Western blot.Results In comparison with the blank control group,the PRT,BUT,lacrimal index,AQP5 immunoactivity,contents of VIP and AQP5,and protein ex⁃pression levels of VIP,cAM

关 键 词：针刺 水液缺乏型干眼 泪腺 血管活性肠肽/环磷酸腺苷/蛋白激酶A/水通道蛋白5信号通路 

分 类 号：R246.8[医药卫生—针灸推拿学]