化湿润睛汤通过调控NF-κB信号通路抑制炎症缓解去势大鼠干眼病

Research of Huashi Runjing Decoction(化湿润睛汤)in the Treatment of Alleviating Castrated Rat Xerophthalmia Through Regulating the NF-κB Signalling Pathway to Inhibit Inflammation

目的评价化湿润睛汤对去势干眼病模型大鼠的疗效,并从NF-κB信号通路研究其可能的抗炎机制。方法将75只6周龄健康雄性SD大鼠随机分为正常组、假手术组、模型组、阳性药物治疗组、化湿润睛汤治疗组,共5组,每组15只。正常组不做任何处理;假手术组只切开腹腔而不切除睾丸及附睾;模型组、阳性药物治疗组、化湿润睛汤治疗组这3组实验大鼠切开腹腔并切除双侧睾丸及附睾。建立去势雄性大鼠干眼模型1周后,对各组大鼠分别通过角膜荧光素染色(fluorescein staining,FL)、泪膜破裂时间(break-up time,BUT)、泪液分泌试验(Schirmer I-test,SIT)检测进行干眼模型评估,待各组实验大鼠伤口基本愈合之后开始用药。用药4周后通过FL、BUT、SIT检测对各组实验大鼠进行药效学评价;通过放射免疫的方法测定各组实验大鼠血清睾酮水平;通过ELISA法检测各组实验大鼠血清中TNFα、IL-1α和IL-6水平;通过HE染色观察各组实验大鼠泪腺组织中病理形态学改变情况;通过免疫组化和Western blot检测各组实验大鼠泪腺组织中NF-κB信号通路主要因子(IKKα、IKKβ、IκB、NF-κB-p65)蛋白质表达的改变。结果(1)FL、BUT、SIT的检测结果表明,化湿润睛汤治疗后改善了去势干眼病大鼠的角膜荧光素染色情况,延长其内膜破裂时间及增加泪液分泌量;(2)放射免疫法检测结果表明,化湿润睛汤治疗后增加了去势干眼病大鼠血清中的睾酮质量浓度;(3)ELISA法检测结果表明,化湿润睛汤治疗后降低了去势干眼病大鼠血清中炎症因子TNF-α、IL-1α和IL-6水平;(4)HE泪腺染色结果表明,化湿润睛汤治疗后改善了去势干眼病大鼠泪腺组织中腺泡萎缩程度,增加了胞质内酶原颗粒的数量;(5)免疫组化和WB检测结果表明,化湿润睛汤治疗后降低了去势干眼病大鼠泪腺组织中NF-κB信号通路主要因子IKKα、IKKβ、IκB、NF-κB-p65的蛋白表达水平。结论化湿润睛汤可能通过抑制泪腺组织NF-κB信号通路,减少炎症因子的表达,进而缓解去势大鼠干眼病的炎症反应。

Objective To evaluate the therapeutic effect of Huashi Runjing Decoction(化湿润睛汤)on castrated xerophthalmia of model rats,and to study its possible anti-inflammatory mechanism from NF-κB signaling pathway.Methods Seventy-five 6-week-old healthy male SD rats were randomly divided into normal group,sham operation group,model group,Huashi Runjing Decoction treatment group and positive drug treatment group,with 15 rats in each group.The normal group did not do any processing;In the sham operation group,only abdominal cavity was open without testis and epididymis resection;Model group,Huashi Runjing Decoction treatment group,positive drug treatment group,the three groups of rats were open abdominal cavity and excised bilateral testis and epididymis.One week after the establishment of the castrated male rat xerophthalmia model,the xerophthalmia model of the rats in each group was evaluated by fluorescein staining(FL),break up time(BUT),and Schirmer I-test(SIT).The drug was started after the wound of the experimental rats in each group was basically healed.After 4 weeks of administration,pharmacodynamic evaluation was conducted on experimental rats in each group by FL,BUT and SIT tests;Determination of serum testosterone level of experimental rats in each group by radioimmunoassay;The levels of TNF-α,IL-1αand IL-6 in serum were detected by ELISA;Immunohistochemistry and Western blot were used to detect the protein expression changes of major factors of NF-κB signalling pathway(IKKα,IKKβ,IκB,NF-κB-p65)in lacrimal tissues of experimental rats in each group.Results(1)The detection results of FL,BUT and SIT showed that after the treatment of Huashi Runjing Decoction,the corneal fluorescein staining of castrated xerophthalmia rats was improved,the rupture time of their inner membrane was prolonged and the amount of tear secretion was increased.(2)The results of radioimmunoassay showed that the serum testosterone concentration of rats with castrated xerophthalmia was increased by Huashi Runjing Decoction after treatment.(3)ELISA results showed that Huashi Runjing Decoction reduced the levels of inflammatory cytokines TNF-α,IL-1αand IL-6 in serum of rats with castrated xerophthalmia after treatment.(4)HE staining of lacrimal glands showed that Huashi Runjing Decoction improved acinar atrophy in lacrimal tissues of rats with castrated xerophthalmia,and increased the number of plasmogen particles in the cytoplasm.(5)Immunohistochemical and WB detection results showed that Huashi Runjing Decoction reduced the protein expression levels of the main factors of NF-κB signaling pathway IKKα,IKKβ,IκB and NF-κB-p65 in lacrimal tissues of rats with castrated xerophthalmia.Conclusions Huashi Runjing Decoction may reduce the expression of inflammatory factors by inhibiting NF-κB signaling pathway in lacrimal tissue,thus alleviating the inflammatory response of castrated rats with xerophthalmia.