密蒙花总黄酮对去势雄鼠干眼症泪腺TGF-β_1及其基因表达的影响

Effect of buddleja officinali flavone on TGF-β_1 expression in lacrimalgland of castrated rats

目的观察密蒙花总黄酮对去势雄鼠导致干眼症泪腺TGF-β1及其基因表达的影响。方法90只健康1月龄SPF级Wistar雄性大鼠切除双侧睾丸及附睾建立干眼症模型;30只匹配大鼠切开阴囊而不切除睾丸作为假手术组。手术1周后,雄激素治疗组用丙酸睾酮注射液1 mg/kg股四头肌内注射,每3日1次(30只模型鼠);密蒙花总黄酮治疗组以密蒙花总黄酮0.02 g/kg与生理盐水混悬液灌胃,每日1次(30只模型鼠);正常组(30只正常鼠)、假手术组、模型组(30只)以生理盐水灌胃,每日1次。造模1、3、5个月后各组随机抽取10只大鼠处死,取双眼泪腺组织免疫组织化学法检测TGF-β1的表达,采用实时定量PCR(real-time PCR)法荧光定量检测TGF-β1mRNA的表达。结果造模5个月后,正常组和假手术组大鼠泪腺组织细胞中几乎无TGF-β1阳性表达,而模型组、雄激素治疗组和密蒙花总黄酮治疗组大鼠泪腺组织细胞中TGF-β1大量阳性表达,表现为大量棕黄色颗粒沉着。正常组、假手术组大鼠泪腺组织细胞中TGF-β1的平均光密度值(OD)高于模型组,差异有统计学意义(P<0.05);密蒙花总黄酮治疗组和雄激素治疗组大鼠泪腺组织细胞中TGF-β1的平均OD值大于模型组,差异有统计学意义(P<0.05);密蒙花总黄酮治疗组的大鼠泪腺组织细胞中TGF-β1的平均OD值小于雄激素治疗组,差异有统计学意义(P<0.05)。TGF-β1mRNA在不同组大鼠泪腺组织细胞中的表达差异比较与TGF-β1表达相同(P<0.05)。结论密蒙花总黄酮可抑制去势雄鼠干眼症泪腺炎症反应,上调TGF-β1mRNA的表达以增加TGF-β1的合成是其作用途径之一。

Background There is increasing evidence that sex hormones play a key role in the formation and development of dry eye.As a traditional Chinese medicine,buddlejae may be a potentially effective therapy for gonadal hormone-associated dry eye.ObjectiveThis study attempts to assess the role of buddleja officinali flavone on the expression of transforming growth factor-β（TGF-β1） and TGF-β1 mRNA in lacrimal gland of castrated rats and discuss the therapeutic effects of buddleja officinali flavone on dry eye caused by a gonadal hormone level imbalance.Methods Bilateral orchidectomy and epididymectomy were performed in 90 SPF 1-month-old male Wistar rats to create the animal models of dry eye,and only scrotum was selected in 30 matched rats as sham group.Thirty normal male Wistar rats served as normal control groups.In one week after operation,testosterone propionate（1 mg/kg） was intramuscularly injected in 2-day interval for 5 months in 30 model rats,and 0.02g/kg of buddleja officinali flavone＋normal saline solution was administered by gavage once per day in other 30 model rats.Only normal saline solution was administered by gavage in 30 normal rats,30 model rats and 30 sham rats.Ten animals from different groups respectively were sacrificed and the lacrimal gland were obtained at 1 month,3 and 5 months after model established,and expression of TGF-β1 protein in lacrimal gland was detected by immunohistochemistry.The use of the animals followed the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission of China.The expression of TGF-β1 mRNA in lacrimal gland was analyzed by semi-quantitative real-time PCR.ResultsThe immunochemistry revealed that fewer positive cells showed the yellow staining for TGF-β1 in the cytoplasm of lacrimal gland in normal group and sham group in 5 months after operation.However,lots of positive cells for TGF-β1 were found in lacrimal gland in model group,testosterone group and buddleja officinalis flavone group.The OD value of positive cells for TGF-β1 was significantly decreased in various time points in model group compared with normal group and sham group（P0.05） and enhanced in testosterone group and buddleja officinali flavone group in comparison with model group（P0.05）,and that in the buddleja officinali flavone group was lower than the testosterone group（P0.05）.The expression of TGF-β1 mRNA in the lacrimal gland followed the same trend.ConclusionBuddleja officinalis flavone inhibits the inflammation of lacrimal gland in castrated rats through increasing the expression of TGF-β1 and TGF-β1 mRNA.