Authors: Long Da, Zhou Yasha, Li Ping, Liu Jiaqi, Peng Jun, Tan Hanyu, Peng Qinghua
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Background
Researches showed that the increase of intraocular pressure (IOP) in glaucomatous eye is associated with the increasing resistance to aqueous humor outflow effects of transforming growth factor-β (TGF-β) and CD44.Qingguangan is a traditional Chinese medicine and used to treat glaucoma.However, its mechanism of lowing-IOP effect is not elucidated.
Objective
This study was to investigate the lowing-IOP effect and mechanism of qingguangan granule in DBA/2J mouse, a spontaneous glaucoma model mice.
Methods
Ten 3 month-old female DBA/2J mice with normal IOP were chosen as control group, and 20 spontaneous ocular hypertension mice aged 9 months were randomized into high IOP group and qingguangan-treated group, with 10 mice for each group.The qingguangan (2.5 g/kg) was administered by gavaging twice per day for consecutive 15 days in the qingguangan-treated group, and normal saline solution was used in the same way in the control group and high IOP group.IOP was measured by anterior chamber injection/suction system at a perfusion rate of 2.5 and 5.0 μl/min, respectively, and the coefficient of aqueous outflow facility (C value) and outflow resistance (R value) were calculated.Another 60 3-month-old DBA/2J mice were randomized into blank control group gavaged with normal saline solution and high-, middle-and low-dose qingguangan groups gavaged with 25.00, 12.50 and 6.25 g/kg drugs, respectively, and the mouse serum containing drugs was extracted 7 days after treatment.The scleral tissue with trabecular meshwork were obtained for the culture of trabecular meshwork cells and the cells were identified by immunohistochemistry of fibronectin (FN), laminin (LN) and neuronspecific enolase (NSE). TGF-β was added into the medium for 24 hours with the final concentration of 0, 5, 10, 20, 50 and 100 ng/ml, and MMT chromatometry was employed to detect the cell vitality.The cells pre-treated with 20 ng/ml TGF-β were treated with different concentration of drug serum for 24, 48 and 72 hours, and the level of TGF-β2receptor in cell supernatant and the expression of CD44 protein in the cells were detected by ELISA and Western blot assay, respectively.
Results
The IOPs with perfusion both 2.5 μl/min and 5.0 μl/min in the qingguangan-treated group and the control group were significantly lower than those in the high IOP group (all at P<0.01). Compared with the high IOP group, the C value was significantly reduced (2.35±1.34 vs.1.08±0.36) and the R value was evidently elevated (0.64±0.55 vs.1.05±0.47) in the qingguangan-treated group (all at P<0.01). Cultured cells were spindle-shaped with the positive response to FN, LN and NSE antibody.The cell vitality was lower in the 5, 10 and 20 ng/ml TGF-β group than that in the 0 ng/ml TGF-β group (all at P<0.05). Compared with the blank control group, the TGF-β2receptor content in the supernatant and the related expression level of CD44 protein in the cells were elevated in the TGF-β-treated group (all at P<0.01), and TGF-β2 receptor contents and CD44 expression levels in the TGF-β+ high dose drug serum group was significantly lower than those in the TGF-β group and TGF-β+ low dose drug serum group 24, 48 and 72 hours after culture (all at P<0.01).
Conclusions
Qingguangan can lower IOP of spontaneous glaucoma mice by affecting aqueous humor dynamics.Serum containing qingguangan down-regulates the expressions of TGF-β2 receptors and CD44 in trabecular meshwork cells in vitro.