The Experimental Investigation On Tetrandrine Inhibiting Haze After Epi-LASIK And Its Mechanism

Objective: This study was to investigate the therapeutic effect of Tetrandrine on corneal haze formation and the expression of transforming growth factor beta 2(TGF-β2) and connective tissue growth factor(CTGF) in the cornea after epipolis laser in situ keratomileusis (Epi-LASIK) to better understand its efficacy.Methods: Twenty-seven New Zealand white rabbits were performed bilateral -10.00 diopters epipolis laser in situ keratomileusis (Epi-LASIK), then randomly divided into 3 groups by self-control, NC group, Tetrandrine group, Fluoromethalone group. And there were eighteen eyes in each group. Corneal haze grades were evaluated by slim light at 0.5-, 1-, and 2 month postoperative intervals. Nine rabbits were killed at each time point and six corneas were excised in each group, and then cut into two pieces equally. One was embeded and sliced for histological assay, and the other was snap frozen in liquid nitrogen, followed by storage at -80℃until analysis. PartⅠ: Histopathologic study of the inhibitive effect of tetrandrine on corneal haze formation after Epi-LASIK in rabbits: Paraffin sections were examined by optical microscope and sirius red dyeing analyses, and ultrathin sections were detected by transmission electron microscope. PartⅡ: Mechanism of suppression of tetrandrine on corneal haze formation after Epi-LASIK in rabbits: paraffin sections were used to measure TGF-β2 and CTGF proteins by immunohistochemical analyses, and the frozen corneas were used to detect their mRNA transcripts by reverse transcription-polymerase chain reaction(RT-PCR).Results: PartⅠ: Corneal haze grades and collagen typeⅢexpression at 0.5- and 1 month after surgery were significant differences among the groups(P<0.05), Comparing with the NC group, The Corneal haze grades and collagen typeⅢexpression in Tet group and FML group were apparently lower(P<0.01), but there was no significant difference between Tet and FML groups(P>0.01). And there was no significant differences among the three groups at 2 month postoperatively as well(P>0.05). Collagen type I expression among the groups were no significant differences too(P>0.05).The optical and transmission electron microscope examination discovered that the structure of the epithelial layer in each group was intact. And the anterior stromal proliferative reactions between Tet and FML groups were apparently weaker than that in NC group, which suggested that both Tet and FML eye drops have inhibitive effect on corneal haze formation. PartⅡ: RT-PCR procedure outcomes showed that the levels of TGF-β2 mRNA in Tet and FML groups were apparently lower than that in NC group (P<0.01), but there was no significant difference between Tet and FML groups(P>0.01); The levels of CTGF mRNA in Tet and FML groups were apparently lower than that in NC group at 0.5 month postoperatively(P<0.01), and there was no significant difference between Tet and FML groups(P > 0.01), but it could not be detected at 1- and 2 month postoperative intervals. Immunohistochemistry outcomes showed that the levels of TGF-β2 and CTGF were all apparently lower than that in NC group (P<0.01) at each time point postoperatively, and there was no significant difference between Tet and FML groups(P>0.01), which was similar to the transcriptional levels. The expression of TGF-β2 began to ascend at 0.5 month, and reached a peak at 1 month, then descended at 2 month postoperatively. And significant differences were found in the expression of CTGF within the three groups(P<0.01), which decreased continually along the time.Conclusions Tet could inhibit rabbit corneal haze formation after Epi-LASIK by suppressing the proliferation of keratocytes and decreasing the expression levels of collagen typeⅢ,and regulators such as TGF-β2 and CTGF, which were similar to FML.