The Study On The Effects Of The Qijingmingmu Decoction And Collagen Cross-linking On The Conjunctival Collagen

Objective:In this study,2×2 factorial design was used to observe the effects of Qijingmingmu Decoction and conjunctival collagen cross-linking(CXL)on conjunctival collagen and to investigate the effect of Qijingmingmu Decoction combined with conjunctival CXL for the treatment of conjunctivochalasis(CCh).The same study design was also applied in the animal experiments to verify the effects of Qijingmingmu Decoction and CXL on the conjunctival collagen.Methods:1.One hundred and sixty four patients with CCh were randomly divided into four groups(41 paitents in each group):control,TCM(Traditional Chinese Medicine),CXL and TCM+CXL group.The patients in the control group received no intervention while those in the TCM group were orally administered Qijingmingmu decoction;the patients in the CXL group underwent conjunctival CXL,and the patients in the TCM+CXL group were given both treatment regimens mentioned above.The loose conjunctival tissue was excised after 3 months.The examinations of anterior-segment optical coherence tomography(AS-OCT),confocal laser scanning microscope(CLSM),light microscopy(LM)and transmission electron microscopy(TEM)were conducted and the collagen levels in conjunctiva were investigated by RT-PCR,western blot and immunohistochemistry before experiment and at post-experiment 3 months when ocular surface disease index(OSDI)and TCM syndrome scale were also evaluated,whereby the treatment effects were evaluated and compared among the four groups.2.Forty New Zealand model rabbits of conjunctivochalasis were randomly assigned to four groups(10 rabbits in each group):control,TCM,CXL and TCM+CXL group.The rabbits in the control group received no intervention while the rabbits in the TCM group were intragastrically administered Qijingmingmu decoction the rabbits in the CXL group underwent CXL on conjunctiva while those in the CM+CXL group were given both treatment regimens mentioned above.The loose conjunctival tissue was excised after 3 months.The examinations of AS-OCT,CLSM,LM and TEM were conducted and collagen levels in conjunctiva were investigated by RT-PCR,western blot and immunohistochemistry before experiment and at post-experiment 3 months.Results:Part ? Clinical trialThe four groups were matched for sex and CCh duration.1.Efficacy indexes:1.1 OSDI scale scores:No differences among the 4 groups before the treatments while there was significant difference among the 4 groups after treatments(F=30.58,P<0.001)with the TCM+CXL group being the most effective,then the TCM group and the CXL group.1.2 TCM syndrome scores:No significant differences were found among the four groups while there were significant differences among the four groups(F=18.04,P<0.001)with the TCM+CXL group being the most effective,then the TCM group and the CXL group.2.Clinical indexes:2.1 No differences in the conjunctival epithelial thickness measured by OCT were found in the four groups between baseline and 3-month visit(F=0.054,P=0.984)while there were significant differences in both the thickness of conjunctival stroma(F=51.064,P<0.001)and full conjunctival thickness(F=42.084,P<0.001)between baseline and 3-month follow-up.A significant change in the thickness of the lamina propria and the full conjunctival layer occurred to the four groups at 1,2,and 3 months follow-up(P<0.001).At the follow-up of 1,2,and 3 months,Least Significant Difference(LSD)in the four groups and the main effect test showed that the thickness of the lamina propria and whole layer of the TCM+CXL group and CXL group was significantly higher than that of the control group and the TCM group.2.2 On the examinations of confocal microscopy:The collagen fibers of the lamina propria in the control group were sparsely arranged,and loosely arranged in disorder in the TCM group.The collagen fibers of the lamina propria in the CXL group and the TCM+CXL group were denser than in the control group.3.Experimental indexes3.1 On the examinations of HE staining and Masson staining:The collagen fibers of the lamina propria in the control group were sparsely arranged and disordered;the collagen fibers of the lamina propria in the TCM group were loosely arranged and the number of cells was marginally increased compared with the control group;the collagen fibers of the lamina propria in the CXL group and the TCM+CXL group conjunctiva are denser and the number of cells is higher than in the control group.3.2 On the examinations of TEM:Significant difference in the diameter of conjunctival collagen fibers was found among the control group,CXL group and TCM+CXL group(P<0.001)with the CXL having the most significant effect.The main effect test showed that the diameter of conjunctival collagen fibers of the TCM+CXL group and CXL group was significantly higher than that of the control group and the TCM group.The main effect test demonstrated a significant change could occur to the collagen fibers.3.3 RT-PCR analysis:Type ? and ? collagen mRNA were statistically different among the four groups(F?=213.029,F?=887.175,P<0.001).The between-group comparison has shown no significant difference in Type ? or ?collagen mRNA between the control group and the CXL group while there was a significant difference between the TCM group and TCM+CXL gourp(P<0.001).TCM treatment can increase the relative expression of type ? and ?collagen mRNA and TCM combined with CXL treatment was more efficient in up-regulating its expression.The simple effect test showed that Type ? and ?collagen mRNA of the TCM+CXL group and TCM group was significantly higher than that of the control group and the CXL group.By the simple effect test,the increase in the expression of type ? or ? collagen mRNA was found after TCM treatment,which was more significant when combined with CXL.3.4 The relative expression levels of type ? and ? collagen detected by WB were significantly different among the four groups(F?=278.85,F?=79.32,P<0.001).The between-group comparison has shown significant difference(P<0.01).TCM and CXL treatment can increase the relative expression of type ?and ? collagen and TCM combined with CXL treatment was more efficient in increasing its expression.Both the main effect and simple effect test showed that the relative expression levels of type ? and ? collagen of the TCM+CXL group and TCM group was significantly higher than that of the control group and the CXL group.3.5 On immunohistochemistry examinaitons:The difference in the average optical density of type ? and ? collagen in conjunctiva among the four groups was significantly different(F?=166.426,F?=170.78,P<0.001).There was no significant difference between the control group and CXL group or between the TCM and TCM+CXL group(P>0.05)while there were significant difference between the other two groups(P<0.001).The main effect test showed that the average optical density of type I and III collagen in conjunctiva of the TCM+CXL group and TCM group was significantly higher than that of the control group and the CXL group.Part ? Animal experimentIn this study,the rabbit model in the four groups were matched for age,gender and CCh duration1.There was no significant difference in the thickness of conjunctival epithelial layer among the four groups between baseline and 3-month follow-up(F=0.506,P=0.681)while a significant difference in the thickness of the lamina propria(F=8.859,P<0.001)and the whole conjunctival thickness(F=7.703,P<0.001)were found between baseline and 3-month follow-up.Significant differences in the lamina propria thickness and the whole conjunctiva thickness were also found among the four groups at 1,2 and 3 months(P<0.001).At all the time points,the lamina propria thickness and the whole conjunctival thickness in the TCM+CXL group and CXL group was significantly higher than in the control group or in the TCM group(P<0.001).2.On the Confocal microscopy examinations:The collagen fibers of the lamina propria in the control group were disordered and the increased number of the collagen fibers of the lamina propria was found in the TCM group.The collagen fibers of the lamina propria in the CXL group were more compactly arranged than in the control group.The densest conjunctival collagen fibers were found in the TCM+CXL group.3.On the examinations of HE staining and Masson staining:the conjunctival collagen fibers in the control group were loose and not continuous.The number of collagen fibers in in the TCM group was increased,which were thick and densely packed.4.On the examinations of TEM:A difference in the diameter of conjunctival collagen fibers was found between the control group and CXL group,which is most significant when compared with it in the TCM+CXL group.A significant difference in the diameter of conjunctival collagen fibers was found among TCM group,CXL group,and TCM+CXL group(P<0.001)with it being the biggest in the CXL group investigated by the main effect test.5.On the examinations of RT-PCR:A difference in the level of type ? and ?collagen mRNA was observed among the four groups(F?=2182.228,F?=1029.534,P<0.001);No significant difference in the level of type ? and ?collagen mRNA was found between the control group and the CXL group while there was a significant difference in it between the other groups(P<0.001).TCM treatment can increase the expression of type I and III collagen mRNA and TCM combined with CXL treatment being the most efficient detected by the simple effect test.6.On the examinations of WB:A difference in the level of type ? and ?collagen was observed among the four groups(F?=104.81,F?=83.55,P<0.001);No significant difference in the level of type I collagen was found between the TCM and TCM+CXL group(P>0.05)while there were significant difference in the level of type ? and ? collagen was found between the other two groups(P<0.01).Both the main effect and simple effect test showed that TCM treatment can increase the expression of type ? and ?collagen and TCM combined with CXL treatment being the most efficient.7.On the examinations of Immunohistochemistry:A significant difference in the average optical density of type ? and ? collagen was found among the four groups(F?=331.077,F?=207.257,P<0.001);There was no significant difference in it between the control group and CXL group or between the TCM and TCM+CXL group(P>0.05)while there were significant difference between the other two groups(P<0.001).TCM treatment can increase the expression of type ? and ? collagen and TCM combined with CXL treatment being the most efficient by the main effect test.Conclusions:1.Both Qijingmingmu decoction and conjunctival collagen cross-linking are effective in improving the symptoms of CCh.2.Qijingmingmu Decoction can increase the expression of collagen in conjunctival tissue,and collagen cross-linking can help to increase the conjunctival collagen fibers in diameter.3.Collagen can be used as a new target for the treatment of CCh.4.Qijingmingmu decoction can up-regulate the expressions of collagen I and III that may be one of the mechanisms underlying the role playing in the treatment for CCh.5.Qijingmingmu decoction can effectively treat CCh when combined with conjunctival collagen cross-linking.