Experimental Study On Anti-apoptotic Effect Of Qishen Ointment Combined With Local Corneal Oxygen Therapy On Dry Eye Model Mice

Objective: To observe the effects of Qishen ointment combine with oxygen therapy on apoptosis and related protein expression in lacrimal gland of dry eye mice model induced by benzalkonium chloride.And explore the mechanism of Qishen ointment combine with oxygen therapy ihibite lacrimal gland cell apoptosis in dry eye mice model.Method: By using the random number table,48 SPF BALB/c mice are divided into 6groups: the control group,model group,sodium hyaluronate group,oxygen therapy group,Qishen ointment group,Qishen ointment combine with oxygen therapy group,Each group contains 8 mice.In addition to the control group,the other 5 groups are induced with 0.2%benzalkonium chloride solution intraocularly instill,5ul/eye,twice per day,continue for 6weeks to establish the dry eye mice model.After 6 weeks of modeling,text the mice with the tear secretion amount(S?T),break-up time(BUT),and corneal fluorescein staining(FL).After successful modeling,the model group is not treated.The sodium hyaluronate group is given 0.1% sodium hyaluronate eye drops 5 u L/eye,3 times per day.The oxygen therapy group underwent corneal oxygenation.After routine eye cleansing,the mice are anesthetized with 1% chloral hydrate,using a self-made oxygen mask which one end is attach to the mouse eye,and the other end is connected to medical oxygen.The oxygen humidification bottle is half full with injection water.The oxygen flow rate is 4 L/min,given 20 min/day.The Qishen ointment group is treated with self-made Qishen ointment gavage,0.34 ml/time,once per day.The Qishen ointment combine with oxygen therapy group is treated with both corneal oxygenation and Qishen ointment gavage.All Administrations above are applied for 3 weeks.2 hours after the last intervention,all group are anesthetized with 1% chloral hydrate by intraperitoneal injection,measured the tear secretion,break-up time,and stain corneal fluorescein.Then the bilateral lacrimal glands of the mice are immersed in a 4%paraformaldehyde solution with tweezers.The expression level of bax,bcl-2 and caspase-3 in lacrimal gland tissue is detected by immunohistochemistry(ICH)and immunoblotting(WB).The apoptosis rate of lacrimal gland cells is detected by TUNEL.Results:1.After the model is established,the mice in each group is tested.The results shows that:comparing with the control group,the SIT and BUT of the other 5 groups are significantly decreased,and the FL detection was significantly increased(p<0.01),and the difference is statistically significant.2.After treatment,the SIT,BUT,and FL of each group of mice is detected.SIT test result shows: Qishen ointment combine with oxygen therapy group> Qishen ointment group>sodium hyaluronate group> oxygen therapy group> model group.There are significant differences between groups(p<0.05)except the difference between model group and oxygen therapy group(p>0.05)and difference between Qishen ointment group and Qishen ointment combine with oxygen therapy group(p>0.05).BUT test results shows: Qishen ointment combine with oxygen therapy group> oxygen therapy group> Qishen ointment group>sodium hyaluronate group> model group.There are significant differences between groups(p<0.05)except the difference between model group and sodium hyaluronate group(p>0.05).FL test result shows that: Qishen ointment combine with oxygen therapy group< oxygen therapy group< Qishen ointment group< sodium hyaluronate group< model group.There are significant differences between groups(p<0.05)except the difference between model group and sodium hyaluronate group(p>0.05).3.Detected by TUNEL staining,the apoptosis of each group shows: control group< Qishen ointment combine with oxygen therapy group< Qishen ointment group<sodium hyaluronate group<model group,There are significant differences between groups(p<0.05)except the difference between model group and sodium hyaluronate group(p>0.05),and the difference between Qishen ointment group and Qishen ointment combine with oxygen therapy group(p>0.05).4.Each group is tested for apoptosis proteins bax,bcl-2 and caspase-3 in lacrimal gland cells by ICH and WB.The results of bax and caspase-3 shows: Control group<Qishen ointment combine with oxygen therapy group<Qishen ointment group<sodium hyaluronate group<model group,there are significant differences between groups(p<0.05)except the difference between model group and sodium hyaluronate group(p>0.05),and the difference between Qishen ointment group and Qishen ointment combine with oxygen therapy group(p>0.05).The results of bcl-2 shows: Control group> Qishen ointment combine with oxygen therapy group> Qishen ointment group> sodium hyaluronate group> model group,there are significant differences between groups(p<0.05)except the difference between model group and sodium hyaluronate group(p>0.05),and the difference between Qishen ointment group and Qishen ointment combine with oxygen therapy group(p>0.05).Conclusion:1.The oxygen therapy can improve the signs of dry eye mice: increase BUT,and reduce FL;2.The Qishen ointment can improve the signs of dry eye mice: increase BUT,and reduce FL;3.The oxygen therapy is better than Qishen ointment in increasing BUT,and reducing FL;and the combination of the two shows better effects than using alone.4.Oxygen therapy has the effect of anti-apoptosis of lacrimal gland cells in mice with dry eyes,and the mechanism of action may be by down-regulating bax and caspase-3;up-regulating the bcl-2 protein expression.5.Qishen ointment has the effect of anti-apoptosis of lacrimal gland cells in mice with dry eyes,and the mechanism of action may be by down-regulating bax and caspase-3;up-regulating the bcl-2 protein expression.6.In terms of the anti-apoptosis effect of lacrimal gland cells in mice with dry eyes,Qishen ointment is superior to oxygen therapy,but there is no statistical difference between Qishen ointment alone,oxygen therapy and Qishen ointment combine with oxygen therapy.