Effects Of Turtle Shell Extract On Proliferation And Apoptosis Of IgG4-associated Orbital Disease Fibroblasts

Objective:To observe the effect of P6 on the proliferation and apoptosis of fibroblasts and explore the possibility of preventing and treating lacrimal gland fibrosis by traditional Chinese medicine and its extract.Methods:From October 2021 to August 2022,6 patients with IgG4-ROD and lacrimal gland removal were selected from our hospital.The fibrotic lacrimal gland was taken,digested with 0.2%type II collagenase,and then cultured and identified.At the same time,the acetic acid turtle shell tablets were purchased,and Xiongsha^[34]and other methods were used to grind the turtle shell into powder.After Ultrasound extraction,P6 with molecular weight less than 6000k Da was extracted by dialysis.Take the 4th generation cells and divide them into 4 groups:blank control group,P6 intervention group with different concentrations（mass concentrations of 25mg/L,50mg/L,100mg/L,200mg/L）,and 10ug/L TGF-β+P6group（P6 mass concentration is the same as before）,10ug/L TGF-βGroup.Each group is equipped with four compound wells,which are laid on a 96-well plate.After standing for 48 hours in a 37℃constant temperature incubator,CCK-8 reagent is added to detect the cell proliferation rate,and the optimal concentration is selected according to the results.Then the cells are divided into four groups,including one control group and three experimental groups（P6 single addition group,TGF-βsingle addition group,P6+TGF-βGroup）,the apoptosis of each group fibroblasts was detected by flow cytometry.Results:1.Cell proliferation was measured by CCK-8 method:compared with the blank group,all concentrations of P6 could inhibit fibroblast proliferation,the difference was statistically significant（P<0.05）,adding TGF-βafter that,only 25mg/L P6 still significantly inhibited cell proliferation.TGF-β+different concentrations P6 group compared with P6 single addition group,the cell proliferation rate of the four groups was slightly increased,but the difference was not statistically significant（P>0.05）.2.Cell apoptosis was measured by flow cytometry:compared with the blank group,the apoptosis rate of P6 single addition group was significantly higher,the difference was statistically significant（P<0.05）;TGF-βThe apoptosis rate in group decreased（P<0.05）.The apoptosis rate of P6 single addition group was significantly higher than that of TGF-β+P6 group,the difference was statistically significant（P<0.05）.Conclusion:1.TGF-βcan promote the proliferation of fibroblasts.In the experiment,all concentrations of P6 can inhibit the proliferation of fibroblasts,but 25 mg/L of P6 has the best effect on inhibiting cell proliferation and can reverse TGF-βand promote proliferation.2.P6 can promote the apoptosis of fibroblasts and reverse TGF-βit is a new direction to treat IgG4-ROD lacrimal gland fibrosis.