| Objective To observe the effects of different concentrations of IL-1? on the proliferation and migration of rabbit retinal pigment epithelial cells,and to study the inhibitory effects of different concentrations of volatile Oil of frankincense and dexamethasone on IL-1?-mediated proliferation and migration of rabbit retinal pigment epithelial cells and the effects of its NF-?B signaling pathway.Therefore,the anti-inflammatory effects of the volatile oil of frankincense and dexamethasone and its molecular mechanism were discussed.Methods MTT assay was used to detect the effect on rabbit RPE cells proliferation of different concentrations of IL-1?(2.5g/L,5g/L,10g/mL,15g/L,20g/L,30g/L)after 24 h,48h and 72 h,and the optimal concentration and time of action were screened.At the same time,differrent concentrations of volatile oil of Frankincense(0.15mg/mL,0.3mg/mL,0.45mg/mL,0.6 mg/mL,0.75mg/mL)and positive control drug dexamethasone(50mg/L,100mg/L,200mg/mL,400mg/L,600mg/L)on IL-1?-mediated proliferation of rabbit RPE cells.were detected by MTT assay.Flow cytometry was used to detect the effect of IL-1?-mediated apoptosis of different concentrations of volatile Oil of frankincense and dexamethasone.According to the MTT test results and the apoptotic rate test results,the high-,medium-,and low-concentration groups(0.15mg/ml,0.3mg/mL,0.6mg/mL)and the positive control drug dexamethasone high,medium,low concentration groups(50mg/L,100mg/L,400mg/L)were screened.Flow cytometry was used to detect the effects of low,medium and high concentrations of volatile oil of frankincense and dexamethasone on the proliferation cycle of rabbit RPE cells under the action of IL-1?.Scratch experiments were performed to observe the effects of low,medium and high concentrations of volatile oil of frankincense and dexamethasone on IL-1?-mediated cell migration.The effects of low,medium and high volatile oils of Frankincense and dexamethasone on the expression of NF-?Bp65,p-I?B? and COX-2 in rabbit RPE cells were detected by Western Blot.Immunocytochemical staining was used to observe the effects of volatile oil of Boswellia on the expression of NF-?Bp65,p-I?B? and COX-2 in rabbit RPE cells induced by IL-1?.Results1.MTT assay was used to detect the proliferation of rabbit RPE cells after 24h?48h and 72 h of different concentrations of IL-1?:Compared with the serum-free DMEM group,IL-1? cells in each phase the survival rate increased significantly,especially at the concentration of 10g/L(P<0.01).When the concentration was less than 10g/L,the proliferation of IL-1? in rabbit RPE cells was concentration-dependent.In the 24 h phase,except for 20g/L IL-1?,30g/L IL-1? group,the other groups were statistically significant compared with the serum-free DMEM group(P<0.05 or P<0.01);48h phase,there were significant differences between IL-1? and serum-free DMEM in each concentration group(P<0.05 or P<0.01).At 72 hours,the difference between IL-1? and serum-free DMEM was statistically significant(P<0.01).Comparison between different phases in the same treatment group:The cell survival rate of each treatment group increased with time,that is,IL-1? has a time-dependent effect on the proliferation of rabbit RPE cells.Subsequent experiments were performed on IL-1? at a concentration of 10g/L in rabbit RPE cells.2.MTT method to detect the effect of different concentrations of volatile oil of frankincense and dexamethasone on the survival rate of rabbit RPE cells with IL-1? for 24 hours:10g/L IL-1? control group and serum-free DMEM control group positive promote rabbit RPE cells Proliferation,the difference was statistically significant(P<0.01),except for the 0.2%DMSO vehicle control group(P>0.05),the other treatment groups significantly inhibited the proliferation of rabbit RPE cells compared with the IL-1? group,It was statistically significant(P<0.01),and with the increase of concentration,the inhibitory ability of the concentration of the volatile oil of the frankincense and dexamethasone on the cell proliferation was more significant and concentration-dependent.3.Annexin-VFITC/PI counterstaining FCM detection of different concentrations of volatile oil of boswellia and dexamethasone for 24 h on the apoptosis rate of rabbit RPE under the action of IL-1?:Comparison of early apoptotic rate:10g/L IL-1? control Group and serum-free The DMEM blank control group had a significant decrease in the early apoptotic rate,and the difference was statistically significant(P<0.01),except 0.2%DMSO vehicle control group and 50mg/L dexamethasone(P>0.05),the early apoptotic rate of the treatment group were significantly increased,the difference was statistically significant(P<0.05 or p<0.01),and with the increase of concentration,the concentration of the volatile oil of the frankincense and the dexamethasone in the concentration group,the early apoptotic rate of cells was gradually increased in a concentration-dependent manner.The comparison of late apoptotic rate:10g/L IL-1? control group and serum-free DMEM blank control group compared with other periods of apoptotic rate decreased significant,the difference was statistically significant(P<0.01),except 0.2%DMSO vehicle control In the group(P>0.05),the apoptosis rate of the other treatment groups was significant Increased with the IL-1? group,the difference was statistically significant(P<0.01),and with the increase of concentration,the concentration of the volatile oil of the frankincense group and dexamethasone group the late apoptotic rate of the cells was gradually increased In a concentration-dependent manner.4.PI staining was used to detect the effects of different concentrations of Boswellia volatile oil and dexamethasone on the cell cycle of rabbit RPE under the action of IL-1? for 24 hours: Compared with the serum-free DMEM control group,the 10g/L IL-1? control group showed a significant decrease in the proportion of cells in the G1/G0 phase,S phase and G2/M the proportion of cells increased significantly,and the difference was statistically significant(P<0.01),Indicating that IL-1? promoted the proliferation of rabbit RPE cells by inducing G1/G0 phase cells Change to S and G2/M period.Except for the 0.2%DMSO vehicle control group,the proportion of G1/G0 cells in the other treatment groups was significantly higher than that in the IL-1? group(P<0.01).As the concentration increased,the number of G0/G1 cells in different concentrations of frankincund volatile oil and dexamethasone increased.As the concentration increases,the proportion of cells in the S and the G2/M phase decreases.Except for the high-concentration frankincense volatile oil group S phase and IL-1? group S phase,there was no significant difference(P>0.05),there were significant differences between the other groups in the S and G2/M phases and the IL-1? group(P<0.05 or P<0.01),showed that G1/G0 RPE cells slowed the cell proliferation cycle,it indicated that the volatile oil of frankincense and dexamethasone blocked the RPE cells of rabbits after the action of IL-1? in the G1/G0 phase,slowing the cells into the proliferative cycle,inhibition of cell division and proliferation,thereby promoting apoptosis.5.Scratch test was carried out to detect the effects of different concentrations of boswellia volatile oil and dexamethasone on the migration of rabbit RPE cells with IL-1? for 24 h and 48h:The migration of 10g/L IL-1? control group and serum-free DMEM control group at 24 h and 48 h the healing rate increased significant,and the difference was statistically significant(P<0.01),indicating that 10g/L IL-1? can significantly promote the healing of rabbit RPE cells and promote their migration.Except 0.2%DMSO vehicle control group(P>0.05),the migration healing rate of other treatment groups was significant lower than that of IL-1? group,the difference was statistically significant(P<0.01),and with the concentration The migration and healing rate of the volatile oils of various concentrations of frankincense and dexamethasone were gradually decreased,which was concentration-dependent.6.Western blot was used to detect the expression of NF-?Bp65 in cytosolic protein and nuclear protein in each group,and the nuclear translocation rate was calculated:The NF-?Bp65 protein was expressed in cytoplasm and nucleus in a blank control group.Compared with the blank control group,the 10g/L IL-1? control group showed more protein expression in the nucleus and a significant higher nuclear translocation rate,the difference was statistically significant(P<0.01),indicating that 10g/L IL-1? can significantly promote the transfer of NF-?Bp65 protein from cytoplasm to nucleus.The nuclear translocation rate of volatile oil of frankincense and dexamethasone was significantly lower in the low,middle and high treatment groups compared with the IL-1? group,the difference was statistically significant(P<0.01),and with the increase of concentration,the nuclear translocation rate of the frankincense volatile oil group and the dexamethasone group decreased gradually,which was concentration dependent.7.The expression of p-I?B? cytoplasmic protein and the expression of COX-2 in total protein were detected by western blot: The blank control group p-I?B? protein in the cytoplasm and COX-2 in total protein expressed a small amount,there was no significant difference compared with the vehicle control group(P>0.05).Compared with the blank control group,the p-I?B? and COX-2 protein expressions were significantly increased in the 10g/L IL-1? control group,and the differences were statistically significant(P<0.01),indicating that 10g/L IL-1? can significantly increase the expression of p-I?B? and COX-2 protein,there was no significant difference in the expression of COX-2 protein IL-1? in the low concentration group of frankincense volatile Oil(P>0.05).The other treatment groups and dexamethasone high,medium and low concentration groups were significantly inhibited compared with IL-1? group,the expressions of p-I?B? and COX-2 protein were statistically significant(P<0.01),and the protein expression decreased gradually with the increase of the concentration of volatile oil and dexamethasone.8.Immunocytochemical staining for detection of NF-?Bp65,p-I?B? and COX-2 protein expression in rabbit RPE cells:NF-?Bp65:NF-?Bp65 was expressed in a small amount in the cytoplasm and nucleus of the control group,showing a weak positive expression,and more expression in the cytoplasm.The 10g/L IL-1? control group was compared with the control of NF-?Bp65 was significantly increased in cytoplasm and nucleus,and the expression of NF-?Bp65 in some nuclei was more than that in cytoplasm,the difference was statistically significant(P<0.01).The vehicle control group there was no significant difference in the IL-1?control group(P>0.05).The expression of NF-?Bp65 in the high,middle and low treatment groups of frankincense volatile oil and dexamethasone was significant decreased compared with IL-1? group,the difference was statistically significant(P<0.01),with the increase of concentration,the expression of NF-?Bp65 decreased gradually in the volatile oil of frankincense and the dexamethasone,which was concentration dependent.p-I?B?:p-I?B? was expressed in a small amount in the cytoplasm and nucleus in the blank control group,and the expression in the cytoplasm was more.The 10g/L IL-1? control group was compared with the blank control group,the expression of p-I?B? was significantly increased,and it was strongly positive,the difference was statistically significant(P<0.01).There was no significant difference between the vehicle control group and the IL-1? control group(P>0.05).However,the expression of p-I?B? was significantly decreased in the high-,medium-and low-treatment groups of the frankincense and dexamethasone groups,and the difference was statistically significant(P<0.01),and with the increase of concentration,the expression of p-I?B? decreased gradually in the volatile oil of frankincense and the dexamethasone groups,which was concentration-dependent.COX-2:COX-2 in the blank control group showed a small amount of expression in the cells,which was weakly positive.Compared with the blank control group,the 10g/L IL-1? control group showed a significant increase in cytoplasmic expression and was strongly positive,statistical significance(P<0.01),there was no significant difference between the vehicle control group and the IL-1? control group(P>0.05).The expression of COX-2 in bovine volatile oil and dexamethasone was significant lower in the middle and low treatment groups than in the IL-1? group,the difference was statistically significant(P<0.01),and the concentrations of the volatile oil of the frankincense increased with the increase of concentration.The expression of COX-2 in the group and the dexamethasone group decreased gradually,which was concentration-dependent..Conclusions1.IL-1? can promote the proliferation of rabbit RPE cells.The concentration of 10g/L is the most proliferative effect.When the concentration is less than 10g/L,the proliferation of rabbit RPE cells is dose-and time-dependent.2.The intervention of frankincense volatile oil and dexamethasone for 24 hours significantly inhibited IL-1?-mediated proliferation of rabbit RPE cells,and the inhibition was more significant with increasing concentration.3.The intervention of frankincense volatile oil and dexamethasone for 24 h significantly increased the early and late apoptotic rate of IL-1?-mediated rabbit RPE cells,and the apoptotic rate increased with the increase of concentration.4.IL-1? promotes the proliferation of rabbit RPE cells by inducing G1/G0 phase cells to transform into the S phase and G2/M phase.the volatile oil of frankincense and dexamethasone blocked the RPE cells of rabbits after the action of IL-1? in the G1/G0 phase thus inhibited cell proliferation and there was concentration-dependent.5.Both the volatile oil of frankincense and dexamethasone could significantly inhibit the migration of rabbit RPE cells mediated by IL-1?,and the migration healing rate decreased with the increase of concentration.6.Both the volatile oil of frankincense and dexamethasone can actually inhibit the nuclear transfer of NF-?Bp65 and the expression of P-I?B? and COX-2 protein increases induced by IL-1? intervention,and as the concentration increases,the inhibition is more pronounced. |
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