[Effects of Hedyotis diffusa Extract on Epidermal Growth Factor Induced Proliferation, Apoptosis, and TNF-α Induced...

Objective To observe the effects of Hedyotis diffusa extract (HDE) on the prolifera- tion, apoptosis, and inflammatory factors of HaCaT cells, and to explore its possible molecular mecha- nisms. Methods HaCaT cells were divided into 3 groups, the vehicle group, the control group, and 3 dose HDE groups. No epidermal growth factor (EGF) or HDE was added in the vehicle group. EGF was added with no HDE treatment in the control group. HDE (25, 50, 100 mg/mL) and EGF were added in the 3 HDE groups to co-culture HaCaT cells. The effects of HDE on EGF-induced proliferation of HaCaT cells were de- tected using CCK-8 assay. The effects of HDE on the growth cycle and apoptosis rate of HaCaT cells were measured using flow cytometry. Moreover, protein expression levels of Ki67, Bcl-xL, clAP1 , procaspase- 3, and cleaved caspase-3 were determined using Western blot. In addition, levels of IL-6, IL-8, and IL-10 in the supernatant were detected using ELISA. The level of phosphoration of NF-κB p65 (p-p65) was meas- ured using Western blot. Results Compared with the vehicle group, the absorbance of HaCaT cells and the expression level of Ki67 increased (P <0. 05, P <0. 01) ; levels of p-p65, IL-6, and IL-8 were elevated (P <0. 05, P <0. 01); IL-10 level was lowered (P <0.01) in the control group. Compared with the control group, the absorbance of HaCaT cells and the expression level of Ki67 decreased (P <0.05, P <0.01) ; levels of p-p65, IL-6, and IL-8 were reduced (P <0. 05, P <0. 01); IL-10 level was elevated (P <0. 05, P < 0.01) in the 3 dose HDE groups. Meanwhile, the apoptosis rate of HaCaT cells increased more in the 3 dose HDE groups than in the control group (P <0. 05, P <0. 01). The percentage of HaCaT cells at G1 phase was 58. 51 %, 73.12%, and 79. 95% in 25, 50, and 100 mg/mL HDE groups, respectively, showing statisti- cal difference when compared with that in the control group (52. 85%; P <0. 05, P <0. 01). The percentage and apoptosis rate of HaCaT cells at G1 phase were elevated more in 50 and 100 mg/mL HDE groups than in 25 mg/mL HDE group (P <0. 01). Besides, the percentage and apoptosis rate of HaCaT cells at G1 phase were elevated more in 100 mg/mL HDE group than in 50 mg/mL HDE group (P <0. 05). Compared with the control group, protein expression levels of Bcl-xL and cIAP1 were reduced in 100 mg/mL HDE group (P < 0. 01). There was no statistical difference in caspase-3 total amount (P >0. 05), but cleaved caspase-3 ex- pression increased with statistical difference (P <0. 01). Conclusion HDE inhibited the proliferation of HaCaT cells possibly by arresting HaCaT cell growth at G1 phase, promoted the apoptosis of HaCaT cells by stressing protein expressions of Bcl-xL and cIAPI , and elevating protein expressions of cleaved caspase-3, and suppressed inflammatory response of HaCaT cells via regulating NF-κB expression.