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Objective To evaluate the effect of camptothecin on the autophagy of human primary keratinocytes (HPKs).Methods HPKs were isolated from foreskin tissues of healthy males by a two-step digestion method,and the third-passage cells were used for following experiments.These HPKs were randomly divided into several groups:experimental groups treated with camptothecin at concentrations of 200 nmol/L,2 and 6 μ mol/L separately,and a control group treated with 0.1％ dimethyl sulfoxide (DMSO).After 24-and 48-hour treatment,cell counting kit-8 (CCK-8)assay was conducted to estimate the proliferative activity of HPKs.Flow cytometry was performed to detect cell apoptosis after 24-hour treatment,and Western blot analysis to measure the expression of autophagy-associated proteins such as microtubule-associated protein 1 light chain-3 (LC3) and p62.Some other HPKs were treated with 2 μmol/L camptothecin for 24 hours.Indirect immunofluorescence assay was performed to observe changes in LC3 expression,and transmission electron microscopy to observe the ultrastructure of autophagosomes,so as to further validate the inductive effect of camptothecin on autophagy.Results The inhibitory effect of camptothecin on the proliferation of HPKs gradually increased along with the increase of camptothecin concentration,and there was a significant difference in the proliferation inhibition rates among the experimental groups and control group at 24 hours (F =152.9,P ＜ 0.01).Additionally,the proliferation inhibition rates were significantly higher in the 2-,6-μmol/L camptothecin groups than in the control group (t =12.09,18.76,both P ＜ 0.01),but there was no significantly difference between the 200-μmol/L camptothecin group and control group (t =2.24,P ＞ 0.05).At 48 hours,there was still a significant difference in the proliferation inhibition rates among the experimental groups and control group (F =123.8,P ＜ 0.01),and all the experimental groups showed increased proliferation inhibition rates compared with the control group (all P ＜ 0.01).At 24 hours,the cell apoptosis rates also significantly differed among the control group,200-nmoYL,2-μmol/L and 6-μmol/L camptothecin groups (2.30％ ± 1.68％,15.90％ ±2.14％,29.33％ ± 3.51％,35.28％ ± 3.05％,respectively;F =89.57,P ＜ 0.01),and all the three experimental groups showed higher cell apoptosis rates compared with the control group (all P ＜ 0.01).After 24-hour treatment with 2 or 6 μmol/L camptothecin,the protein expression of LC3]Ⅱ were significantly up-regulated,but the protein expression of p62 was significantly down-regulated.Indirect immunofluorescence assay showed that the percentage of autophagosome-positive cells was significantly higher in the 2-μmol/L camptothecin group than in the control group (60.16％ ± 8.78％ vs.38.96％ ±13.12％,t =3.003,P ＜ 0.05).After 24-hour treatment with 2 μmol/L camptothecin,autophagosomes and autolysosomes were observed in HPKs with a transmission electron microscope.Conclusion Camptothecin at concentrations of 2 and 6 μmol/L can increase the autophagy level in HPKs,meanwhile,inhibit cell proliferation and induce cell apoptosis.
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Objective To evaluate effects of camptothecin on the autophagy of HaCaT cells.Methods Some cultured HaCaT cells were divided into several groups to be treated with camptothecin at concentrations of 5,10,25,50,100 and 200 nmol/L,and 0.1％ dimethyl sulfoxide (DMSO) (control group),respectively.Cell counting kit-8 (CCK-8) assay was conducted to estimate the proliferative activity of HaCaT cells after 24-and 48-hour treatment,flow cytometry to evaluate cell apoptosis after 24-hour treatment,and Western blot analysis to measure the expression of autophagy-related proteins microtubuleassociated protein 1 light chain 3 (LC3) and p62.Some HaCaT cells were divided into 2 groups to be treated with 10 nmol/L camptothecin and 0.1％ DMSO for 24 hours,respectively.Then,indirect immunofluorescence assay (IFA) was performed to determine the LC3 expression.Results Camptothecin at low concentrations of 5 and 10 nmol/L had no significant effects on the proliferation and apoptosis of HaCaT cells.Compared with the control group,the cellular proliferative rates were significantly inhibited by (31.23 ± 1.00)％,(54.21 ± 8.10)％ and (66.75 ± 10.70)％ in the 50-,100-and 200-nmol/L camptothecin groups after 24-hour treatment respectively,and by (25.81 ± 5.99)％,(44.35 ± 5.32)％,(65.81 ± 8.28)％ and (73.23 ± 9.59)％ in the 25-,50-,100-and 200-nmol/L camptothecin groups after 48-hour treatment respectively (all P ＜ 0.001).After 24-hour treatment,the apoptosis rates were significantly higher in the 50-,100-and 200-nnol/L camptothecin groups (14.46％ ± 2.38％,19.15％ ± 1.59％,29.88％ ± 1.37％,respectively) than in the control group (3.80％ ± 0.13％,all P ＜ 0.001).After 24-hour treatment with 5 and 10 nmol/L camptothecin,the protein expression of LC3 Ⅱ was significantly up-regulated,while p62 protein expression was significantly down-regulated:IFA showed that the percentage of autophagosome-positive cells was significantly higher in the 10-nmol/L camptothecin group than in the control group after 24-hour treatment (36.67％ ± 4.55％ vs.6.23％ ± 0.92％,t =6.546,P =0.003).Conclusions Camptothecin at low concentrations of 5 and 10 nmol/L can induce autophagy of HaCaT cells,but has no obvious effects on cell proliferation and apoptosis.Camptothecin at concentrations of 50,100 and 200 nmol/L can inhibit cell proliferation,promote cell apoptosis,and decrease autophagy levels.
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Hyperbaric oxygen (HBO) therapy is the inhalation of 100％ oxygen at a pressure higher than one atmosphere absolute (ATA),and has been used as an auxiliary therapy for various skin diseases.It has been proved that HBO can increase the oxygen content in skin tissues,accelerate aerobic metabolism of skin,promote epithelial regeneration and wound healing,relieve adverse stimuli on peripheral nerves and sensors in the skin,inhibit apoptosis of neurons,enhance the function of regulatory T cells,alleviate inflammation,and mobilize vascular stem/progenitor cells (SPCs) from the bone marrow to peripheral blood and ulcer tissues.At present,HBO has been widely applied in the auxiliary treatment of psoriasis,atopic dermatitis,postberpetic neuralgia,chronic refractory cutaneous ulcer,pyoderma gangrenosum,fungal infection,vascular embolization after cosmetic facial filling,and some other skin diseases.