Effects of camptothecin on autophagy of human primary keratinocytes / 中华皮肤科杂志

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.

Effects of camptothecin on the autophagy of HaCaT cells / 中华皮肤科杂志

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.

Hyperbaric oxygen for the treatment of skin diseases / 中华皮肤科杂志

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.

Novel camptothecin loaded nanogel system :formulation optimization and in vitro transdermal permeability study / 药学实践杂志

Objective To prepare a novel drug delivery system camptothecin loaded nanogel (CPT-PPO gel),and inves-tigate its contents,physical and chemical properties and in vitro transdermal permeability.Methods The solvent evaporation method was utilized to prepare core-shell nano-drug delivery systems (CPT-PLGA-PAMAM,CPT-PP),in which the Poly lac-tic-co-glycolic acid (PLGA ) was used to load camptothecin as the nucleus and the polyamidoamine (PAMAM ) G3.0 was wrapped in the surface of PLGA as the shell.Then the oleic acid (OA) was connected to CPT-PP to obtain the surface modified drug delivery system (CPT-PLGA-PAMAM OA,CPT-PPO).HPLC was used to determine the content of camptothecin in nanoparticles,transmission electron microscopy was applied to identify the nanoparticles morphology,and laser analyzer was used to determine the particle size.The hydroxypropyl methylcellulose (HPMC) was added as the base for the preparation of the nanogel (CPT-PPO gel) at last.The Franz-diffusion cell was used to determine the permeation rate of nanogel in vitro. Results The resulting CPT-PPO gel was stable at 4℃,the average particle size was (246.7 ± 5.4) nm and the encapsulation efficiency was up to (78.7 ± 6.9)%.Comparing to the normal gel,(CPT gel),the cumulative penetration amount and the re-tention amount in the skin of the nanogels (CPT-PPO gel,CPT-PP gel) were significant higher (P＜0.01),the retention and cumulative penetration amount of CPT-PPO gel was significant higher than that of CPT-PP gel (P＜0.05).Conclusion After modified by OA,CPT-PPO gel can increase the cumulated amount and absorption in skin and can be used as a carrier of CPT in the new formulation for topical treatment of psoriasis.

Effects of camptothecin on the expression of hypoxia-inducible factor-1α in HaCaT cells / 中华皮肤科杂志

Objective To estimate the effects of camptothecin (CPT) on the expression of hypoxia-inducible factor-1α (HIF-1α) in HaCaT cells under hypoxic conditions (2％ O2),and to explore the potential therapeutic mechanism of topical CPT for psoriasis.Methods Some HaCaT cells were classified into 6 groups:5 test groups cultured in Dulbecco's modified Eagle's medium (DMEM) with the presence of CPT at 12.5,25,50,100 and 200 nmol/L respectively,and 1 control group cultured in DMEM with the presence of dimethyl sulfoxide (DMSO).All the 6 groups of cells were cultured under normoxic conditions for 12,24,48 or 72 hours or under hypoxic conditions for 12 hours.Cell counting kit-8 (CCK-8) assay was conducted to estimate the proliferation of HaCaT cells after the normoxic culture,and Western blot to quantify the protein expression of HIF-1α after the hypoxic culture.Some HaCaT cells were classified into a normoxia group (21％ O2) and a hypoxia group (2％ O2),and each group was divided into a CPT (100 nmol/L)-treated subgroup and a non-intervention subgroup (treated with the vehicle).After 12-hour culture,real-time fluorescencebased quantitative PCR was performed to measure the mRNA expression of HIF-1α.Statistical analysis was carried out by using Levene'.s test,one-way analysis of variance,Dunnett-t test and factorial analysis with the SPSS16.0 software.Results After treatment with CPT at 12.5-200 nmol/L for 12-72 hours,the proliferation of HaCaT cells was inhibited in a concentration-and time-dependent manner.More concretely,the cell proliferation rates were inhibited by 17.66％ ± 6.46％,33.11％ ± 4.63％ and 56.31％ ± 1.69％ respectively in HaCaT cells after 12-hour treatment with 200 nmol/L CPT as well as 24-hour treatment with 100 and 200 nmol/L CPT compared with the control group at the corresponding time points (all P ＜ 0.05).The protein expression level of HIF-1 α was significantly decreased in HaCaT cells after 12-hour treatment with CPT at 12.5,25,50,100 and 200 nmol/L under hypoxic conditions compared with the control group (0.348 ± 0.065,0.261 ± 0.112,0.115 ± 0.043,0.045 ± 0.024 vs.1.445 ± 0.329,all P＜ 0.05).The mRNA expression level of HIF-1α (expressed as △Ct) in the CPT-treated subgroup and non-intervention subgroup was-5.575 ± 0.29 and -5.451 ± 0.21 respectively in the normoxia group,significantly higher than that in the hypoxia group (-6.543 ± 0.57 and -6.203 ± 0.31 respectively,F =29.856,P ＜ 0.05),while there was no significant difference between the CPT-treated and non-intervention subgroups (F =1.667,P ＞ 0.05).Conclusions CPT at 100 nmol/L could inhibit the expression of HIF-1α protein,but had no obvious effect on that of HIF-1α mRNA.

Effectiveness of chest orthosis in early treatment of chest trauma / 中国综合临床

Objective To evaluate the clinical effect of the chest orthosis on patients with multiple rib or(and) sternal fracture in early period of closed chest trauma.Methods Patients with multiple fractured of ribs or (and) sternal fracture were divided into control group (n =16)treated with the traditional fixation methods ( thoracic girdle added with folding towels) and experimental group ( n =30 ) treated with chest orthosis between January 2009 and December 2011.Correlated parameters of these patients in the two groups,including pain,indexes of blood gas analysis,pulmonary complications and hospitalization time were evaluated.Results There were significant differences on visual analogue scale(VAS) [ (4.45 ±2.23) vs (8.15 ±2.02),t =2.921,P ＜0.01 ],blood gas analysis including PaO2 [ 88.16 ± 9.12) mm Hg vs (77.22 ± 6.24 ) mm Hg,t =2.413,P ＜0.05] andPaCO2[ (40.91 ±3.40)mm Hg vs (46.06 ±5.40)mm Hg,t =2.335,P＜0.05] between experimental group and control group.The incidence rate of pulmonary complications in experimental group was significantly lower than that in control group [ 17％ ( 5/30 ) vs 44％ ( 7/16 ),x2 =23.478,P ＜ 0.05 ].And hospitalization time in experimental group was significantly shorter compared with control group[ (7.26 ± 4.17) d vs ( 14.26 ±3.53)d,t =2.430,P ＜0.05].Conclusion The chest orthosis in early treatment of chest trauma can reduce the pain and improve the condition of patients,and it is a simple,effective and cheap method with significant clinical effect.

Effect of using laparoscopic trocar for rapidly closed thoracic drainage on serious pneumothorax / 中国综合临床

Objective To explore a rapid,simple and effective therapy for serious pneumothorax which could be used in pre-hospital and in-hospital first-aid.Methods Sixty-seven patients were randomly divided into the observation group and control group .Patients in the observation group were treated using laparoscopic trocar for rapidly closed thoracic drainage,and patients in the control group were treated by using the traditional large caliber drainage tube and the intercostal incision method of conventional closed thoracic drainage.The operation time,remaining time of drainage,length of stay,effective rate,and complications,including of postoperative pain,hemorrhage,subcutaneous emphysema and infection were observed in both groups. Results The total effective rate was 94.1%(32/34) in the observation group,which was significantly higher than that in the control group(90.9%,30/33)(x2=1.876,P＞0.05).No significant difference was found on the remaining time of drainage and length of stay between the two groups(remaining time of drainage:[4.56±1.65]d vs.[6.26±3.45]d;length of stay:(6.0±2.6)d vs.(6.7±2.2)d ,t=1.335 and 0.779,respectively,Ps＞0.05).The operation time of using laparoscopic trocar was significantly lower than that of the control group((5.00±1.28)min vs.(15.00±4.03)min,t=3.031,P＜0.05).The incision length was(0.95±0.11)cm in the observational group,which was significantly lower than that in the control group((2.41±0.52)cm ,t=2.585,P＜0.05).Postoperative pain occurred in 14.7%(5/34) of patients in the observational group,which was significantly lower than that in the control group(87.9%(29/33))(t=2.983,P＜0.05).In the observational group no hemorrhage and infection occurred,whereas in the control group the hemorrhage and infection rate was 36%(12/33) and 33%(11/33),respectively(x2=5.880 and 3.687,respectively,Ps＜0.05). Conclusion The use of laparoscopic trocar for rapidly closed thoracic drainage in the treatment of serious pneu-mothorax is simple,easy,convenient,effective and reliable,with few complications.This therapy is suitable for using in pre-hospital and in-hospital first-aid.