ABSTRACT
Objective:To investigate the effect of ultrasound combined with 4-hydroxyphenyl-retinamide (4-HPR) lipid microbubbles on type Ⅰ collagen α1 chain (COL1A1) protein expression in keloid-derived fibroblasts.Methods:In vitro cultured keloid-derived fibroblasts were divided into 3 groups: control group receiving conventional culture with incomplete Dulbecco′s modified Eagle′s medium (DMEM) , 4-HPR lipid microbubble group cultured with incomplete DMEM containing 15 mg/L 4-HPR lipid microbubbles, and ultrasound + 4-HPR lipid microbubble group cultured with incomplete DMEM containing 15 mg/L 4-HPR lipid microbubbles under ultrasound treatment. After 24-hour treatment, reverse transcription (RT) -PCR and Western blot analysis were performed to determine the mRNA and protein expression of COL1A1 in keloid-derived fibroblasts in each group. Intergroup comparison was carried out by using t test. Results:The mRNA relative expression level of COL1A1 was 1.00 ± 0.18, 0.69 ± 0.15 and 0.35 ± 0.18 in the control group, 4-HPR lipid microbubble group and ultrasound + 4-HPR lipid microbubble group respectively, and the protein relative expression level of COL1A1 was 0.93 ± 0.03, 0.74 ± 0.07 and 0.44 ± 0.06 in the above 3 groups respectively. Moreover, the mRNA and protein expression of COL1A1 was significantly lower in the 4-HPR lipid microbubble group and ultrasound + 4-HPR lipid microbubble group than in the control group ( P < 0.05 or 0.001) , and lower in the ultrasound + 4-HPR lipid microbubble group than in the 4-HPR lipid microbubble group ( P < 0.05) . Conclusion:Ultrasound combined with 4-HPR lipid microbubbles could markedly inhibit the mRNA and protein expression of COL1A1 in keloid-derived fibroblasts.
ABSTRACT
Objective To evaluate effects of 4-hydroxyphenyl retinamide (4-HPR) in different vehicles on the proliferation and apoptosis of human keloid fibroblasts (HKFs).Methods A film-ultrasonic dispersion method was used to prepare 4-HPR liposome solution and 4-HPR microbubbles.Primary HKFs were in vitro treated with the 4-HPR liposome solution at different concentrations of 0-80 mg/L for 6-48 hours,and the proliferative activity of HKFs was evaluated by methyl thiazolyl tetrazolium (MTT) assay.Some other HKFs were divided into 3 experimental groups to be treated with 15 mg/L 4-HPR solution (4-HPR solution group),15 mg/L 4-HPR liposome solution (4-HPR liposome solution group) and 15 mg/L 4-HPR microbubbles (4-HPR microbubble group),respectively,and each group was divided into ultrasonic-treated and-untreated subgroups.HKFs without treatment served as control group.After 24-hour treatment,MTT assay was conducted to evaluate the proliferative activity of HKFs in the above groups,flow cytometry to detect apoptosis of HKFs in all groups except the 4-HPR solution group.Results The 4-HPR liposome solution and 4-HPR microbubbles were successfully prepared.MTT assay showed inhibitory effects of 4-HPR liposome solution at concentrations of 1-80 mg/L on the proliferation of HKFs,and the proliferation inhibition rate was positively associated with the drug concentrations (r =0.633,P ＜ 0.01).After the ultrasonic treatment,inhibitory effects on the proliferation of HKFs significantly differed among the 4-HPR microbubble group,4-HPR solution group and 4-HPR liposome solution group (P ＜ 0.01).The 4-HPR liposome solution group and the 4-HPR microbubble group both showed significantly increased apoptosis rates (21.81％ ± 3.73％,39.79％ ± 1.61％,respectively) compared with the control group (6.18％ ± 0.61％,both P ＜ 0.01).Conclusion The 4-HPR microbubbles are successfully prepared,and 4-HPR in different vehicles all can promote HKF apoptosis and suppress HKF proliferation,among which,4-HPR microbubbles in combination with ultrasonic treatment have stronger inhibitory effects than the 4-HPR liposome solution.
ABSTRACT
Objective To establish a simple and efficient method for developing a keloid model in nude mice with human keloid-derived fibroblasts.Methods Twenty-seven female BALB/c nude mice were randomly divided into five groups with 5,5,5,8 and 4 mice in group A,B,C,D and E respectively.The mice in group A,B and C were inoculated with 0.1 ml of suspension containing human keloid-derived fibroblasts at concentrations of 1.0 × 104,3.0 × 104 and 5.0 × 104 per microliter Matrigel,respectively,at the right axillary fossa.The tumors that formed in one mouse in group C were taken out,and cut into several parts measuring 5 mm × 5 mm × 5 mm in size,which were then subcutaneously transplanted into the right axillary fossa of mice in group D.The mice in group E were subcutaneously injected with 100 μl of Matrigel and served as the control group.The formation of tumor in mice was observed by naked eyes,and the size of tumors was measured until day 30 after tumor formation in group A,B and C as well as after tumor transplantation in group D.Mice were sacrificed on day 30 after tumor formation,and histopathologic examination was performed to analyze histological features of transplanted tumors and pathological changes in visceral organs such as heart,liver,spleen,lung and kidney.Results The tumor formation rate was consistently 100％ in group A,B and C,and the time required for tumor formation was (90.20 ± 3.96),(61.00 ± 2.92) and (39.60 ± 3.20) days in group A,B and C respectively.There was a significant difference in tumor volume on the 30th day after tumor formation between group A,B and C ((288.34 ± 25.29) vs.(1 370.63 ± 105.24) vs.(1 940.98 ± 184.37) mm3,F =138.74,P ＜ 0.05).The size of implanted tumor mass in group D firstly increased,then gradually decreased,but began to continuously increase since the 14~ day,and tumor finally formed in 7 out of 8 mice.There was no evidence of tumor formation in group E.Histopathologic examination showed uniform histological manifestations,which were similar to those of human scar,in tumor tissues from mice in group A,B,C and D.Neither pathological changes nor metastases were observed in visceral organs of these mice.Conclusion Keloid-bearing nude mouse model can be established by subcutaneous inoculation with human keloidderived fibroblasts,or by subcutaneous transplantation of tumor masses of a certain size that have formed in nude mice.
ABSTRACT
Objective To estimate the influence of pepper and alcohol on irritant contact dermatitis induced by cinnamaldehyde in rabbit ears.Methods Thirty-two Japanese large-ear white rabbits were used in this study,and equally divided into 4 groups to receive intragastric infusion of sodium chloride physiological solution twice daily (control group),sodium chloride physiological solution once and 50％ alcohol solution once daily (alcohol group),10％ pepper solution once and sodium chloride physiological solution once daily (pepper group),50％ alcohol solution once and 10％ pepper solution once daily (mixture group),for 7 days.The dose of these solutions for intragastric infusion was uniformly 8 ml/kg.After the appearance of symptoms of irritant contact dermatitis (including increase in water intake,dry stool,vasocongestion of auricle of ears),the back skin of 4 mice in each group was injured and served as the injured skin subgroup,and the back skin of the other 4 mice in each group remained uninjured and served as the intact skin subgroup.Then,skin irritation test was carried out according to the Hygienic Standard for Cosmetics on both sides of the back of these rabbits with 2％ cinnamaldehyde (irritant area) and 70％ alcohol (control area) respectively.Skin reaction at the irritated sites was observed and scored at 1,24,48 and 72 hours after the irritation.Analysis of variance was conducted to assess the differences in reaction intensity between these groups.Results After 7 days of intragastric infusion,the symptom score was 0.25 ± 0.46 in the control group,significantly lower than that in the pepper group (5.38 ±0.74,P＜ 0.01),alcohol group (7.25 ± 0.71,P＜ 0.01) and mixture group (12.75 ± 0.70,P＜ 0.01).In rabbits with intact skin,the intensity of irritant skin reaction was significantly stronger in the mixture group than in the pepper group at 24 and 48 hours (F =28.44,30.33,respectively,both P ＜ 0.05),while in rabbits with injured skin,the irritant skin reaction was more intense in the alcohol group and mixture group than in the pepper group at 24,48 and 72 hours (F =197.12,94.54,87.63,respectively,all P ＜ 0.01).Conclusions Pepper and alcohol alone or in combination at the tested concentration can induce irritation symptoms in rabbits,and both of them can enhance the response of skin to irratation by cinnamaldehyde.
ABSTRACT
Objective To evaluate the effect of spicy foods on allergic contact dermatitis caused by cinnamic aldehyde.Methods Sixty mice were equally divided into 3 groups,i.e.,blank control group,cinnamic aldehyde group and spicy food+cinnamic aldehyde group.Spicy foods (including alcohol and bush redpepper fruit) were intragastrically given for 7 days to induce irritation in the mice of spicy food+cinnamic aldehyde group.Then,the mice of innamic aldehyde group and spicy food +cinnamic aldehyde group were challenged by cinnamic aldehyde on the skin.The difference in allergic reaction intensity was compared between the cinnamic aldehyde group and spicy food +cinnamic aldehyde group.Results After 7-day intragastric administration of spicy foods,increased scores of symptom and sign were observed in the spicy food+cinnamic aldehyde group compared with the blank control group receiving intragastric sodium chloride physiological solution (P ＜ 0.01 ).Intense allergic reactions were induced by cinnamic aldehyde,and the reaction intensity score was significantly higher in mice irritated with spicy foods than in those with sodium chloride physiological solution at 24 and 48 hours after the challenge,but was similar at 72 hours between the two groups regardless of a significant difference in pathological score (P ＜ 0.05).Conclusions The mixture of alcohol and bush redpepper fruit at the concentration used in this experiment could induce irritation in mice,and increase the intensity of allergic reaction caused by cinnamic aldehyde.