| Purpose: This research aimed to simulated ultraviolet irradiation and observing the influences of skin tissues IL-6?TNF-??IL-15 and its receptor,MAPK/ERK1/2 signal pathway,PI3K/Akt signal pathway as well as the related inflammatory factors.This paper explore the mechanism of Lu-dangshen in anti-photoaging on the skin to provide new expreimental basis for medical cosmetology.Materials and methods: 90 healthy mice from Kunming of SPF were randomly divided into namely blank control group,model control group,lu dangshen high dosage control group,lu dangshe middle dosage control group,lu dangshen low dosage control group,VE control group,Anti-IL-15 control group,MAPK inhibitor control group and PI3 K inhibitor control group.Except blank control group,the other eight groups(include model control group,lu dangshen low dosage control group,lu dangshen middle dosage control group,lu dangshen high dosage control group,VE control group,Anti-IL-15 control group,MAPK inhibitor control group and PI3 K inhibitor control group)were conducted with skin photoaging model preparation.These eight groups were irradiated by 2 40 w UVA tubes and 2 40 w UVB tubes.which were placed side by side in a self-made simulated daylight box to irradiate mice back skin.Shave off the back long hair and fine hair of 1.5 × 1.5 per square centimeter,once for every two or three days.The shaved mice were put into box and irradiated simultaneously,three times a week,totally 14 weeks.After successful modeling,the intragastric route was used for continuous administration for 4 weeks,once a day.Mice in blank control group were given intragastric administration of saline,and model control group were aiso given intragastric administration of saline;Mice in lu dangshen groups(include high dosage control group,middle dosage control group,and low dosage control group)were administrated with different concentrations of lu dangshen(include low concentration,medium concentration,and high concentration);Mice in VEcontrol group were given vitamin E drops which was used as positive control group;Mice in anti-IL-15 control group was administrated with corresponding formulation,Mice in MAPK inhibitor control group was administrated with corresponding formulation,and Mice in PI3 K inhibitor control group was also administrated with corresponding formulation.At the 19 th week,all the mice were killed for full-thickness skin in the middle of the back,and frozen them below 70? for tests.Level of IL-6?TNF-??IL-15 in mice skin tissues were tested with ELISA;Common expression level of IL-15?IL-15 R,common expression level of IL-15 and IL-15 R in the skin tissues were tested with IF.And the expression of IL-15?IL-15R?MAPK?P-MAPK?PI3K?P-PI3K?ERK1/2?P-ERK1/2?Akt?P-Akt proteins were tested with Western-blot.The expression of IL-15?IL-15R?MAPK?P-MAPK?PI3K?P-PI3K?ERK1/2?P-ERK1/2?Akt?P-Akt proteins were also tested with IHC.Results:1.Skin photoaging model preparationIrradiation intensity of UVA was 261.79 J per square centimeter in total,and irradiation intensity of UVB was 6.54 J per square centimeter in total.The intensity values were in accordance with the requirements of intensity of ultraviolet radiation of skin photoaging experiment.After fourteen weeks irradiation,the mice became tired and weak,and usually rolled themselves in a ball with less movement.They had dried and lusterless skin,rough epidermis with thick and deep texture,dark red skin color,and sometimes presented ecchymosis.These phenomena comply with the standards of skin photoaging model.That means successful modeling.2.ELISA tests results: the expression level of IL-6?TNF-??IL-15 in blank control group were low.(1)Expression level of IL-6: Compared with blank control group,the expression of IL-6 in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,the expression of IL-6 in lu dangshen groups(include high dosagecontrol group,middle dosage control group,and low dosage control group)decreased greatly,probability is less than 0.01(P<0.01);Compared with VE group,the expression of IL-6 in lu dangshen low dosage group increased significantly,probability is less than 0.01(P<0.01);Compared with VE group,the expression of IL-6 in lu dangshen middle dosage group increased,probability is less than 0.05(P<0.05),while compared with VE group,that in lu dangshen high dosage group showed no significant variation.(2)Expression level of TNF-?: Compared with blank control group,the expression of TNF-?in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,the expression of TNF-? in lu dangshen groups(include high dosage control group,middle dosage control group,and low dosage control group)decreased greatly,probability is less than 0.01(P<0.01);Compared with VE group,the expression of TNF-? in lu dangshen low dosage group increased significantly,probability is less than 0.01(P<0.01);Compared with VE group,the expression of TNF-?in lu dangshen middle dosage group increased,probability is less than 0.05(P<0.05),while compared with VE group,that in lu dangshen high dosage group showed no significance.(3)Expression level of IL-15: Compared with blank control group,the expression of IL-15 in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,the expression of IL-15 in lu dangshen groups(include high dosage control group,middle dosage control group,and low dosage control group)decreased greatly,probability is less than 0.01(P<0.01);Compared with VE group,the expression of IL-15 in lu dangshen low dosage group increased significantly,probability is less than 0.01(P<0.01);Compared with VE group,the expression of IL-15 in lu dangshen middle dosage group increased,probability is less than 0.05(P<0.05),while compared with VE group,that in lu dangshen high dosage group showed no significant variation.3.IF tests results(1)Expression of IL-15,IL-15 R and common expression of IL-15 and IL-15 R present positive in all groups.(2)Expression level of IL-15: Expression level of IL-15 in blank control group weredecreased;Compared with blank control group,expression level of IL-15 in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,expression level of IL-15 in Lu dangshen groups(include high dosage control group,middle dosage control group,and low dosage control group)decreased significantly,probability is less than 0.01(P<0.01);Compared with VE group,expression level of IL-15 in Lu dangshen low dosage group and middle dosage group increased greatly,probability is less than 0.01(P<0.01);While compared with VE group,that in Lu dangshen high dosage group showed no significance.(3)Expression level of IL-15R: Expression level of IL-15 R in blank control group were decreased;Compared with blank control group,expression level of IL-15 R in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,expression level of IL-15 R in Lu dangshen groups(include high dosage control group,middle dosage control group,and low dosage control group)decreased significantly,probability is less than 0.01(P<0.01);Compared with VE group,expression level of IL-15 R in Lu dangshen low dosage group and middle dosage group increased greatly,probability is less than 0.01(P<0.01);While compared with VE group,that in Lu dangshen high dosage group showed no significance.(4)Expression level of common expression of IL-15 and IL-15R: Expression level of common expression of IL-15 and IL-15 R in blank control group were decreased;Compared with blank control group,expression level of common expression of IL-15 and IL-15 R in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,expression level of common expression of IL-15 and IL-15 R in Lu dangshen groups(include high dosage control group,middle dosage control group,and low dosage control group)decreased significantly,probability is less than 0.01(P<0.01);Compared with VE group,expression level of common expression of IL-15 and IL-15 R in Lu dangshen low dosage group and middle dosage group increased greatly,probability is less than 0.01(P<0.01);While compared with VE group,that in Lu dangshen high dosage group showed no significance.4.Results of Western-blot tests(1)Expression of IL-15?IL-15 R protein(1)Expression of IL-15 protein: Compared with blank control group,the expression of IL-15 protein in model control group were significantly higher,probability is less than 0.01(P<0.01);Compared with model control group,the expression of IL-15 protein in Chinese medicine dose group decreased,probability is less than 0.01(P<0.01);Compared with expression of anti-IL-15 antibody control group,expression of IL-15 protein in Chinese medicine dose group increased significantly,probability is less than 0.05(P<0.05).(2)Expression of IL-15 R protein: Compared with blank control group,the expression of IL-15 R protein in model control group were significantly higher,probability is less than 0.01(P<0.01);Compared with model control group,the expression of IL-15 R protein in Chinese medicine dose group decreased,probability is less than 0.01(P<0.01);Compared with expression of anti-IL-15 antibody control group,expression of IL-15 R protein in Chinese medicine dose group increased significantly,probability is less than 0.05(P<0.05).(2)Expression of MAPK?P-MAPK protein(1)Expression of MAPK protein: Compared with blank control group,the expression of MAPK protein in model control group were significantly higher,probability is less than 0.01(P<0.01);Compared with model control group,the expression of MAPK protein in Chinese medicine dose group decreased,probability is less than 0.01(P<0.01);Compared with expression of anti-IL-15 antibody control group,expression of MAPK protein in Chinese medicine dose group increased significantly,probability is less than 0.01(P<0.01).(2)Expression of P-MAPK protein: Compared with blank control group,the expression of P-MAPK protein in model control group were significantly higher,probability is less than0.01(P<0.01);Compared with model control group,the expression of P-MAPK protein in Chinese medicine dose group decreased,probability is less than 0.01(P<0.01);Compared with expression of anti-IL-15 antibody control group,expression of P-MAPK protein in Chinese medicine dose group increased significantly,probability is less than 0.01(P<0.01).(3)Expression of PI3K?P-PI3 K protein(1)Expression of PI3 K protein : MAPK protein in model control control group expressed significantly more than this in the blank group,probability is less than 0.01(P<0.01);Compared with model control group,the expression of PI3 K protein in Chinese medicine dose group decreased,probability is less than 0.01(P<0.01);Compared with expression of anti-IL-15 antibody control group,expression of PI3 K protein in Chinese medicine dose group increased significantly,probability is less than 0.01(P<0.01).(2)Expression of P-PI3 K protein:P-PI3 K protein in model control group expressed significantly more than this in the blank group,probability is less than 0.01(P<0.01);Compared with model control group,the expression of P-PI3 K protein in Chinese medicine dose group decreased,probability is less than 0.01(P<0.01);Compared with expression of anti-IL-15 antibody control group,expression of P-PI3 K protein in Chinese medicine dose group increased significantly,probability is less than 0.01(P<0.01).(4)Expression of ERK1/2?P-ERK1/2 protein(1)Expression of ERK1/2 protein: Compared with normal mice,the expression level of ERK1/2 protein in model control group increased significantly,probability is less than0.01(P<0.01);Compared with model control group,the expression level of ERK1/2 protein in Chinese medicine dose group decreased significantly,probability is less than 0.01(P<0.01);Compared with MAPK inhibitor control group,expression level of ERK1/2 protein in Chinese medicine dose group increased,probability is less than 0.01(P<0.01).(2)Expression of P-ERK1/2 protein: Compared with normal mice,the expression level of P-ERK1/2 protein in model control group increased significantly,probability is less than0.01(P<0.01);Compared with model control group,the expression level of P-ERK1/2 protein in Chinese medicine dose group decreased significantly,probability is less than 0.01(P<0.01);Compared with MAPK inhibitor control group,expression level of P-ERK1/2 protein in Chinese medicine dose group increased,probability is less than 0.01(P<0.01).(5)Expression of Akt?P-Akt protein(1)Expression of Akt protein: Compared with normal mice,the expression level of Akt protein in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,the expression level of Akt protein in Chinese medicine dose group decreased significantly,probability is less than 0.01(P<0.01);Compared with MAPK inhibitor control group,expression level of Akt protein in Chinese medicine dose group increased,probability is less than 0.01(P<0.01).(2)Expression of P-Akt protein: Compared with normal mice,the expression level of P-Akt protein in model control group increased significantly,probability is less than 0.01(P<0.01);Compared with model control group,the expression level of P-Akt protein in Chinese medicine dose group decreased significantly,probability is less than 0.01(P<0.01);Compared with MAPK inhibitor control group,expression level of P-Akt protein in Chinese medicine dose group increased,probability is less than 0.01(P<0.01).5.Results of IHC tests(1)Expression of IL-15?IL-15 R protein(1)Expression of IL-15 protein: The average optical density of IL-15 protein in model control group were significantly higher than those in the blank control group,probability is less than0.01(P<0.01);The average optical density of IL-15 protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than 0.01(P<0.01);The average optical density of IL-15 protein in Chinese medicine dose group were evidently higher than those in anti-IL-15 antibody control group,probability is less than 0.05(P<0.05).(2)Expression of IL-15 R protein: The average optical density of IL-15 R protein in model control group were significantly higher than those in the blank control group,probability is less than 0.01(P<0.01);The average optical density of IL-15 R protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than0.01(P<0.01);The average optical density of IL-15 R protein in Chinese medicine dose group were evidently higher than those in anti-IL-15 antibody control group,probability is less than0.05(P<0.05).(2)Expression of MAPK?P-MAPK protein(1)Expression of MAPK protein: The average optical density of MAPK protein in model control group were significantly higher than those in the blank control group,probability isless than 0.01(P<0.01);The average optical density of MAPK protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than0.01(P<0.01);The average optical density of MAPK protein in Chinese medicine dose group were evidently higher than those in anti-IL-15 antibody control group,probability is less than0.01(P<0.01).(2)Expression of P-MAPK protein: The average optical density of P-MAPK protein in model control group were significantly higher than those in the blank control group,probability is less than 0.01(P<0.01);The average optical density of P-MAPK protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than0.01(P<0.01);The average optical density of P-MAPK protein in Chinese medicine dose group were evidently higher than those in anti-IL-15 antibodycontrol group,probability is less than 0.01(P<0.01).(3)Expression of PI3K?P-PI3 K protein(1)Expression of PI3 K protein: The average optical density of PI3 K protein in model control group were significantly higher than those in the blank control group,probability is less than0.01(P<0.01);The average optical density of PI3 K protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than 0.01(P<0.01);The average optical density of PI3 K protein in Chinese medicine dose group were evidently higher than those in anti-IL-15 antibody control group,probability is less than 0.01(P<0.01).(2)Expression of P-PI3 K protein: The average optical density of P-PI3 K protein in model control group were significantly higher than those in the blank control group,probability is less than 0.01(P<0.01);The average optical density of P-PI3 K protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than0.01(P<0.01);The average optical density of P-PI3 K protein in Chinese medicine dose group were evidently higher than those in anti-IL-15 antibody control group,probability is less than0.01(P<0.01).(4)Expression of ERK1/2?P-ERK1/2 protein(1)Expression of ERK1/2 protein: The average optical density of ERK1/2 protein in modelcontrol group were significantly higher than those in the blank control group,probability is less than 0.01(P<0.01);The average optical density of ERK1/2 protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than0.01(P<0.01);The average optical density of ERK1/2 protein in Chinese medicine dose group were evidently higher than those in MAPK inhibitor control group,probability is less than0.01(P<0.01).(2)Expression of P-ERK1/2 protein: The average optical density of P-ERK1/2 protein in model control group were significantly higher than those in the blank control group,probability is less than 0.01(P<0.01);The average optical density of P-ERK1/2 protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than 0.01(P<0.01);The average optical density of P-ERK1/2 protein in Chinese medicine dose group were evidently lower than those in MAPK inhibitor control group,probability is less than 0.01(P<0.01).(5)Expression of Akt?P-Akt protein(1)Expression of Akt protein: The average optical density of Akt protein in model control group were significantly higher than those in the blank control group,probability is less than0.01(P<0.01);The average optical density of Akt protein in Chinese medicine dose group were evidently lower than those in model control group,probability is less than 0.01(P<0.01);The average optical density of Akt protein in Chinese medicine dose group were evidently higher than those in PI3 K inhibitor control group,probability is less than 0.01(P<0.01).(2)Expression of P-Akt protein: The average optical density of P-Akt protein in model control group were significantly higher than those in the blank control group,probability is less than0.01(P<0.01);The average optical density of P-Akt protein in Chinese medicine dose group were evidently lowerer than those in model control group,probability is less than0.01(P<0.01);The average optical density of P-Akt protein in Chinese medicine dose group were evidently higher than those in PI3 K inhibitor control group,probability is less than0.01(P<0.01).Conclusions1.Skin tissues will release many inflammatory factors with exposure of UV irradiation,among them IL-15 and its receptor can respectively activate Protein kinase ERK1/2?AKT through MAPK and PI3 K signal transduction pathways.Which leads to increase expression of MMPs,lower the conduction pathway of transforming growth factor ?/smad,perhaps photoaging on the skin of the main mechanism.2.Traditional Chinese medicine of Lu dangshen has function of anti-photoaging on the skin.One of the possible mechanisms of lu dangshen in anti-photoaging is that it can directly or indirectly lower the expressions of related inflammatory factors and consequently reduce the effect of inflammatory factors and their receptors on the genes and proteins of MAPK and PI3 Ksignal transduction pathways. |
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