Effect Of Acne Mixture On Sebaceous Gland Secretion And Inflammatory Related Signal Pathway In Rabbit Ear Acne Model

onePurpose:In this study, in vivo experiments designed to investigate the role of Golden Hamster sebaceous glands and serum sex hormone in the pathogenesis of acne;Study on acne mixture of golden hamster sebaceous overlapping the number of leaves, spot the size of the sebaceous glands and serum testosterone, estradiol influence.Mechanism of acne treatment of acne vulgaris mixture of preliminary exploration, provide experimental basis for clinical application.Material and method:Using abdominal gland spot of golden hamster as a model,acne agent as the experimental factors,clearing acne drug capsule as a positive control,gland spot of golden hamster, serum testosterone, estradiol levels as effect indicators,conduct in vivo tests.Experimental golden hamsters were randomly divided into model group, low dose group of traditional Chinese medicine, traditional Chinese medicine dose group, high dose group, clearing acne capsule group. The control group was filled with 2ml physiological saline,low dose group of Chinese high school in different proportions concentrated acne agent 2ml gavage,Clearing acne clearing acne capsule group with 2ml diluted to oral capsules,once a day,extract material in the experiment on day 14,Blood was collected 3-5ml method by removal of the eye,After collecting blood finished with cervical way to kill off the animals,In bright light, with a vernier caliper measurement of the largest diameter of the right side of the plaque and the maximum longitudinal diameter, and then immediately remove the golden hamster side stomach gland spot tissue fixation and staining, the number of overlapping leaf gland spot under a microscope, ELISA was used to detect serum testosterone and estradiol levels.Results1. Sebaceousoverlapping the number of leaves in the microstructure:Compared with the model group, Chinese high, significantly reducing sebaceous overlapping the number of leaves, the low-dose group, clearing acne capsule group, the difference was statistically significant(P <0.01); compared with the heat acne capsule group, Chinese high, medium no significant difference(P>0.05), low-dose group sebaceous overlapping the number of leaves.2. Gland spot Size: no size gland spot in each group before treatment was significant difference(P > 0.05). After treatment, compared with the model group, Chinese high, middle, both sides of the gland spot area of low dose group was significantly reduced, the difference was statistically significant(P<0.01); compared with the heat acne capsule group, traditional Chinese medicine, low dose gland spot group significantly increased, the difference was statistically significant(P<0.05, P<0.01), and high dose groups compared to its significantly reduced, the difference was significant(P<0.05)3. testosterone levels: Compared with the model group, Chinese high, medium and low dose group, testosterone levels clearing acne capsule group decreased significantly, the difference was statistically significant(P<0.01); compared with the heat acne capsule group, traditional Chinese medicine, testosterone levels rise in the low dose group, the difference was statistically significant(P<0.01), and high dose groups compared to its significantly lower, the difference was significant(P<0.05).4. The level of estradiol: Compared with the model group, Chinese high dose group, clearing acne capsule estradiol levels were significantly increased, the difference was statistically significant(P<0.01), female Chinese low-dose group diol increased, the difference was statistically significant(P<0.05); compared with the heat acne capsule group, traditional Chinese medicine, low levels of estradiol dose group reduced, the difference was statistically significant(P<0.01), high dose group compared with its significant increase, the difference was statistically significant(P<0.05).Conclusion1.Acne mixture by inhibiting the proliferation and secretion of sebaceous gland cells, thereby enabling the sebaceous glands spots, so as to achieve the effect of treating acne.2. Acne mixture by lowering the golden land in rat serum testosterone levels, increased estradiol levels, thereby enabling the sebaceous glands secretion, hyperplasia of function loss.3. The function of the acne mixture inhibit the secretion of sebaceous gland hyperplasia and strengthened with the increase of drug delivery dosage, good concentration-response relationship.twoPurpose:Subject of the present study was to acne rabbit ear model, assuming that the ERK, signaling pathway NF-κB, p38 MAPK, Ah R / ARNT as research centers, the use of modern biology experimental techniques, ERK, NF-κB, p38 MAPK were measured signals its passage under the control of TNF-α, IL-6, IL-8 protein levels, Ah R/ARNT and CYPIAI directly under the control of, GSTA1 protein expression levels, to explore the pathogenesis of acne related signaling pathways in which role in order to fully explore and reveal acne agent in the regulation of ERK, NF-κB, p38 MAPK, multiple links Ah R/ARNT signaling pathway mechanism of action, provide experimental basis for a safe and effective medicine to treat acne, lay a theoretical study of late-stage clinical basis.Material and method:The method of using treated coal tar acne rabbit ear model, respectively, in the rabbit ears side ear canal opening 2cm × 2cm range, with a glass rod coated with coal tar daily 1 times, each about 0.5ml, 2 weeks. Modeling on the 14 th day take ear skin biopsy acne lesions, with 10% formalin-fixed and paraffin wax-embedded blocks and then slice each specimen requires serial sections 4 and HE staining, then sliced and dried, and thereafter under the microscope, it is determined after successful model for subsequent experiments. Acne agent is experimental factors, heat acne positive drug capsules to p38 MAPK NF-κB Ah R/ARNT signaling pathway associated protein, for the effect indicators in vivo tests. The 48 rabbits were randomly divided into 6 groups, namely control group, model group, low dose group of traditional Chinese medicine, traditional Chinese medicine dose group, high dose group, clearing acne capsule group, model control group were given normal saline 20 ml, medicine High school low-dose group in different proportions concentrated mixture 20 ml oral acne, clearing acne capsule group diluted to 20 ml clearing acne capsules orally, once a day for 14 days of treatment all were killed, ear skin tissue was observed following indicators: immunization staining TNFR1, TNF-α, IL-6, IL-8, ERK, NF-κB, p38 MAPK protein expression, Western blot assay Ah R, CYP1A1, GSTA1 expression.Results1. Building 14 days after rough surface visible to the naked eye rabbit ears, thickening, sample have acne skin lesion; Histopathologic changes visible hyperkeratosis, skin and hair follicle epithelial hyperplasia of granular layer, the stratum spinosum still obvious, follicular infundibulum and still see the Angle of material accumulation, infundibulum to expand such as pot, the leather still see more inflammatory cells infiltration.2. Immunohistochemical assay results:2.1 Protein levels of TNFR1:Blank control group the average optical density of TNFR1 protein value is negative, model control group the average optical density value of TNFR1 protein expression is strong positive. Immunohistochemical determination of the average optical density value results:compared with blank control group, model control group TNFR1 significantly higher average optical density value, statistically significant difference(P<0.01); Compared with model control group, high, medium and low dose group TNFR1 significantly reduce the average optical density value, statistically significant difference(P<0.01); Compared with heat acne capsule group, traditional Chinese medicine, low dose group of TNFR1 in higher value, the average optical density differences statistically significant(P<0.05, P<0.01), while Chinese medicine high dose group of TNFR1 average optical density value is lower, the difference is statistically significant(P<0.01).2.2 Protein levels of TNF-α: Blank control group the average optical density of TNF-αprotein value is negative, model control group the average optical density value of TNF-αprotein expression is strong positive. Immunohistochemical determination of the average optical density value results: compared with blank control group, model control group TNF-αaverage optical density value rise significantly, statistically significant difference(P<0.01); Compared with model control group, traditional Chinese medicine of high, middle dose group of TNF-αvalue significantly lower average optical density, statistically significant difference(P<0.01), low dose group of TNF-αvalue significantly lower average optical density, statistically significant difference(P<0.05); Compared with heat acne capsule group, Chinese medicine, the low dose group of TNF-αvalue increases, the average optical density differences statistically significant(P<0.01), while Chinese medicine high dose group compared with heat acne capsule group there was no statistically significant difference.2.3 Protein levels of IL-6:Blank control group the average optical density of IL-6 protein value is negative, model control group the average optical density value of IL-6 protein expression is strong positive. Immunohistochemical determination of the average optical density value results: compared with blank control group, model control group IL-6 significantly higher average optical density value, statistically significant difference(P<0.01); Compared with model control group, high, medium and low dose group IL- 6 average optical density value decreased significantly, statistically significant difference(P<0.01); Compared with heat acne capsule group, traditional Chinese medicine, low dose group of IL-6 in higher average optical density value, statistically significant difference(P<0.01), while Chinese medicine high dose group of IL-6 average optical density value is lower, the difference is statistically significant(P<0.05).2.4 Protein levels of IL-8: Blank control group the average optical density of IL-8 protein value is negative, IL-8 protein model control group to represent the average optical density value of strong positive. Immunohistochemical determination of the average optical density value results: compared with blank control group, model control group IL-8 significantly higher average optical density value, statistically significant difference(P<0.01); Compared with model control group, high, medium and low dose group IL-8 average optical density value significantly reduced, statistically significant difference(P<0.01); Compared with heat acne capsule group, Chinese medicine group, low dose of IL-8 higher average optical density value, statistically significant difference(P<0.05, P<0.01), while Chinese medicine high dose group compared with the heat acne capsule group is significantly lower, the difference was statistically significant(P<0.05).2.5 Protein levels of ERK: Blank control group ERK protein value is negative, the average optical density model control group the average optical density value of ERK protein expression is strong positive. Immunohistochemical determination of the average optical density value results: compared with blank control group, model control group ERK significantly higher average optical density value, statistically significant difference(P<0.01); Compared with model control group, traditional Chinese medicine of high, middle dose group of ERK average optical density value significantly reduced, statistically significant difference(P<0.01), and low dose group compared with Chinese traditional medicine is not statistically significant; Compared with heat acne capsule group, Chinese medicine, the low dose group of ERK increased the average optical density value, the difference is statistically significant(P<0.01), while Chinese medicine high dose group of ERK average optical density value is lower, the difference is statistically significant(P<0.05).2.6 Protein levels of NF-κB: Blank control group the NF-κB protein value is negative, the average optical density model control group NF-κB protein predominate the average optical density values are expressed as strong positive. Immunohistochemical determination of the average optical density value results: compared with blank control group, model control group the NF-κB significantly higher average optical density value, statistically significant difference(P<0.01); Compared with model control group, traditional Chinese medicine of high, middle dose group of NF-κB predominate the average optical density value significantly reduced, statistically significant difference(P<0.01), low dose group the NF-κB level average optical density value is reduced, statistically significant difference(P<0.05); Compared with heat acne capsule group, Chinese medicine, the low dose group of the NF-κB value increased, the average optical density differences statistically significant(P<0.01), while Chinese medicine high dose group of the NF-κB average optical density value is lower, the difference is statistically significant(P<0.05).2.7 Protein levels of p38MAPK: Blank control group p38 MAPK protein value is negative, the average optical density model control p38 MAPK proteins are expressed as the average optical density value of strong positive. Immunohistochemical determination of the average optical density value results: compared with blank control group, model control group p38 MAPK significantly higher average optical density value, statistically significant difference(P<0.01); Compared with model control group, high, medium and low dose group p38 MAPK average optical density value significantly reduced, statistically significant difference(P<0.01); Compared with heat acne capsule group, low dose group of p38 MAPK higher average optical density value, statistically significant difference(P<0.01), Chinese medicine dose group compared with no statistically significant difference, high dose group p38 MAPK compared to significantly reduce the average optical density value, statistically significant difference(P<0.05).3. Western blot method test results3.1 Protein levels of Ah R: Comparison with control group, Ah R water level model group significantly increased, the difference was statistically significant(P<0.01);Compared with the model group, high medicine, Ah R expression, the low-dose group levels were significantly lower, the difference was statistically significant(P<0.01); compared with the heat acne capsule group, traditional Chinese medicine, the expression levels of Ah R low dose increase, the difference was statistically significant(P<0.01), while the high dose Ah R expression level of the group will be cut, the difference was statistically significant(P<0.05).3.2 Protein levels of CYP1A1: blank control group, CYP1A1 levels of model group significantly increased, the difference was statistically significant(P<0.01); compared with the model group, Chinese high expression levels in the low dose group CYP1A1 significantly lower, the difference was statistically significant(P<0.01); compared with the heat acne capsule group, traditional Chinese medicine, the expression level of the low-dose group of CYP1A1 increase, the difference was statistically significant(P<0.01), while the high dose group compared to, CYP1A1 significantly lower clearing acne capsule group, the difference was statistically significant(P<0.05).3.3 Protein level GSTA1: the blank control group, GSTA1 level model group significantly increased, the difference was statistically significant(P<0.01); compared with the model group, Chinese high, medium, GSTA1 expression level of the low dose group was significantly lower, the difference was statistically significant(P<0.01); in traditional Chinese medicine, GSTA1 expression level of the low dose group significantly increased, the difference was statistically significant(P<0.05, P<0.01), while the high dose group and clearing acne capsule group GSTA1 expression level was significantly reduced, the difference was statistically significant(P<0.05).Conclusion1. Acne mixture can lower serum TNF-α, IL- 8, IL- 6 inflammatory factor protein expression levels, inhibit acne inflammation development.2. The acne mixture can cut p38 MAPK, ERK and NF-κB protein expression levels, jamming signal p38 MAPK, ERK and nf-kappa B signaling pathway.3. The serum levels of inflammatory factors by p38 MAPK, ERK and regulation of the NF-κB signaling pathway mixture is one of the ways to treat acne acne by reducing inflammation factor levels, inhibit p38 MAPK, ERK and NF-κB signaling pathway.4. Acne mixture can interfere with the Ah R/ARNT signaling pathways and directly under the control of CYP1A1, GSTA1 level, regulate endocrine, interfere with acne development process.5. Acne mixture lower serum inflammatory factors, control p38 MAPK, ERK and NF-κB and Ah R/ARNT the function of the signal path with the increase of drug delivery dosage gradually strengthen, the good effects of relationship.