| Sebum plays an important physiological role in human skin,including thermoregulation,control of water evaporation,moisturizing and antibacterial defense.However,excessive sebum secretion can lead to seborrheic diseases,such as seborrheic alopecia,acne and so on.These diseases can cause not only physical damage,such as permanent scars,but also psychological disorders,which have a serious negative impact on the quality of life.Clinically,most topically used sebum inhibiting medicines are chemical drugs,such as antibiotics,isoretinoic acid and benzoyl peroxide,which can cause mild to serious side effects,such as dryness,peeling,rebound,drug resistance and dyslipidemia.In addition,because estrogen has a strong effect on inhibiting the production of sebum,hormone drugs such as spironolactone and oral contraceptive have been proved to be effective.However,estrogen and progesterone drugs should be carefully selected according to women age,body mass index and cardiovascular risk,and long-term use of drugs can also cause side effects,and are not suitable for men,in order to overcome the limitations of the above drugs.It is particularly important to develop a safe and effective new preparation to inhibit sebum secretion.Sterol regulatory element binding protein-1(SREBP-1)is essential for anabolic steroids,such as triglycerides(TG)and free fatty acids(FFA).It can inhibit the sebum production by decreasing the activation of SREBP-1.In this study,the SREBP-1inhibiting anti-sebum polypeptide(SREi)was selected as active substance.Because SREi is water-soluble and easy to degrade,it is difficult to penetrate the stratum corneum,so it is very important to prepare a suitable drug delivery system to prepare its new preparation and verify its effectiveness.On this basis,according to the report that sex hormone played a regulatory role in regulating sebum secretion,phytoestrogen genistein(Gen)and polyethylene glycol estrone(ES)fragment(ES-DSPE-PEG2000)were co-loaded in the carrier system to achieve a better effect of inhibiting sebum secretion.In order to solve the above problems,the ordinary deformable liposome hydrogel loaded with SREi(SREi-ADL3-Gel)was prepared by thin film hydration method,and characterized by high performance liquid chromatography(HPLC)and Malvern particle size analyzer.The results showed that the particle size of anionic flexible liposome(SREi-ADL3)with 4.40 mg/m L sodium deoxycholate(SDCh)as surfactant was uniform,and the entrapment efficiency was 92.62 ± 6.32%.The SREi-ADL3-Gel with carbomer as gel matrix showed a three-dimensional network structure under scanning electron microscope,and SREi-ADL3 was uniformly distributed in the hydrogel.The in vitro release results showed that SREi-ADL3-Gel had a certain sustained release effect,and could enhance the stability of SREi-ADL3 and reduce the degradation rate of polypeptides.In addition,the cumulative penetration of SREi-ADL3-Gel measured by Franz diffusion cell was 4.62 times higher than that of free SREi.In the pharmacodynamic experiment of SREi-ADL3-Gel determined in the golden hamster model,the sebaceous gland area on the back of golden hamster was measured every day with different preparations of SREi for 21 days.The results showed that compared with the control group,0.1mg/m L SREi-ADL3-Gel could significantly reduce the area of sebaceous gland(** p < 0.0001)and the levels of TG and FFA in sebaceous gland(** p < 0.0001).The results of Western blot and q PCR showed that compared with free SREi,the protein and m RNA expression levels of SREBP-1,Fatty acid synthase(FAS)and Acetyl–coenzyme A(Co A)carboxylase 1(ACC1)were also significantly decreased in SREi-ADL3-Gel group(** p < 0.01),indicating that SREiADL3-Gel has a good inhibitory effect on sebum production.The possible mechanism was to enhance the inhibitory effect of SREi on SREBP-1,thus down-regulating the protein and m RNA expression of downstream FAS and ACC1.In vitro cytotoxicity test,acute percutaneous toxicity test,multiple skin irritation test and skin allergy test proved that SREi-ADL3-Gel had a good skin safety.Based on the results of SREi-ADL3-Gel studies,ES-DSPE-PEG2000 modified deformable liposomal hydrogel(ES-Gen/SREi-ADL-Gel)coloaded with Gen and SREi was prepared by film hydration method.Molecular docking method was used to predict the binding ability of Gen to estrogen receptor(ER)and the change of ES-DSPEPEG2000 binding site to ER,which was prepared by chemical coupling method from estrone.The optimal prescription and preparation process of ES-Gen/SREi-ADL was established by response surface optimization method(RSM).The results showed that the entrapment efficiency of SREi and Gen was more than 85.0%,the particle size was132.75 ± 5.29 nm and uniformly distributed,and the release behavior of ES-Gen/SREiADL-Gel was good,which conformed to the first-order kinetic model.By observing the morphology and drug content of ES-Gen/SREi-ADL-Gel stored for 6 months,it was proved that it had a good stability.The skin permeability of ES-Gen/SREi-ADL-Gel was detected by Franz diffusion cell,and the cumulative skin permeability of SREi and Gen in ES-Gen/SREi-ADL-Gel for 12 hours was 25.02% and 28.27%,which was 3.20 and 4.88 times higher than that of free solution,respectively.In the pharmacodynamic experiment of ES-Gen/SREi-ADL-Gel,the golden hamster model was used to prove the expression of G protein coupled estrogen receptor(GPER),androgen receptor(AR)and estrogen receptor β(ERβ)in the sebaceous gland of golden hamster,but almost no expression of estrogen receptor α(ERα),which indicated that the sebaceous gland model of golden hamster was suitable for the design of ES-Gen/SREi-ADL-Gel system.After adaptive feeding for 1 week,54 golden hamsters were randomly divided into 9 groups with 6 hamsters in each group.Normal saline,retinoic acid ointment,ES-ADL-Gel,free SREi,free Gen,SREi-ADL,Gen/SREi-ADL,ES-Gen/SREi-ADL and ES-Gen/SREi-ADL-Gel were given for 21 days.Sebaceous glands were taken for hematoxylin and eosin(HE)staining,Oil red O staining,Western blotting,q PCR,TG,FFA and testosterone(T)levels.Serum was taken for TG,FFA and T levels,and main organs for organ coefficient.The results showed that ES-Gen/SREi-ADL-Gel containing 0.05 mg/m L SREi and Gen had a better inhibitory effect on sebum compared with the control group.After 21 days of administration,the sebaceous gland area of golden hamsters decreased by 35.17%(**p< 0.0001),and the levels of TG and FFA in sebaceous glands decreased by 58.32% and51.25% respectively(*p < 0.001).The results of Western blotting and q PCR suggested that the mechanism of ES-Gen/SREi-ADL-Gel inhibiting sebum might be through increasing the protein and m RNA expression of GPER and ERβ in sebaceous glands and reducing the protein level of AR,so as to inhibit the activation of PI3K/Akt pathway and the inhibition of AMPK pathway caused by androgen binding with AR,and inhibiting m TOR phosphorylation,so as to inhibit SREBP-1 activation and sebum production.In addition,ES-Gen/SREi-ADL-Gel was proved to be a safe nanopreparation by cytotoxicity test,hemolysis test and multiple skin irritation experiments.In summary,this study confirmed that ES-DSPE-PEG2000 modified deformable liposomal hydrogel coloaded with Gen and SREi met the requirements of skin administration,and its effectiveness was proved by pharmacodynamic experiments in golden hamsters.The possible effect of inhibiting sebum secretion was that polypeptide SREi directly inhibited the activation of SREBP-1,Gen and ES-DSPE-PEG2000 activated the transcription and translation of ER-related genes by binding to ER.It could deactivate the AR protein in the cells,inhibit the phosphorylation of PI3K/Akt/m TOR and up-regulate the phosphorylation of AMPK,thus inhibiting the activation of SREBP-1.Together,they inhibited the activity of SREBP-1,downregulated the levels of ACC1 and FAS,and inhibited sebum secretion.The safety experimental results of ES-Gen/SREi-ADL-Gel showed that it had a good safety,which provided an important theoretical basis for ES-Gen/SREi-ADL-Gel to become a new nano-preparation for inhibiting sebum secretion. |
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