Effects Of Tanshinone ⅡA On Sebaceous Gland Hyperplasia Of Golden Hamsters And Its Inhibition Of HaCaT Cell Lipid Synthesis Pathway

Chapter OneTopical Application of Sulfotanshinone Sodium Suppresses Sebaceous Hyperplasia in Syrian HamstersBackgroundAcne vulgaris is a common dermatologic disorder of the pilosebaceous unit. It usually affects almost everybody during the life. The pathogenesis of acne is complex but dependent on four key factors including androgen-mediated stimulation of sebaceous gland activity, follicular hyperkeratinization, colonization of the bacterium Propionibacterium acnes, and inflammation. The sebaceous gland is a major organ invovoled in acne incidence. Excessive sebum secretion elicited by androgen plays an critical part in the pathogenesis of acne vulgaris. Therefore, inhibition of the the sebaceous gland activity to reduce the sebum secretion is an important way of acne treatment.Tanshinone is one of the representative liposoluble constituent extracted from Salvia mitiorrhiza, a kind of traditional Chinese medicine. In recent years, many clinical reports show that oral administration of tanshinone is effective and safe for the treatment of acne, seborrheic dermatitis and other skin disorders related to excessive sebum secretion. It is thought to have the inhibition effect on sebum secretion, but little is known about the specific machanism of its anti-sebum effect. Tanshinone has more than10kinds of monomers, one of which is Tanshinone II A. The pharmacological effects of Tanshinone ⅡA have been extensively studied. Therefore, we select the topical agent of Tanshinone ⅡA for study, as well as the sebaceous gland spots of Syrian hamster Flank Organ as the animal model, which is now recognized as the ideal model for studying the suppressive action of drugs on sebaceous hyperplasia.ObjectiveIn this study, we aim to observe the effect of topical tanshinone ⅡA sulfonate (STS) on sebaceous hyperplasia in animal models, including the changes of spots size, organizational structure, proliferation and apoptosis of sebocytes, so as to provide experimental basis for its clinical application on acne treatment.Methods1. Grouping and drug treatment:20adult male Syrian hamsters were randomly divided into four groups (n=5). STS was applied to sebaceous gland spots in the right flank organ trice daily, while those in the left were treated with normal saline as control. Parameters were examined before,10days,20days and30days after the beginning of the topical treatment.2. Sebaceous spots measuring:After the hamsters were anesthetized by intraperitoneal injection of sodium pentobarbital, a vernier caliper was utilized to measure the size of sebaceous gland spots in the bright light.3. Histological observation and immunohistochemical analysis:The sebaceous gland spots biopsies were placed in10%phosphate-buffered formalin, then dehydrated in ascending concentrations of ethanol, cleared in xylene and embedded in paraffine. After conventional treatment,4μm thickness of tissue sections were made for regular hematoxylin-eosin staining or immunohistochemical staining.4. Detection of the sebocytes apoptosis:TUNEL assay was used to detect the apoptosis of sebaceous gland cells at each time point.Results 1. Changes of sebacous gland spots size after STS treatment:At the baseline, no significant difference was observed in the size of sebaceous glands spots between the two sides of the flank organ(all P>0.05). Compared with normal saline, STS significantly reduced the size of the sebaceous gland spots on day30(P<0.05).2. Changes of histological structure of sebaceous glands spots after STS treatment:Before medication, the sebaceous glands beneath the epidermis of both sides were lobulated, intact and tightly arranged, some of which were opening in the follicular orifice. After STS treatment, sebaceous glands were loosely arranged with decreased quantity and volume, and obviouly atrophic on day30in the right flank organ of hamsters.3. Changes of proliferation and apoptosis of sebaceous gland cells after STS treatment:A decrease was observed in the expression of PCNA in STS treated sebcaceous gland cells compared with those treated with normal saline(P<0.01). STS also incduced an enhancement of apoptosis in sebaceous gland cells, which was more apparent on day20, and the degree of apoptosis was higher in the central area than in the peripheral area of sebaceous glands (P＜0.01).ConclusionsTanshinoneⅡA sulfonate (STS) can reduce the size and alter the microstructure of sebaceous gland spots, and inbihit the hyperplasia of sebaceous glands, which may be through directly reducing the cell proliferation and induing the enhancement of cell apoptosis of sebaceous gland. Chapter TwoTanshinone IIA Inhibits the Dihydrotestosterone-Induced SREBP-lc Pathway in HaCaT cells via Activation of PI3K/AKT Signaling PathwayBackgroundIn recent years, with in-depth study on the mechanism of lipid metabolism, the sterol regulatory element binding protein(SREBP) pathway becomes a research focus. SREBPs are important nuclear transcription factors that regulate the synthesis of cholesterol, fatty acids and triglycerides. As suggested by their name, SREBPs bind sterol response elements in the promoters of genes involved in lipogenesis, including fatty acid synthase, long-chain fatty acyl elongase, stearoyl CoA desaturase, HMG CoA synthase. The SREBP family consists of three isoforms:SREBP-1a, SREBP-1c, and SREBP-2. SREBP-la controls the synthesis of fatty acid and cholesterol synthesis, while SREBP-lc is preferentially invovled in the regulation of fatty acid synthesis. SREBP-2is believed to be more active in cholesterol metabolism. SREBP-1c constitutes90%of SREBP-1, which is a key regulator of fatty acid metabolism. SREBPs have been shown to be regulated by androgens in the hamster ear sebaceous model(Rosignoli et al.,2003). Besides sebacous gland, kerationcyte is another origin of epidermal surface lipid. Harrison et al.(2007) reported that SREBP-1is expressed in keratinocytes as well as in sebocytes, and plays an important role in lippgenic gene regulution.Currently, tanshinone is widely used for clinical treatment of acne, seborrheic dermatitis and other diseases related to excessive sebum secretion. It’s proved to be quite effective with relatively few side effects. Our previous study showed that topical application of sulfotanshinone sodium could suppress sebaceous hyperplasia in Syrian hamsters. Another study pointed out that Tanshinone ⅡA played the role of anti-sebum by inhibiting sebocytes proliferation and lipid synthesis or indirectly downregulating the expression of androgen receptor mRNA in sebocytes. The above studies have confirmed the anti-sebum effect of Tanshinone Ⅱ A, but it’s still unclear about the specific mechanisms.ObjectiveThe goal of this study was to further investigate the mechanism about how Tanshinone ⅡA played the role of anti-sebum. We studied it from the following aspects:(1) the effect of dihydrotestosterone on the expression of SREBP-1c in human HaCaT cells;(2)the regulatory role of PI3K/AKT and MAPK signaling pathway in the DHT-induced expression of SREBP-1c and its target genes in HaCaT cells;(3) the effect of Tanshinone ⅡA on DHT-induced SREBP-1c pathway and possible signaling mechanism.Methods1. Cell culture:Human immortalized skin ketatinocyte cell line HaCaT cells were cultured in DMEM supplemented with10%fetal bovine serum and1%penicillin-streptomycin. At70-80%confluence, cells were used for experiment.2. Experiment grouping and treatment of cells:Ⅰ:To observe the effect of DHT on the expression of SREBP-1c, HaCaT cells were treated with different concentrations of DHT(0,10,100, and1000nmol/L respectively). Ⅱ:To examine the regulatory effect of PI3K/AKT and ERK/MAPK pathways on DHT-induced expression of SREBP-1c and lipogenic enzyme genes in HaCaT cells, DHT at a concentration of100nmol/L was taken as the stimulus quantity, and random groups were created:control group, DHT group(100nmol/L), LY294002+DHT group, PD98059+DHT group. Ⅲ:To observe the effect of Tanshinone ⅡA on the DHT-induced SREBP-lc pathway, HaCaT cells were divided randomly into5groups:control group, DHT group(100nmol/L), Tanshinone ⅡA1.25μmol/L+DHT group, Tanshinone ⅡA2.5μmol/L+DHT group, Tanshinone ⅡA5μmol/L+DHT group.3. Detection of SREBP-lc and lipogenic enzyme genes:Cells were grouped according to experimental grouping requirements. Then Real-Time PCR was used to detect the expression of SREBP-lc mRNA, lipid synthase (FAS, ACS, SCD, of HMGCR) mRNA in HaCaT cells.4. Detection of SREBP-lc protein and related signal proteins:Western blot was employed to detect the expression of SREBP-1、p-AKT、p-ERK、p-P38、p-JNK proteins.5. Detection of lipid synthesis:Oil red staining method was used to observe the lipid changes in HaCaT cells. Flow cytometry was applied to detect the lipid synthesis after Nile red staining.Results1. Effects of DHT on the expression of SREBP-lc in HaCaT cells:The expression levels of SREBP-lc mRNA and protein in HaCaT cells were low in the unstimulated group(0nmol/L group). When stimulated with DHT at concentrations of10,100and1000nmol/L respectively for24hours, the expression levels of SREBP-lc mRNA and protein were significantly increased in a dose-dependent manner, showing statistical differences (P＜0.05), compared with the control group.2. Effects of DHT on PI3K/AKT pathway and MAPK pathway in HaCaT cells: After DHT stimulation at different concentrations (10,100,1000nmol/L) for24hours, the expression of p-AKT protein and p-ERK protein were significantly enhanced in a dose-dependent manner (P<0.05), compared with the0nmol/L group. However, there was no significant change in p-P38or p-JNK expression in the MAPK pathway.3. Effects of PI3K inhibitor (LY294002) and MEK inhibitor (PD98059) on the DHT-induced expression of SREBP-lc in HaCaT cells:After LY294002(50μM) and PD98059(50μM) were respectively pretreated HaCaT cells for40min, the expression of p-AKT and p-ERK proteins were both distinctly decreased, showing a statistical difference (P＜0.05), compared with the DHT(100nmol/L) group. The expression of SREBP-lc mRNA and protein were also decreased significantly compared with the DHT group (P<0.05) after LY294002pretreatment, however, there were no significant differences in the expression levels of SREBP-lc mRNA and protein(P>0.05) after PD98059pretreatment.4. Effects of PI3K inhibitor (LY294002) and MEK inhibitor (PD98059) on the expression of lipid synthetase related genes in HaCaT cells:FAS, ACS, SCD and HMGCR are all important enzymes with a role in lipid synthesis, whose gene transcription is regulated by SREBP-lc. DHT (100nmol/L) can induce obvious increases in the transcription of FAS, ACS, SCD and HMGCR, differences of which showed statistical significance (P<0.05) when compared with the blank control group. After pretreatment with LY294002for40min, the induction effect of DHT on the expression of FAS, ACS, SCD and HMGCR mRNA can be significantly inhibited (p<0.05), but pretreatment with PD98059had no inhibitory effect(p>0.05).5. Effects of Tanshinone ⅡA on the DHT-induced SREBP-lc in HaCaT cells: While DHT(100nmol/L) might increase SREBP-1c expression in HaCaT cells compared with the blank control group, Tanshinone IIA pretreatd with different concentrations of1.25、2.5、5μmol/L can reduce the expression of SREBP-1c mRNA and protein to different degrees(P<0.05), compared with the DHT group. 6. Effects of Tanshinone IIA on the expression of lipid synthetase related genes in HaCaT cells:DHT (100nmol/L) can induce clear increases in the transcription of FAS, ACS, SCD and HMGCR, differences of statistical significance (P<0.01) when compared with the blank control group. After pretreatment with Tanshinone IIA (2.5μmol/L) for24h, the induction effect of DHT on the expression of FAS, ACS, SCD and HMGCR mRNA can be significantly inhibited (p<0.05).7. Effect of Tanshinone ⅡA on DHT-induced lipid synthesis in HaCaT cells: HaCaT cells were pretreated with Tanshinone IIA (2.5μmol/L) for24hours, and then DHT(100nmol/L)was added to stimulate the cells for24hours. It was observed that while in comparison with the blank control group, DHT might stimulate HaCaT cells to synthesize more red lipids, and after pretreatment with Tanshinone IIA the lipids were significantly fewer. Flow cytometry after Nile red staining, showed that the average fluorescence intensity of the DHT group was significantly increased (P<0.05)compared with the blank control group. The average fluorescence intensity of the Tanshinone ⅡA+DHT group decreased compared with the DHT group (P＜0.05).8. Effects of Tanshinone IIA on DHT-induced phosphorylation of AKT in HaCaT cells:Compared with the blank control group, DHT(100nmol/L) can distinctly increase the level of phosphorylation of AKT(P<0.05). After the pretreatment with Tanshinone IIA(2.5μmol/L) for24h, the expression of p-AKT protein in HaCaT cells was decreased significantly(P<0.05) compared with the DHT group, an effect similar to that of LY294002.ConclusionsDHT can activate the SREBP-lc pathway in HaCaT cells, specifically by up-regulating the expression of SREBP-lc and its target genes (FAS, ACS, SCD and HMGCR), and eventually increasing lipid synthesis and secretion. The PI3K/AKT signaling pathway has important regulatory effects on lipid synthesis and secretion induced by DHT. Tanshinone II A, playing a role similar to the PI3K inhibitor, can significantly inhibit the DHT-induced SREBP-lc pathway. This study indicates that Tanshinone IIA can inhibit lipid secretion caused by an excess of androgenic hormones, and that it can be used for clinical treatment of skin diseases associated with excessive sebum secretion, such as acne vulgaris, seborrheic dermatitis, et al.