Triptolide inhibits IFN-γ signaling via the Jak/STAT pathway in HaCaT keratinocytes.

Interferon-gamma (IFN-γ) signaling in keratinocytes plays an important role in IFN-γ-mediated skin inflammation involved in psoriasis. Blocking IFN-γ signal transduction in keratinocytes could be a strategy for controlling inflammatory skin disorders. Tripterygium wilfordii Hook. F. (T. wilfordii) has been used effectively in psoriasis treatment in China. Its therapeutic mechanism on IFN-γ-dependent inflammation has not been elucidated. Triptolide is one of main components of T. wilfordii's antiinflammatory and immune effects. This study aimed to explore the effects of triptolide on an rhIFN-γ-stimulated human keratinocyte cell line (HaCaT) in culture. The expression of IFN-γ receptor α (IFN-γRα), phospho-Janus kinase2 (pJak2), phospho-signal transducer and activator of transcription 1 (pSTAT1) and suppressor of cytokine signaling 1 (SOCS1) was detected by western blotting. The expression of intercellular adhesion molecule-1 (ICAM-1) on the HaCaT cell surface was determined by cell-surface ELISA. The results demonstrated that triptolide inhibited the expression of IFN-γRα (IC50 = 1.37 × 10⁻8 M), pJak2 (IC50 = 2.82 × 10⁻9 M) and pSTAT1 (IC50 = 1.29 × 10⁻9 M) in HaCaT cells. The expression of SOCS1 was up-regulated (ED50 = 3.32 × 10⁻¹¹ M). Triptolide also significantly reduced the expression of ICAM-1 on the HaCaT cell surface (IC50 = 5.82 × 10⁻¹° M). This study suggests that triptolide may contribute to the therapeutic value of T. wilfordii by modulating the IFN-γ signal pathway in IFN-γ-dependent skin inflammatory diseases, including psoriasis.

Effects of curcumin on mRNA expression of cytokines related to Tou-like receptor 4 signaling in THP-1 cells / 中华皮肤科杂志

Objective To investigate the effects of curcumin on mRNA expression of cytokines related to Toll-like receptor (TLR) 4 signaling in THP-1 cells.Methods After pretreatment with different concentrations (50,25,12.5 mg/L) of curcumin or dexamethasone for 12 hours,THP-1 cells were stimulated by lipopolysaccharide (LPS.1 mg/L) for 4 hours followed by the collection of cells.Then total RNA was isolated from these cells and subjected to reverse transcription-polymerase chain reaction (RT-PCR) for the detection of mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),interleukin-1 receptorassociated kinase (IRAK1) and nuclear factor (NF)-κB.THP-1 cells without pretreatment or stimulation served as negative control,and those only stimulated with LPS served as LPS group.Results After stimulation with LPS (1 mg/L) for 4 hours,the mRNA expressions of TRAF6,IRAK1 and NF-κB were significantly upregulated in THP-1 cells compared with negative control cells (f=38.69,39.13,23.99,all P<0.01).Curcumin of 50 mg/L and 25 mg/L significantly inhibited the mRNA expressions of TRAF6.IRAK 1 and NF-κB upregulated by LPS with an inhibition rate of more than 50% (all P<0.0 1).Conclusions Certain concentrations of curcumin can inhibit the mRNA expressions of TRAF6.IRAK1 and NF-κB.which demonstrates the regulatory effect of curcumin on the mRNA expressions of TLR4 signaling pathway-associated cytokines.

Effects of triptolide on interferon-γ signaling in a human keratinocyte cell line HaCaT / 中华皮肤科杂志

Objective To investigate the effects of triptolide on the expression of a series of proteins associated with interferon-γ (IFN-γ)signaling in HaCaT keratinocytes.Methods After pretreatment with difrerent dosages of triptolide(10-10-10-7 mol/L),HaCaT cells were stimulated by recombinant human IFN-γ(rhIFN-γ,500 U/mL)for various periods followed by the collection of cells.Then,total protein was extracted from these cells and subjected to Western blotting for the detection of expression of interferon-γ receptor α(IFN-γRα),phosphorylated Janus kinase 2(pJAK2)and suppressor of cytokine signaling (SOCS1).Results Triptolide at the concentrations of 10-8 mol/L and 10-7 mol/L significantly inhibited the IFN-γRα expression upregulated by rhIFN-γ(both P＜0.05).The expression of pJAK2 induced bv rhIFN-γ was also suppressed by triptolide at the concentrations of 10-9 moI/L and 10-8 mol/L(both P＜0.05).The inhibition of triptolide on IFN-γRα and pJAK2 expression was dose-dependent and the 50%inhibitory concentrations(IC50 value)were 1.37×10-8 mol/L and 2.83×10-9 mol/L,respectively.On the contrary,triptolide upregulated the expression of SOCS1 stimulated by rhIFN-γ at the concentrations of 10-10,10-9 and 10-8 mol/L(P＜0.05,0.05,0.01,respectively)with the 50%effective dosage(ED50 value)at 3.32 × 10-11 mol/L.Conclusions By inhibiting the expression of IFN-γRα as well as phosphorylation of JAK2 and upregulating the expression of SOCS1,triptolide inhibits the phosphorylation of STAT-1,resulting in the inhibition of genetic transcription of multiple inflammatory factors induced by IFN-γ signaling in HaCaT keratinocytes,and the inhibition probably contributes to the efficacy of triptolide in the treatment of IFN-γ-dependent inflammatory skin disorders,such as psoriasis.

Effects of 13-hexyl-berberine hydroehloride and 13-hexyl-palmatine hydroehloride on the activation of nuclear factor-kappa B and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in a human keratinooyte cell line, Hat?aT stimulated by tumor n / 中华皮肤科杂志

Objective To investigate the effects of 13-hexyl-berbefine hydroehlofide (HB-13) and 13-hexyl-paimatine hydrochloride (HP-13) on the activation of nuclear factor-kappa B (NF-kB) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in a human keratinocyte cell line, HaCaT stimulated by tumor necrosis factor alpha (TNF-α). Methods HaCaT cells were cultured in the presence of various concentrations (0.39, 0.78, 1.56 μg/mL) of HB-13 or HP-13 for 120 minutes followed by the stimulation with recombinant human TNF-α for 120 minutes (in phosphorylatEd-IkB-α test) or 15 minutes (in phosphorylated-p38 test). Then, HaCaT cells were disrupted, total protein was extracted, and the expressions of phosphorylated I B-α and phosphorylated p38 were detected with Western blot. HaCaT cells receiving neither pretreatment nor stimulation served as blank control, untreated HaCaT cells stimulated by rhTNF-α as stimulator control, and HaCaT cells pretreated with turmeric root tuber and stimulated by rhTNF-α as positive control. Results From 0.39 to 1.56 μg/mL, both HB-13 and HP-13 significantly inhibited the expression of p-IkB-α in HaCaT cells stimulated by rhTNF-α, and a nonsignificant dose-dependent trend was observed for their inhibitory effect, with the ICo value being 0.441 μg/mL for I-IB-13 (r = -0.990, n = 3, P > 0.05) and 0.832 μg/mL for HP-13 (r = -0.992, n = 3, P > 0.05). In contrast, neither 1-113-13 nor HP-13 within the experiment concentration range had a significant effect on the expression of p-p38 in HaCaT cells stimulated by rhTNF-α (P > 0.05). Conclusions Within the experimental concentration range, both HB-13 and HP-13 can inhibit the activation of NF-kB in HaCaT cells induced by TNF-α signal, but neither of them suppress the phosphorylation of p38MAPK induced by TNF-α signal in HaCaT cells.

The Effects of Keratinocyte Growth Factor and Its Receptors Antisense Oligonucleotide on Prolifera- tion of HaCat Cells / 中华皮肤科杂志

Objective To investigate the effects of keratinocyte growth factor (KGF) and KGF receptor (KGFR) antisense oligonucleotide (ASODN) on cell cycle and apoptosis of HaCat cells. Methods HaCaT cell, an immortalized keratinocyte cell strain, was cultured in vitro. Flow cytometry was used to measure the cell cycle and apoptosis mediated by KGF and ASODN. Results The rates of S phase and apoptosis in the group treated with KGF increased significantly than those in the control group (both P

Effect of Antipsoriatic Drugs(Tripdiolide etc.)on Leukotriene A4Hydrolase Activity / 中华皮肤科杂志

Objective To investigate the effects of some compounds,bestatin,tripdiolide,etc,on leukotriene A4hydrolase activity.Methods LTB4,product of leukotriene A4hydrolase,was determined by reverse phase high performance liquid chromatography(PR-HPLC).Bestatin,an inhibitor of LTA4hydrolase was used as a positive control.Results Tripdiolide could inhibit LTA4hydrolase activity in a dose-depen-dent pattern.The50%inhibitory concentration values(IC 50 )was2.58?10 -5 ?g/mL.The other compounds,an-thralin,cyclosporin A,tretinoin,calcipotriene,clobetasol propionate,methotrexate,and erythromycin,had no effects.Conclusions Tripdiolide possibly has anti-inflammatory effect through down-regulation of leukotriene A4hydrolase in psoriasis.