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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 × 10â»8 M), pJak2 (IC50 = 2.82 × 10â»9 M) and pSTAT1 (IC50 = 1.29 × 10â»9 M) in HaCaT cells. The expression of SOCS1 was up-regulated (ED50 = 3.32 × 10⻹¹ M). Triptolide also significantly reduced the expression of ICAM-1 on the HaCaT cell surface (IC50 = 5.82 × 10⻹° 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.
Assuntos
Diterpenos/farmacologia , Interferon gama/metabolismo , Queratinócitos/efeitos dos fármacos , Fenantrenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Compostos de Epóxi/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Janus Quinase 2/metabolismo , Queratinócitos/metabolismo , Fosforilação , Receptores de Interferon/metabolismo , Fator de Transcrição STAT1/metabolismo , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Tripterygium/química , Receptor de Interferon gamaRESUMO
BackgroundThe obsessive-compulsive disorder (OCD) features complexity in etiological factors and high heterogeneity in clinical manifestations. OCD patients with different ages of onset vary in clinical symptoms and etiology. However, current studies on inpatients with early- and late-onset OCD are limited. ObjectiveTo explore the differences in clinical characteristics between early- and late-onset OCD inpatients as well as the factors affecting the onset age of OCD, so as to provide references for early screening and treatment of OCD patients. MethodsThis study was based on collected medical records of 540 patients with OCD who received inpatient treatments at the Affiliated Brain Hospital of Nanjing Medical University between March 2012 and March 2023. Patients with onset age above 18 were placed into early-onset group (n=310) and the others into late-onset group (n=230). Then differences in demographic data and clinical symptoms between two groups of patients were compared. Binary logistic regression was used to analyze the factors that affect the onset age of OCD. ResultsObserving the demographic data, there were significant differences between the two groups in the results in gender, marital status, family history of mental illness, ratio of comorbidities with other mental illnesses, occupational composition, education level and types of obsessive-compulsive symptoms (χ2=22.302、170.556, 9.224, 13.624, 242.277, 59.791, 7.231, P<0.05 or 0.01). Also, the results in ages of onset and hospitalization between two groups were significantly different (Z=-19.915, 16.831, P<0.01). In terms of clinical symptoms, the early onset group had a higher proportion of symptoms including obsessive thinking (χ2=11.998, P<0.05), ordering (χ2=7.731, P<0.05) and rituals (χ2=7.714, P<0.05), while the proportion of obsessive checking (χ2=8.204, P<0.05) and washing (χ2=7.506, P<0.05) symptoms were relatively low. In terms of risk factors, there were several independent risk factors that influence the onset age of OCD inpatients, including comorbid neurodevelopmental disorder, comorbid affective disorder, family history of schizophrenia and family history of affective disorder (OR=19.587, 1.830, 3.065, 4.431, P<0.05). Among them, comorbid neurodevelopmental disorder was the core influencing factor, and female gender was a protective factor for early-onset patients (OR=0.417, P<0.01). ConclusionThere are differences in demographic data and clinical symptom characteristics between early- and late-onset OCD inpatients, and comorbid neurodevelopmental disorder plays as a core risk factor affecting the onset age of OCD inpatients. [Funded by Jiangsu Province Key Research and Development Plan for Social Development Special Project(number, BE2021616) ; Jiangsu Province Social Development General Project (number, BE2022678); Key Project of Nanjing Medical Science and Technology Development Fund (number, ZKX20029)]
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ObjectiveTo investigate the role of STAT3 in hepatocyte proliferation after acetaminophen (APAP)-induced hepatocellular injury in mice. MethodsNormal mouse AML12 hepatocytes were cultured in vitro and were stimulated by APAP (1, 2.5, 5, 10, and 20 mmol/L) for 12, 24 or 48 hours, and the hepatocytes treated with an equal volume of phosphate buffered saline were established as control group. After the optimal stimulation concentration and duration of action were screened out, AML12 hepatocytes were treated with AG490 (10, 50, and 100 μmol/L). The CCK-8 assay was used to measure the viability of AML12 hepatocytes; RT-PCR was used to measure the mRNA expression levels of PCNA, CyclinD1, and Ki67 in AML12 hepatocytes, and Western blot was used to measure the protein expression levels of STAT3, p-STAT3, PCNA, and CyclinD1. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsAfter 24 and 48 hours of APAP treatment, compared with the control group, all concentration groups had a significant reduction in the viability of AML12 hepatocytes (all P<0.05), with a viability of 0.717±0.0271 and 0.752±0.0141, respectively, when the concentration of APAP was 2.5 mmol/L, which was significantly different from that in the control group (all P<0.05) and met the conditions of subsequent experiment. Compared with the control group, the 24-hour APAP (2.5 mmol/L) group had significant reductions in the mRNA expression of PCNA, CyclinD1, and Ki67 (all P<0.01); compared with the 24-hour APAP group, the 48-hour APAP (2.5 mmol/L) group had significant increases in the mRNA expression of PCNA, CyclinD1, and Ki67 (all P<0.01); therefore, a model of hepatocyte regeneration after in vitro AML12 hepatocyte injury was established by stimulation with APAP 2.5 mmol/L for 48 hours. After the addition of AG490, there was no significant difference in viability between the control group and the 10 and 50 μmol/L AG490 groups, and the other groups had a significant reduction in viability (all P<0.01); compared with the APAP group, the AG490 (50 μmol/L)+APAP group and the AG490 (100 μmol/L)+APAP group had a significant reduction in viability (P<0.01); therefore, 50 μmol/L AG490 was selected as the concentration for subsequent experiment. Compared with the control group, the APAP group had a significant increase in the protein expression level of p-STAT3 (P<0.01), while the AG490 group and the APAP+AG490 group had a significant reduction (both P<0.05); compared with the APAP group, the APAP+AG490 group had significant reductions in the protein expression levels of PCNA and CyclinD1 and the mRNA expression levels of PCNA, CyclinD1, and Ki67 (all P<0.05). ConclusionSTAT3 participates in hepatocyte proliferation after APAP-induced hepatocyte injury in mice, while AG490, as an STAT3 inhibitor, can inhibit hepatocyte proliferation after APAP-induced hepatocyte injury by inhibiting the phosphorylation of STAT3.
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Objective To study the role of C3a and C5a in focal segmental glomerulosclerosis (FSGS) patients. Methods (1) A total of 66 patients with FSGS confirmed by renal biopsy were selected, including 18 cases of tip lesion, 11 cases of perihilar, 22 cases of not otherwise specified (NOS), 10 cases of cellular, and 5 cases of collapsing FSGS. The normal renal tissue resected from patients with kidney tumor was taken as a negative control. The expression of C3a and C5a in renal tissues was detected by immunohistochemistry. (2) Serum and urine samples from these 66 FSGS patients were collected, and serum and urine samples from 10 healthy adult selected from the same physical examination center in the same term were used as normal controls. The levels of C3a and C5a in serum and urine were detected by enzyme - linked immunosorbent assay (ELISA). Results (1) Immunohistochemical results showed that C3a and C5a were deposited in glomerulus of FSGS patients, and no deposition in normal renal tissues. The semi - quantitative score showed that kidney C3a score was significantly correlated with serum creatinine (r=0.547, P<0.001) and 24 h urine protein (r=0.329, P=0.007) in FSGS patients, and kidney C5a score was also significantly correlated with serum creatinine (r=0.415, P<0.001) and 24 h urine protein (r=0.414, P<0.001) in FSGS patients. (2) The levels of serum C3a and C5a in FSGS patients were higher than those in healthy adults (both P<0.05), but there was no significant difference among the five pathological types (P>0.05). The levels of urinary C3a/urinary creatinine, urinary C5a/urinary creatinine were higher in FSGS patients than those in healthy adults (all P<0.05). The levels of urine C3a/urinary creatinine and urinary C5a/urinary creatinine in collapsing FSGS were higher than other FSGS types (all P<0.01), but there was no significant difference among the tip lesion, the perihilar, the not otherwise specified and the cellular (P>0.05). (3) Urinary C3a/urinary creatinine levels were significantly correlated with serum creatinine (r=0.774, P<0.001) and 24 h urine protein (r=0.430, P<0.001) in FSGS patients, and urinary C5a/urinary creatinine levels were also significantly correlated with serum creatinine (r=0.677, P<0.001) and 24 h urine protein (r=0.333, P=0.007) in FSGS patients. Conclusion Complement C3a and C5a may be involved in the pathogenesis of FSGS and may be related to the severity of FSGS.
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Objective To evaluate the effect of honokiol on the activation of transient receptor potential (TRP) channel in rat spinal dorsal root ganglion cells and pruritus in mouse models.Methods Healthy male ICR mice aged 4-6 weeks were used to establish histamine-induced and acetone/ether/water (AEW)-induced itching models separately.Totally,mice were randomly divided into 7 groups (histamineinduced model experiment) or 6 groups (AEW-induced model experiment):normal control group and model group both gavaged with sodium chloride physiological solution,solvent group gavaged with sodium carboxymethylcellulose solution,chlorphenamine group (only set up in the histamine-induced model experiment) gavaged with chlorphenamine,50,25 and 12.5 mg/kg honokiol groups gavaged with 50,25 and 12.5 mg/kg honokiol respectively.In the histamine-induced model experiment,the mice were all injected with histamine except the normal control group injected with sodium chloride physiological solution 24 hours after the gavage treatment,while the mice in the AEW-induced model experiment were all topically treated with AEW except the normal control group topically treated with sodium chloride physiological solution for 4 days,followed by gavage with different drugs.The anti-itch effect of each treatment was evaluated by counting the scratching frequency within 30 minutes.Rat spinal dorsal root ganglion (DRG)cells were isolated and subjected to a primary culture.Then,the DRG cells were divided into 6 groups:capsaicin or allyl isothiocyanate (AITC)-induced model group pre-incubated with Hank's balanced salt solution (HBSS),500 μmol/L capsazepine or 10 μmol/L HSC030031 group pre-incubated with capsazepine or HSC030031,solvent group pre-incubated with dimethyl sulfoxide (DMSO),3 honokiol groups preincubated with 7.81,15.63 and 31.25 mg/L honokiol respectively,and Ca2+ fluorescence imaging system was used to observe changes of Ca2+ influx in these cells after capsaicin or AITC stimulation.Statistical analysis was carried out with SPSS 20.0 software by using one-way analysis of variance and Dunnett-t test.Results In the histamine-induced mouse models,the scratching frequency was significantly lower in the 50 and 25 mg/kg honokiol groups than in the model group (21.88 and 21.14 vs.63.70,t =3.48,3.49 respectively,both P =0.003),while no significant difference in the scratching frequency was observed between the 12.5 mg/kg honokiol group and the model group (t =2.01,P =0.062).After the treatment with 50 mg/kg honokiol in the AEW-induced mouse models,the scratching frequency significantly decreased compared with the model group (61.4 vs.101.17,t =0.45,P =0.009),while there were no significant differences among the 25,12.5 mg/kg honokiol groups and the model group (all P > 0.05).Compared with the capsaicin or AITC-induced model group,the increase of Ca2+ fluorescence signal in the DRG cells was significantly inhibited in the 31.25 mg/L honokiol group:at the 45th second,the rate of relative fluorescence intensity change (AF/F0) was 1.11 in the model group,but-0.11 in the 31.25 mg/L honokiol group in the capsaicin-induced model experiment,and 0.56 in the model group,but 0.00 in the 31.25 mg/L honokiol group in the AITC-induced model experiment.Conclusion Honokiol shows an inhibitory effect on mouse models of pruritus induced by histaminergic or non-histaminergic factors,likely by inhibiting Ca2+ influx through activated TRPV 1 and TRPA 1 channels in the DRG cells.
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Objective To investigate mRNA expressions of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 family (APOBEC3s or A3s) as well as expressions and subcellular distribution of major A3 proteins in HaCaT keratinocytes carrying the genome of human papillomavirus type 11 (HPV11.HaCaT),and to evaluate regulatory effects of exogenous interferon-alpha (IFN-α) on the expressions of A3s.Methods The basal levels of A3A,A3B,A3C and A3H mRNA expressions were measured by real-time fluorescence-based quantitative PCR (qRT-PCR) in HPV11.HaCaT cells and normal HaCaT cells.Cultured HaCaT,HPV11.HaCaT and Hela cells were treated with recombinant human IFN-α 2b (rhIFN-α2b) at concentrations of 104,105 and 106 IU/ml for 6,24 and 48 hours separately,and those receiving no treatment served as the normal control groups.Then,qRT-PCR was performed to measure mRNA expressions of A3A,A3B,A3C and A3H in these cells.Immunofluorescence staining was conducted to observe the expression and distribution of A3A protein in cells after the treatment with rhIFN-α2b for 6 hours.Results As qRT-PCR showed,the basal levels of A3A,A3B and A3C mRNA expressions were all significantly higher in HPV11.HaCaT cells than in normal HaCaT cells (all P < 0.05).After stimulation,the mRNA expressions of the four A3 members increased to different extents with the increase in rhIFN-α2b concentrations,and the increase in A3A mRNA was the most significant.Compared with corresponding normal control groups,the mRNA expression of A3A was significantly increased in HaCaT cells (35.77 ± 5.01 vs.1.00 ± 0.05,P < 0.05),HPV 11.HaCaT cells (15.34 ± 2.14 vs.0.99 ± 0.01,P < 0.05) and Hela cells (24.60 ± 5.45 vs.0.97 ± 0.03,P < 0.05) after the treatment with rhIFN-α2b at 106 IU/ml for 6 hours,while the increase in A3B,A3C and A3H mRNA expressions was no more than 9-fold in these cell lines after that.Enhanced staining for A3A was observed in nuclei and cytoplasm of the 3 cell lines after the treatment with rhIFN-α2b at 106 IU/ml for 6 hours.Conclusions HPV11 transfected into HaCaT cells can activate intracellular A3s,especially A3A.IFN-α may play an immunoregulatory role by inducing high levels of A3A expression.
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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.
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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.
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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.