The Study Of Leptin And Leptin Receptor In Pemphigus Patients

ObjectivePemphigus is a common autoimmune bullous dermatosis, and now its patho-genesis remain unknown. Leptin is a cytokine - like hormone that is synthesized and secreted by adipocytes. It can not only promote energy metabolism, decrease food intake, lose weight, but also be a significant member of nerve - endocrine - immunological regulation net. It is an important mesomerism that participates the interaction and the intercommunication between the nutritional state and the immune system. Leptin can educe its biological effect only when binding with specific receptor, and its interaction with cytokine can not be disregard. Leptin can promote the proliferative response of T cell /memory T cell, and it make Th1 cell proliferate much more stronger than does Th2 cell, it may hint that leptin has relationship with the nosogenesis and the development of autoimmune disease. Finding of related disease: there is an abnormal level of leptin in RA, SLE and systemic scleroderma patients, but the study of leptin and its receptor in pemphigus has not been reported.This experiment investigated the diversity of leptin and leptin receptor in the pemphigus patients serum and the role in onset on the level of protein and gene. And to explore the role and relations of leptin and cytokines: IL -2, IL -6, IL -10 and TNF - a in the pathogenic mechanism of pemphigus.Materials and Methods1. Subjects(1) patients: 35 patients with pemphigus who admitted to Department ofDermatology, No. 1 Affiliated Hospital of China Medical University from 2002 to 2005. All the patients, 14 male and 21 female patients, aged 22 - 70, mean age47.95 years. The clinical manifestations of patients were as follows;21 cases of severe patients , 9 cases of moderate and 5 cases of mild patients.(2) controls-. 30 unrelated health blood donors at Iiaoning Provincial Blood Center were selected randomly. They are not significantly different from patients in sex, age and BMI. There are not pemphigus patients in their families.2. MaterialsHuman Leptin RIA Kit ( North Biotechnology Institute, Beijing) and Human Leptin Receptor ELISA Kit(Boster Biotechnology Co. , Ltd. , Wuhan) and RT-PCR Kit (Takara Co. ,Ltd. , Japan)3. Methods(1) RIA: Serum leptin concentrations of patients and controls were measured using Human Leptin RIA Kit according to the manufacturers instructions.(2) enzyme -linked immunoabsorbant assay (ELISA);Serum soluble leptin receptor concentrations of patients and controls were measured using Human Leptin Receptor ELISA Kit according to the manufacturer's instructions.( 3 ) Semiquantitive RT - PCR procedureTo detect the changes of OB - Rb mRNA and OB - Ra mRNA level, total RNA was isolated from 3x10 cells by TriPure reagent, according to the manufacturer's procedures. The compositions of RT -PCR included;lOjxg total RNA, lOxBuffer 1 uJ, RNase Free dH2O 3. 75jxl , Oligo dT - Adaptor primer 0. 5jxl, 25mM MgC12 2jxL, lOmM dNTP luJ ,RNase inhibitor 0.25 jxl , AMV reverse transtriptase 0. 5jxL, in a total volume of 10ui. The reaction condition was;30^ lOmin lcycle;42^ 30min lcycle;99X. 5min lcycle;5*t! 5min 1 cycles;94X, 2min lcycle;94X. 30sec 30cycles;55^ 30sec 30cycles;72t! 4min 30cycles;4*C 5min 30cycles. According to the instructions of the RT -PCR kit. PCR products were electrophoresed on 1.5% agarose gels stained with ethidium bromide. The ethidium bromide - stained PCR products were quantified by densitometry using FlourChem Gel Imaging System. The results were expressed as the ratio between the amount of the OB - Rb and OB - Ra amplica-tion product over the internal control, ￡ actin amplication product for each sample. Experiments were conducted under identical conditions.4. Statistical AnalysisT — test was used for quantitative materials expressed as mean standard deviation (x ± s), All statistical calculation were performed using the spss 11.0 program for windows software, P <0.05 was considered significant.Results1. The serum leptin concentrations in pemphigus patients:(1) The mean serum leptin concentrations in patients with pemphigus was significantly (P <0. 001)higher than that in healthy control subjects.( 2 ) When we divided patients into male and female group, the serum leptin levels in both group were significantly ( P < 0. 01 and P < 0. 05 respectively) higher than in healthy control subjects.(3) The patients with severe pemphigus was not significantly higher than that in the moderate and mild patients (P >0.05).2. The mean serum IL - 2, IL - 6, IL - 10, and TNF - aconcentrations in patients with pemphigus:(1) The mean serum IL - 2 concentrations in patients with pemphigus was significantly (P < 0.05) higher than that in healthy control subjects.(2)IL -10 concentrations in patients with pemphigus was higher than that in healthy control subjects, but there was no statistical significance (P >0.05).(3 ) The mean serum IL - 6 concentration in patients with pemphigus was lower than that in healthy control subjects, but there was no statistical significance (P>0.05).(4) The mean serum TNF - a concentrations in patients with pemphigus was not significantly higher than that in healthy control subjects ( P > 0. 05 ).(5 ) Dependablity analysis indicated: the level of serum leptin was positive correlative with level of IL - 2( r = 0. 33, P < 0.05 ) in patients with pemphigus.3. The mean sLR concentration in patients with pemphigus:(l)The mean sLR concentration in patients with pemphigus was signifi-cantly ( P < 0.005 ) lower than that in healthy control subjects.(2) When we divided patients into male and female group, the sLR levels in both groups were significantly( P <0.05 respectively) lower than in healthy control subjects.(3 ) The mean sLR concentration in patients with severe pemphigus and moderate and mild pemphigus were significantly ( P < 0. 005 and P < 0. 05 respectively) lower than that in healthy control subjects respectively, the patients with severe pemphigus was not significantly lower than that in the moderate and mild patients ( P > 0.05 ).(4)Dependablity analysis indicated: the level of serum leptin was negative correlative with level of sLR(r = -0.446, P <0. 05) in patients with pemphigus.4. The leptin receptor mRNA level in the PBMC of pemphigus patients:(1) The expression of short type Leptin receptor OB - Ra mRNA exists both in the pemphigus patients and the normal contol.( 2 ) The expression of long type Leptin receptor OB - Rb mRNA expressing both in the pemphigus patients and the normal contol.(3 ) The expression of short type leptin receptor OB - Ra mRNA and long type leptin receptor OB - Rb mRNA is weaker than the normal control.(4 ) There is no difference in the expression of short type leptin receptor OB- Ra mRNA and long type leptin receptor OB - Rb mRNA between the severe cases and the moderate or mild cases.(5 ) There is no difference in the expression of short type leptin receptor OB- Ra mRNA and long type leptin receptor OB - Rb mRNA between the male and the female.Conclusions1. The levels of serum leptin was increased, and the levels of sLRwas .decreased in the patients with pemphigus. The level of serum leptin was negative with level of sLR, and there is a negative feedback mechanism between them.2. The levels of serum IL - 2 and IL - 10 were increased and IL -6 wasdecreased in the patients with pemphigus.3. The level of serum leptin was positive correlative with level of IL -2(r = 0.33, P < 0.05 ) in patients with pemphigus.4. The expression of short type leptin receptor OB - Ra mRNA and long type leptin receptor OB - Rb mRNA were weaker than the normal control, there were no significant difference in the expression of short type leptin receptor OB- Ra mRNA and long type leptin receptor OB - Rb mRNA between the severe cases and the moderate or mild cases.