AD ADI AI A (DI I C ) A AI ADI mrna vs p=0.00 A AI ADI IL-4 AI AD IL-4 AI ADI A AI ADI IFN 1 2

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1 Th1/Th g SPF 6 8 SD 8 8 A D I AD AI DI ADI C 1 D AD DI ADI 60mg/kg mmol/l A AD AI ADI 14 I AI DI ADI 20 2U 24 HE RT-PCR mrna ELISA IL-4 IFN-γ A AI ADI 1

2 AD ADI AI A (DI I C ) A AI ADI mrna vs p=0.00 A AI ADI IL-4 AI AD IL-4 AI ADI A AI ADI IFN 1 2

3 Study of the Role of Insulin and Insulin Receptor In Allergic Airway Inflammation of Rats Department of Pulmonary Medicine People s Hospital, Peking University, Beijing, P.R.C MD. Candidate: Ma Yanliang Supervisor: Prof. He Quanying Abstract Objective: It has been observed in several epidemiological studies and in animal models, that bronchial asthma and type 1 diabetes mellitus (IDDM) are rarely associated in the same subject although the mechanism underlying this protection is still unclear. It is well known that the pathogenesis of these two common diseases is both associated with the genetic profile, inflammatory reaction, immunologic system and neuroendocrine mechanism, and there are close relations between these two diseases especially in aspects such as inflammatory reaction, Th1/Th2 imbalance and signaling through insulin and insulin receptor. Rat models of IDDM and allergic airway inflammation were studied to reveal the possible mechanism underlying the inverse relationship between IDDM and asthma and the role of insulin and insulin receptor in allergic airway inflammation. Methods: Sixty-four specific pathogen-free, 6-8 weeks aged, g weighted, male Sprague-Dawley rats were divided into 8 groups as following: group A (asthma); group D (diabetes); group I (insulin treated); group AD (asthma+diabetes); group AI (asthma+insulin treated); group DI (diabetes+insulin treated); group ADI 3

4 (asthma+diabetes+insulin treated); group C (control). Diabetes mellitus was induced in rats of group D, AD, DI and ADI by a single intraperitoneal injection of streptozotocin (STZ 60mg/kg dissolved in 0.1M sodium citrate buffer, ph4.4) and verified by blood glucose concentrations above 11.2mmol/l 24 hours later. Rats of group A, AD, AI and ADI were sensitized by subcutaneous injection of ovalbumin (OVA) and challenged with an aerosolized solution of 1% OVA for 20 min 14 days after sensitization to provoke allergic airway inflammation; rats of group I, AI, DI and ADI received a single subcutaneous injection of 2U insulin 20 min before antigen challenge. Rats were sacrificed 24 hours after antigen challenge, and blood glucose concentration measurements, total and differential leukocyte counts and serum insulin concentration measurements were carried out. Bronchoalveolar lavage (BAL) were performed, total and differential cell counts were determined. Hematoxylin-eosin stained paraffin section of lung tissue were examined to observe histological changes. Immunohistochemistry method was used to describe the distribution of insulin receptor, and the expression of insulin receptor mrna were measured by RT-PCR. Purified peripheral blood mononuclear cells (PBMC) and cells divided from BALF were stained with mouse anti-rat insulin receptor β antibody and flow cytometric dada were acquired. The IL-4 and IFN-γ concentration in BALF was measured with enzyme-linked immunoadsordent assay. Results: After antigen challenge, rats of group A, AI, ADI all exhibited asthma-like symptoms, cells yield from BALF were significantly elevated, the number of eosinophils and neutrophils were significantly increased, histological analysis showed there was a significant edematous reaction in alveolar septa; a heavy accumulation of 4

5 eosinophils and lymphocytes in the submucosa of bronchus and blood vessels; patchy loss of surface epithelium and secretory cells hyperplasia on bronchial mucosa. However, group AD only exhibited mild symptoms, the number of cells yield from BALF were less than that in group A, but more than that in group C, fewer cells were found in the alveolar spaces and the thickening of bronchial and blood vessel walls were insignificant. No significant difference of total and differential blood leukocyte counts was found between the eight groups. Serum insulin levels were significantly lower in groups induced diabetes than that in non-diabetes groups, but higher in groups ADI, AI and A compared with their respective control group (group DI, I, C). Immunohistochemistrical staining for insulin receptor revealed a diffused distribution pattern of the receptor in the lung tissue, the most prominent staining was found in goblet cells, macrophages and eosinophils. Positive cells infiltrating in the alveolar spaces, submucosa of bronchus and blood vessels and bronchial mucosa were increased significantly in groups A, AI and ADI. In groups induced diabetes the expression of insulin receptor mrna was elevated compared with that in non-diabetes groups ( vs p=0.00). Counts of insulin receptor positive cells acquired by flow cytometry analysis showed no significantly difference either in purified peripheral blood mononuclear cells (PBMC) and cells divided from BALF. Higher concentration of IL-4 was found in BALF of groups A, AI, ADI, especially of group AI; IL-4 concentration in BALF of group AD was lower than that of group AI, ADI, but was not significantly different from that of group A and groups without asthma; groups AI and ADI showed higher IFN-γ levels in BALF than other groups. Conclusion: The inhibited allergic airway inflammation in rats rendered diabetic was 5

6 associated with the lack of insulin. After administration of low dose insulin insufficient to alter the hyperglycemia, reversal of the impaired airway responses was attained in diabetic rats and aggravated airway inflammation to antigen provocation presented in non-diabetic rats. Insulin secretion was increased in the presence of inflammation, indicating a possible proinflammation role of insulin. The distribution, concentration of insulin receptor in the lung tissue were altered both in rats with diabetes mellitus and allergic airway inflammation. In the lung of antigen-challenged rats, insulin receptors on the surface of the infiltrating inflammatory cells and bronchial secretory cells were increased and correlated with the degree of inflammatory reaction. Key words: Diabetes Mellitus; Bronchial Asthma; Airway Inflammation; Insulin; Insulin Receptor; Rat 6

7 1 T 1.6 WHO [1 6] 1 BALF [7] [8] [9] 1 [10] [11] [12] [13] 1 1 Th1/Th2 Th1 IDDM Th2 7

8 Th1/Th2 IDDM Th1/Th2 1 IDDM 1 1 IL-4 IFN- 1 Th1/Th2 RT-PCR 1 8

9 6--8 SPF Sprague-Dawley(SD) 200g LD5-2A LTP-C (79HW-1 ) Olympus 752-C ECP3000 (FACSort) (DF-C ) Fisher-Scientific Eppendorf BD 1 PBMCs Percoll RPMI Amersham Biosciences Cambrex 2 BSA 9

10 β BD (FITC) BD 3 HE β Sigma Santa cruz SP RT-PCR Trizol DEPC M-MLV RNA Rnasin Taq DNA dntp PCR DNA Marker EB Invitrogen Promega Promega Promega Promega Tris EDTA 5 (STZ) OVA grade V Sigma Ted T kung /ml 10

11 ( ) 0.1g/ 2 ml 0.9% (PBS ) IL-4 ELISA IFN-γ ELISA Diaclone Diaclone 1 PH mol/l A 1.921g 100ml B 2.941g 100ml A 28ml B 22ml 100ml 2 A 80g 80ml V OVA80mg 1ml OVA1mg 100mg 4 C B ml 1ml %OVA V OVA 100mg 10ml 4 125U/ml U(2ml) 11

12 98ml 4 C g/ml Percoll Percoll ml 1.5M NaCl 18.26ml 0.15M NaCl 137.4ml 6 PBA 0.5g BSA 10g PBS 500ml 4 C PBS 20g 500ml 8 5 TBE Tris 27g 13.75g 0.5M EDTA 10ml 500ml 4 C TBE 1g 10ml 100ml 5µl EB(10mg/ml) SPF 6 8 SD g SPF

13 1. A D I AD AI DI ADI C A D I AD AI DI ADI C 1 ph4.4 15mg/ml 60mg/kg mmol/l 2 A AD AI ADI 4 A 1ml OVA 1mg 100mg1ml B 1ml ml D I DI C 4 1ml B 1ml cm 3 1%OVA L/min 0.6ml/min 3 20 I AI DI ADI 2U 0.05ml A D AD C ml 13

14 OVA mg/kg 2 10ml 6ml 20 C 3 V 2mm 12ml 11 ml 4 RNA 5 4% HE 1 1 5ml PBS Percoll 5ml 45 1cm / ml PBS 5 RPMI /ml 95% 2 BALF 1 BALF 2200 BALF 1500 / C IL-4 IFN-γ 14

15 3RPMI /ml 95% 4100µl /ml /ml BALF 1ml PBA200µl Mouse anti-rat insulin receptor β 10 µl 30 2 PBA 1ml 3 PBA200µl FITC FITC-conjugated goat anti-mouse immunoglobulin specific polyclonal antibody 20 µl 30 4 PBA 1ml PBA 300 µl 4 : HE 1 5min PBS C I 1:75 β 15

16 (Rabbit anti-human insulin receptor β polyclonal antibody) 4 C 6 PBS II II 30 8 PBS PBS DAB 1 H 2 O 2 +1ml DAB RT-PCR 1 RNA Trizol Reagent mg 1ml Trizol Reagent 5 0.2ml 15 4 C 12000rpm,15 20 C 4 C 12000rpm, C 7500rpm,10 DEPC 260nm 280nm OD RNA RNA =OD 260/OD RNA ug/ul =OD /

17 2 CDNA 0.5ml EP RNA 1µg(1-2µl) 1µl 70 C RT 5µl M-MLV 1µl Rnasin 1µl DNTPs DEPC 0.5µl 25µl 37 C 1 95 C5 65 C5 20 C 3 Polymerase chain reaction PCR 5 -gctgagcaaccttaccaa-3 601bp 5 -aaatgtgtacgaactatc -3 β-actin 5 -atcatgtttgagaccttca-3 314bp 5 -catctcttgctcgaagtc-3 PCR 20µl 10 PCR 2 µl DNTPs 1 µl 0.125mM 1 µl 1.25mM Taq DNA 1 µl 0.25U/µl 2 µl 2.5pmol/µl β-actin 2 µl 0.25pmol/µl DNA 1 µl 17

18 10 µl 94 C 1 94 C 1 45 C 1 72 C 3 72 C PCR PCR 10µl 2µl TBE 80V 40 Sx100 β-actin 6 IL-4 IFN-γ ELISA 450nm IL-4 IFN- 7 SPSS10.0 X SD One-Way ANOVA P<

19 D AD DI ADI A I AI C 2 2 ( X SD ) A D * ** ** I AD * ** ** AI DI * ** ** ADI * ** ** C * p < 0.05 ** p < 3 ( X SD ) ( 10 9 /L (%) (%) (%) (%) ( /L) A D I AD AI DI ADI C A I AI C D AD DI ADI vs p 19

20 0.00 p A AI ADI 1 AD D I DI C 4 A AI ADI p < 0.05A AI ADI AD ADI p = ( X SD ) 10 6 /ml (%) (%) (%) (%) A * * * * D I AD AI * * * * DI ADI * * * * C * D I DI C p < 0.05 ADI p < 0.05 A AI ADI p <

21 A AI ADI p < 0.05 AD p < ( 100) ( 400) A D C C I C C C AD A AI A A A ADI A AI A DI C D D C IL-4 IFN IL-4 6 A IL-4 AD AI ADI AI 21

22 IL-4 p < 0.05AD IL-4 AI ADI p < 0.05 A ADI AD AI p < 0.05 IFN 6 AI ADI p < IL-4 IFN (pg/ml X SD ) IL-4 (pg/ml) IFN (pg/ml) A * D I AD AI DI ADI * C * D I DI C p < 0.05 AI ADI p < 0.05 p < (miu/l X SD ) A D I AD AI DI ADI C * * * * p < 0.05 p < 0.05 I p < 0.05 D DI p <

23 vs p=0.00 ADI D DI p < 0.05 AI I C p < 0.05A I C p < 0.05A I A AI ADI D I DI AD 2 RT-PCR PCR 3 β-actin vs p=0.00 AI I p < 0.05 A 8 ( X SD ) A D I AD AI DI ADI C p < 0.05 I p <

24 601 bp β-actin 314 bp A D I AD Marker AI DI ADI C 3 PCR 4 9 ( X SD ) (%) (%) A D I * AD AI DI * ADI C * p <

600 现代药物与临床 Drugs & Clinic 第 31 卷第 5 期 2016 年 5 月 were significantly decreased, but the levels of IL-12 in two groups were significantly increa

600 现代药物与临床 Drugs & Clinic 第 31 卷第 5 期 2016 年 5 月 were significantly decreased, but the levels of IL-12 in two groups were significantly increa 现代药物与临床 Drugs & Clinic 第 31 卷第 5 期 2016 年 5 月 599 喘可治注射液联合细辛脑注射液治疗小儿毛细支气管炎的临床研究唐辉英, 佘定平, 马颐娇深圳市儿童医院呼吸科, 广东深圳 518026 摘要 : 目的探讨喘可治注射液联合