Protective Effects Of Keratinocyte Growth Factor-2 On Cigarette Smoke-induced Lung Injury

Part I:Alteration of Claudin Expressions in Cigarette Smoke-induced Lung InjuryObjective:This experiment established smoking rat model and cell model to investigate claudin expression alterations in smoking-induced lung injury and it’s pssible meaning.Methods:SPF SD rats were randomly divided into control group and smoking group. They received 20 cigarettes for 20-30 minutes each day for a total of 3 months. Samples were collected after executed. Inflammatory cytokine of total proteins in bronchoalveolar lavage fulid was examined. The pathological changes of lung tissues were observed under light microscope via HE and PAS staining. The PAS positive area ratio was calculated in order to evaluate lung injury. The mRNA level of claudin-1,-3,-4,-7 were determined by RT-PCR. We use cigarette smoke extract (CSE) activated 16HBE cell line. The experiment was divided into four groups, including Control group 5%,10%,20%CSE group. The protein level of cells were determined by Western Blot.Results:Rats in smoking group lost weight and lessened resistance. Pathological changes happenned, including airway epithelial necrosis, ciliary structure disappearing, increase of lymphocytes in epithelium. PAS staining and inflammatory factor significantly increased in smoking group. Claudin-1,-4 mRNA levels decreased significantly, while the claudin-3,-7 mRNA levels did not change. In cell model, claudin-1 protein levels decreased significantly and claudin-4 protein levels increased in all CSE groups, compared with Control group. And both changes rised with CSE concentration gradient.Conclusion:1. Airway appeared pathological changes and BALF inflammatory factors increased in smoking rat model.2. Claudin-1、-4 mRNA levels decreased significantly, which may be the reason of decreased barrier function.3. In short-term cell model, claudin-1 protein levels decreased and claudin-4 protein levels increased in CSE group. The bounce phenomenon of claudin-4 protein expression may be a protective effects of cells.Part II:Protective Effects of Keratinocyte Growth Factor-2 on Cigarette Smoke-induced lung InjuryObjective:This experiment established bronchial epithelial cells smoking model to observe protective effects of KGF-2 on CSE-induced injury and claudin protein alteration, which may lay a foundation of KGF-2 for clinical application in lung injury.Methods:We use cigarette smoke extract(CSE) activated 16HBE cell line. Cell viability was determined by MTT method in 10% CSE + KGF - 2 group and 10% CSE group. PI staining was used to analyse percentage of cells in apoptosis phase, G0/G1 phase and S + G2/M phase in P,K,10% CSE + KGF-2 group and 10% CSE group. Flow cytometry was used to detect cell proliferation and Western blot was used to test phosophorylated Akt protein expression. Transepithelial electrical resistance(TEER) was measured in P,K,2Q% CSE,20%CSE+KGF-2 group, while claudin-1, claudin-4 protein expressions were measured too.Results:MTT showed cell viability significantly higher in KGF-2+10%CSE group than in 10%CSE group. The percentage of cells in apoptosis and G0/G1 phase decreased and in S+G2/M phase increased in 10% CSE + KGF-2 group,compared to 10%CSE group. Cell proliferating rate was higher in group with KGF-2. And the effect was overlay in cell activated by CSE. Phosphorylated Akt protein expression was significantly higher in KGF-2+10%CSE group, which may suggest that effects of KGF-2 on proliferation may through PI3K/Akt signaling pathway. TEER was significantly higher in 20%CSE+KGF-2 group than 20%CSE group, suggesting that KGF-2 can enhance cell barrier function. However, this effect was not through altering claudin protein expression, because claudin-1,-4 protein had no significant change between group with or without KGF-2.Conclusion:1. KGF-2 improved airway epithelial cell proliferation and increased number of cells in divisions.2. KGF-2 can enhance airway epithelial barrier function to resist CSE-induced damage.3. Protective effects of KGF-2 on airway epithelial barrier function was not produced by adjusting claudin protein.