Mechanism Of Mechanical Trauma Induces Type Ⅱ Penumonocyte Apoptosisi In Rats

Objects:1. Establishment of Non-lethal Mechanical trauma (MT) model and pulmonary injury model in rats. To establish labetalol intervention rats model using labetalol.2. Non-lethal MT model was used to detect mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.3. Cell model in vitro was used to detect the effects of TNF-αand epinephrine on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.4. pulmonary injury model was used to detect detect the effects of TNF-αand epinephrine on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.Methods:1.The effects of MT on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.Establishment of Non-lethal Mechanical trauma (MT) model in rats. After anesthetized, rats were placed in a Noble-Collip drum (12-in.-diameter) and subjected to 200 revolutions at a rate of 40 rpm to induce a non-lethal MT. TNFαand epinephrine levels were observed by ELISA method. Expression of FoxA1 was detected by RT-PCR and western-blotting in MT group and control group. TUNEL and anti-SP-C staining were used to measure typeⅡpenumonocyte apoptpsis in rat lung tissue following mechanical trauma.2. The effects of TNFαand epinephrine on mRNA and protein expression of FoxA1 in typeⅡpenumonocyte in vitroEstablishment of pulmonary injury model in typeⅡpenumonocyte ,Expression of FoxA1 was detected by RT-PCR and western-blotting in groups. Apoptosis rate of typeⅡpenumonocyte after 100ng/mlTNF-αand 2000ng/ml epinephrine induction was detected by flow cytometry.3. The effects of TNFαand epinephrine on typeⅡpenumonocyte in vivoEstablishment of TNF-α, epinephrine injury rat model : Rats were injected intraperitoneally to TNF-α(2.5ug/kg), epinephrine hydrochloride (1ml/kg). To establish labetalol intervention rats model using labetalol. In the labetalol hydrochloride to rats after gavage 3h, 6h, 24h after the instrument into the Noble-Collip trauma to 40 r / min rotation caused by mechanical trauma of 200 laps.Expression of FoxA1 was detected by western-blotting in three groups. TNFαand epinephrine levels were observed by ELISA method. TUNEL and anti-SP-C staining were used to measure typeⅡpenumonocyte apoptpsis in rat lung tissue Results:1. The effects of MT on mRNA and protein expression of FoxA1 in pulmonary tissues and typeⅡpenumonocyte apoptosis.1.1 The effects of MT on secretion of TNF-αand epinephrine in rat's serum.TNFαand epinephrine levels were observed by ELISA method. Compared with normal of that, TNFαand epinephrine levels were increased at each time point after MT(P<0.01), the highest level was happened at 6h and 8h after MT, (1.7560±0.1311)ng/ml and 135.0898±1.5349(ng/l), separately.1.2 The effects of MT Secondary pulmonary injury on mRNA and protein expression of FoxA1Expression of FoxA1 was detected by RT-PCR and western-blotting in MT group and control group. Compared with control group, mRNA expression of FoxA1 was increased; protein expression of FoxA1 was increased with highest level at 24h after MT(P<0.01).1.3 The effects of MT on typeⅡpenumonocyte apoptosisThe results of TUNEL and SP-C staining showed that apopotosis of typeⅡpenumonocyte increased.2. The effects of TNFαand epinephrine on mRNA and protein expression of FoxA1 in typeⅡpenumonocyte in vitro2.1 The effects of TNFαand epinephrine on mRNA expression of FoxA1 in typeⅡpenumonocyteExpression of FoxA1 was detected by RT-PCR and western-blotting in groups. mRNA and protein expression of FoxA1 was increased with highest level at 24h after MT after TNFαand epinephrine induction(P<0.01).2.1 The effects of TNFαand epinephrine on typeⅡpenumonocyte apoptosisApoptosis rate of typeⅡpenumonocyte after 100ng/mlTNF-αand 2000ng/ml epinephrine induction was detected by flow cytometry. Compared with control goup, apoptosis rate of typeⅡpenumonocyte increased 12h after induction.3. The effects of TNFαand epinephrine on typeⅡpenumonocyte in vivo 3.1 The effects of TNFαand epinephrine on typeⅡpenumonocyteExpression of FoxA1 was detected by western-blotting in groups. Compared with control goup, expression of FoxA1 in TNF-αgroup and epinephrine group increased with highest level at 24h after MT(P<0.01); TNF-αand epinephrine levels were observed by ELISA method. Compared with control group, TNFαand epinephrine levels were increased (P<0.01); The results of TUNEL and SP-C staining showed that apopotosis of typeⅡpenumonocyte increased after injection of TNFαand epinephrine, the highest apoptosis rate was happened 12h after injection.3.2 The effects of labetalol on typeⅡpenumonocyte Protein expression of FoxA1 was detected by western-blotting in groups. Compared with control goup, expression of FoxA1 decreased in labetalol groups(P<0.01); Compared with control goup, TNF-αand epinephrine levels were decreased in labetalol groups(P<0.01); The results of TUNEL and SP-C staining showed that apopotosis of typeⅡpenumonocyte decreased in labetalol groups compared with pulmonary injury groups.Conclusions:1. Mechanical trauma led to increase of TNF-α,epinephrine levels in rat's serum and FoxA1 expression. Mechanical trauma led to apopotosis of typeⅡpenumonocyte.2. FoxA1 expression was increased in typeⅡpenumonocyte after induction of TNF-αand epinephrine. FoxA1 was play a critical role in apopotosis of typeⅡpenumonocyte induced by TNFαand epinephrine.3. Secondary pulmonary injury induced by MT was similar to pulmonary injury caused by excessive TNF-αand epinephrine level, and led to apopotosis of typeⅡpenumonocyte.4. Labetalol inhibited gangliated nerve activity, decreasing epinephrine and TNF-αsecretion, reducing apopotosis of typeⅡpenumonocyte, and lessening pulmonary injury.5. Secondary pulmonary injury induced by MT was related with excessive TNF-αand epinephrine level, Which TNF-a, have mutually reinforcing effects of epinephrine and increased lung injury, and led to apopotosis of typeⅡpenumonocyte.6. Labetalol has a protective effect on Secondary pulmonary injury induced by MT.