| Acute lung injury(ALI) is an acute progressive respiratory failure induced by various reasons, and its serious stage is acute respiratory distress syndrome(ARDS). Its mechanism is very complex. At present, there are no satisfactory therapeutic methods and its mortality is up to 50 percent. Atrial natriuretic peptide(ANP), consisted of 28 amino acids, is an important regulator of the sodium and volume homeostasis. It has strong vasodilating, diuresis and natriuretic effects. In recent years, ANP has been used to treat many diseases, for example hyperpietic, renal insufficiency, Cardiac insufficiency, hydrocephalus and glaucoma, and the curative effect is satisfactory. In recent years, scientists have found that ANP could effectively relieve ARDS, but the mechanism is not clear. Our laboratory has done our best to discover the mechanism. The following is our productions. (1) The intravenous infusion of ANP may efficiently decrease the level of TNF-α , IL-8 and ET-1 in blood and bronchoalveolar lavage fluid (BALF) of ALL (2) ANP could upregulate the expression of aquaporinl(AQP1) in lung tissue ofALI, and facilitate the lung water to be absorbed. (3) ANP could improve the blood air level and reduce the Wet/Dry weight ratio of lung in ALI. In this study, we will examine the protecting effects and the possible mechanisms of ANP on alveolar type II cells (AT- II ) in ALI induced by lipopolysaccharide(LPS).Part I ANP could alleviate ALI and protect the AT- II from being injured by LPS.METHODSExperiment on animals Eighteen rats were divided into 3 groups randomly: Control group(n=6), the rats v/ere injected physiological with saline through jugular; LPS group(n=6), the rats were injected with LPS(2 mg/kg) and physiological saline through jugular; ANP group(n=6), the rats were were injected with LPS(2 mg/kg) and ANP(2 u g/kg) through jugular. Four hours later, killed the rats and collected the BALF. Surface tension(ST), total phospholipids(TPL), total protein (TP) and malondialdehyde(MDA) content of the BALF were measured. The activity of lactate dehydrogenase(LDH) and alkaline phosphatase(A KP) in BALF were detected, and the pathological features of lung were observed by the microscope.Experiment on cells AT- II were suspended in RPMI-1640 supplemented with 15% fetal bovine serum(FBS) (1 X 106 cells/mL). Then AT-II were placed in a 6 well cell culture cluster (0.5 X 106 cells/cm2). After 36 h of incubation, the mediums were changed with the fresh test mediums(2.5 mL/well) of designed composition. AT-II were divided into 3 groups: (1) Control group(n=6), the medium consisted of RPMI-1640 without FBS. Thecell culture mediums were cellected after 4, 12 and 24 h. (2) LPS group(n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS(1 mg/L). The cell culture mediums were also gathered after 4,12 and 24 h. (3) ANP group(n =6), the medium consisted of RPMI-1640 without FBS supplemented with LPS( 1 mg/L) and ANP( 10"8,10'7,10"6 mol/L). After 12 h, the cell culture mediums of each dose were collected, and the cell culture mediums of the ANP (10'7 mol/L) were collected after 4 and 24 h. ST, TPL, AKP, LDH and MDA were examined in the medium of every group. RESULTSExperiment on animals Compared with control group ,LPS could increase the TP content and the activity of LDH in BALF, which can be reversed by ANP. TPL increased and ST decreased in LPS group, and there is no significant difference of TPL or ST between ANP group and control group. ANP could prevent increasing of the activity of AKP and the content of MDA in BALF induced by LPS. So we could conclude that ANP could decrease the injury induced by LPS in ALI, partly through protecting AT- II and promoting the secretion of PS.Experiment on cells AT- II were characterized by AKP staining. The change of the intensity of AKP staining with increaseing time in culture was insignificant before 60 h of culture. The intensity of AKP staining decreased significantly after 96 h of culture. At the 3 time points of 4 h, 12 h and 24 h of culture, ANP could increase the content of TPL in the mediums which had been decreased by LPS in a concentration-dependent and time-dependent manner, and the effect at 12 h time point was the most significant of 3 time points. The trends of the change of LDPL MDA> AKP and ST in mediums were completely opposite to the trend of TPL.CONCLUSIONANP can protect the lung from being injured by LPS in ALL One of the most important mechanisms is that ANP can protect the AT- II and promote the secretion of PS.Part II ANP could protect the AT- II from being injured by LPS through cGMPMETHODSAT- II were suspended in RPMI-1640 supplemented with 15% FBS (1 X 106 cells/mL). Then AT- II were placed in a 6 well cell culture cluster (0.5 X 10 cells/cm ). After 36 h of incubation, the mediums were changed with the fresh test mediums(2.5 mL/well) of designed composition. AT- II were divided into 7 groups: (1) Control group(n=6), the mediums consisted of RPMI-1640 without FBS. (2) LPS group(n=6), the medium consisted of RPMI-1640 without FBS but supplemented with LPS(1 mg/L).(3) L +A group(n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS(1 mg/L) and ANP(10"7 mol/L).(4) IB+L group(n=6), the mediums consisted of RPMI-1640 without FBS supplemented with LPS(1 mg/L) and IBMX (0.2 mmol/L).(5) IB+L+A group(n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS(1 mg/L), ANP(10'7 mol/L) and IBMX(0.2 mmol/L).(6) LY+L group(n=6) , the medium consisted of RPMI-1640 without FBS supplemented with LPS(lmg/L) and LY-83583 (0.01 mmol/L).(7) LY+L+A group(n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS(lmg/L), ANP(10"7 mol/L) and LY-83583(0.01 mmol/L). After 12 h, the mediums of all groups were collected,and the level of cGMP, ST, TPL, AKP, LDH and MDA in them weremeasured.RESULTSCompared with control group( 1.23+ 0.68 pmol/well), both ANP (4.14 ±0.69 pmol/well) and LPS(2.27±1.05 pmol/well) could increase the levels of cGMP of AT- II ,but the effect of ANP was better than LPS. The levels of intracellular cGMP in Both IB+L group(9.08 ± 0.50 pmol/well) and IB+L+A(9.20±0.66 pmol/well) group were obviously increased, while the level of intracellular cGMP in both LY+L group(0.38±0.12 pmol/well) and LY+L+A group(0.41+0.12 pmol/well) were significantly decreased. In the presence of phosphodiesterase inhibitor (IBMX) or the guanylyl cyclase inhibitor (LY-83583), there was no significant difference with intracellular cGMP either with the presence or absence of ANP. These result shew that IBMX or LY-83583 could inhibit ANP raising the level of intracellular cGMP through different mechanisms respectively.Compared with control group, LPS could increase LDH, AKP, MDA and ST of mediums and decrease TPL of mediums, which can be reversed by ANP. Compared with L+A group, the levels of LDH, AKP, MDA and ST in mediums were all increased and TPL of mediums were significantly decreased in LY+L+A group. In IB+L+A group, the trends of the change with LDH, AKP, MDA, ST and TPL in mediums were completely opposite to that in LY+L+A group. LDH, AKP and MDA in mediums of IB+L+A group were all lower than those in IB+L group. Both LDH and ST of mediums in LY+L+A group were lower than those in LY+L, and TPL in mediums of LY+L+A group was higher than that in LY+L. Compared with LPS group, ,LDH, AKP, MDA and ST of mediums were all decreased, while TPL of mediums were...
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