Effect Of Qizhizhoufei Granule On The Proliferation And Biological Activities Of LPS Induced Inflammatory Alveolar Type Ⅱ Epithelial Cells

Objective To explore a appropriate method of primary culture ofalveolartype Ⅱe pithelial cells(AT-Ⅱ)of newborn Wistar rat. By LPS manufacturingcell inflammation model, Qizhizhoufei granule as drug intervention, observe theinflammatory AT-Ⅱ morphology, cell cycle and mitochondrial activity, the influenceof the alveolar surface active protein A(SP-A). To explore the mechanism of action ofits prevention and treatment of COPD,and at the same time, for the development ofresearch on Qizhizhoufei granule provide pharmacodynamic foundation.Methods Separate AT-Ⅱfrom lungs of SPF newborn Wistar rats by trypsincombined collagenase digestion method, purify AT-Ⅱby immune adhesion and stickover and over again method,culture AT-Ⅱin vitro and carry on research of cellgeneration.Do the identification of AT-Ⅱ through electron microscope (SEM). Cellmodel method:10groups of the same growth status and nearly the same number ofcells, two groups without of LPS, two groups with5ug/ml LPS, two groups with10ug/ml LPS, two groups with20ug/ml LPS and two groups with40ug/ml LPS,intervention in one day.The second day,plus100ug/ml Qizhizhoufei granule to one ofthe same concentration of groups with5different concentrations of LPS, interventionin two days,and the other group as contrast. To observe and detect the variation of theAT-Ⅱ cell morphology, lamellar body, mitochondrial, cell cycle, and the expressionof alveolar surface active proteinA(SP-A) of each group.Results (1) The primary culture AT-Ⅱ cells’ growth in good condition.(2) TheLamellar corpuscles are observed under SEM. LPS groups reduced numbers oflamellar body, and its number of Qizhizhoufei granule intervention groups madesomething increase.(3) MTT showed that LPS groups’ AT-Ⅱ with abnormal cellproliferation,and strengthen the its proliferation rate increased with concentration ofLPS.After the intervention of Qizhizhoufei granule, cell proliferation is restrained,proliferation rate declined.(4) The cell cycle detection results showed that LPSgroups’ AT-Ⅱ cell’s percentage of G2phase and S phase is higher.Its proliferationpercentage rises with the increase of concentration of LPS. After the intervention of Qizhizhoufei granule, its percentage of G2phase and S phase is decreased.(5)Rhodamine123dyeing the mitochondria of AT-Ⅱ cell detection results showed thatunder laser confocal microscope, strong activity of mitochondria and highfluorescence intensity in cells of the blank group. With the increase of theconcentration of LPS, fluorescence intensity gradually weakened. After theintervention of Qizhizhoufei granule, fluorescence intensity has been restored.(6)SABC Immunohistochemical detection SP-A results showed:①DAB chromogenic:There are many tan particles can be seen in cells of the blank group. There are moretan particles can be seen in cells of the blank group which having been plusQizhizhoufei granule,and the colour of the tan particles is darker. The tan particles ofLPS groups significantly reduced.With the increase of the concentration of LPS, thenumber of the tan particles gradually decreased, and the colour of the tan particlesgradually become shallow. After the intervention of Qizhizhoufei granule, thenumber of its tan particles in cells increased, and the colour of them darken.②FITCfluorescent dye:High fluorescence intensity in cells of the blank group. There arehigher fluorescence intensity in cells of the blank group which having been plusQizhizhoufei granule.With the increase of the concentration of LPS, fluorescenceintensity gradually weakened. After the intervention of Qizhizhoufei granule,fluorescence intensity has been restored.Conclusion The primary culture AT-Ⅱcells obtained from this experiment havegood growth state,and they are suitable for research in vitro.They have optimalgrowth state within24~72h,it is the best time of experiment research. LPS can spurthe AT-Ⅱ inflammatory reaction and abnormal cell proliferation, and the size ofproliferation is proportional to the strength of LPS concentration. LPS weakens theAT-Ⅱ cells’s SP-A.,and reducing the biological activities of the AT-Ⅱcells.Qizhizhoufei granule has inhibition effect on the abnormal proliferation of theinflammatory by LPS AT-Ⅱcells, helps to improve cell’s ultrastructural, promotesthe SP-A’s expression,and improve the AT-Ⅱ cells’ biological activities.