| Background In general cases, sepsis always leads to multiple organ dysfunction syndrome, MODS, as the one of the fatal causes of ICU patients. Acute Lung Injuryis the major pathological symptom as lung is the most vulnerable target organ during the process. And one of the main pathogenic factors of sepsis is lipopolysaccharide, LPS, which will damage to cells in pulmonary microvasculature via either direct or indirect ways, which increases pulmonary microvascularpermeability, causes inflammation, edema, and ends up with ALI. Increased pulmonary microvascularpermeability aggravates oxygen deficient by letting interstitial space filled with fluid. Second of all, this process also collects mediators of inflammation which deliver harmful factors like protease and oxygen free radical. Also, gathering of mediators of inflammation itself raises the problem of damage to pulmonary microvasculature. So that it is significantly important to seek an effective treatment for sepsis.Penehyclidine Hydrochloride (PHC) is an anticholinergic agents origins from our country and we have itsindependent intellectual property rights,PHC has advantage of selectivity over mAch-receptor.Study shows that PHC can ease the damage to lung of mouse with sepsis through reducing production of peroxide by restraining activation of p38 MAPK and depressing the expression of iNOS. Besides, the trending study of signal communication also shows that ?-arrestins has an intimate relationship with activation of p38 MAPK, and by using it as scaffolding proteinto transfer signal, can connect G protein coupled receptor signal pathway with other signal pathways such as TLR-ILIR and MAPK pathway within cells to alter their functionalities or activities in order to encourage or discourage signal communication accordingly. ?-arrestins include of ?-arrestin-1 and ?-arrestin-2. Other study shows that significant increasing of MAPK activities in embryonic fibroblast of which mouse are lack of ?-arrestin-1. So we can presume that PHC's functionalities of easing ALI damage, protecting Human Pulmonary Microvascular Endothelial Cells and reducing cellular permeability may due to its effect on ?-arrestin-1 in ?-arrestins'family.Objective This study is to explore the effect of PHC on injuryed-PMVECs lead by LPS, discussion of effect of ?-arrestin-1 on LPS-induced HPMECs permeability transformation, and study whether HPMECs is affected by PHC through up-regulation of P-arrestin-1. Results suggest that P-arrestin-1 shRNA expression plasmids are constructed successfully and are transfected into HPMECsMethodsCulture HPMECs, observe growing condition and cell morphology then subculture HPMECs; Make use of the 4th to 6th generation of cells for research; Construct ?-arrestin-1 shRNA expression plasmids, transfect empty plasmids and ?-arrestin-1 shRNA expression plasmids to regular HPMECs and divide them into 2 sets with 4 groups each. Within normal HPMECs set (group C/L/PL/P):add equivalent amount of culture medium to group C; add 0.1?g/ml of LPS to group L; add 2?g/ml of PHC, and additional 0.1?g/ml of LPS after 1 hour to group PL; add 2?g/ml of PHC to group P. Exam all features after 1 hour-treatment of each group is set. Within transfection set (group NL/NP/BL/BP):add 0.1?g/ml of LPS to group NL with empty plasmids transfected cells; add 2?g/ml of PHC with additional 0.1?g/ml of LPS after 1 hour to group NP with empty plasmids transfected cells; add 0.1?g/mlof LPS to group BL with ?-arrestin-1 shRNA expression plasmids transfected cells; add 2?g/ml of PHC with additional 0.1?g/mlof LPS after 1 hour to group BP with with ?-arrestin-1 shRNA expression plasmids transfected cells.Exam all features after 1 hour of each group is set.Results 1) HPMECs purchase from ScienCell Research Laboratories, US, can be cultured in vitro steadily, according to official guide, cell line developed as shape of classic pave stone from 7th generation cells under phase contrast microscope can be used for next step of study. Construct ?-arrestin-1 shRNA expression plasmids by designing, combination 3 shRNA of eligo DNA and restructuring shRNA expression plasmids using shRNA designing software. Examine the expression of ?-arrestin-1mRNAby RT-PCR.2) Normal HPMECsgroups:Compare to Group C, Hsp27, p38 MAPK, NF-?B p65 expression and LDH lever rises in group L, and P-arrestin-1, I-?B expression decreases, along with severe damage to cytoskeleton. Group PL has similar trend with slight severalty. Group P has no statistic meaningful result compare to A except for the increase of ?-arrestin-1 expression. Last but not least, Hsp27, p38 MAPK, NF-?B p65 expression and LDH lever decreases in group PL, and ?-arrestin-1, I-?B expression increases, along with lighter damage to cytoskeleton compare to group L.3) Transfected cellsgroups:Hsp27, p38 MAPK, NF-?B p65 expression and LDH lever decreases in group NP, and ?-arrestin-1, I-?B expression increases, along with lighter damage to cytoskeleton and lower cellular permeability than group NL; group BP has no meaningful static result compare to group BL, they have similar results in both cytoskeleton changes and cellular permeability. While in group BP Hsp27, p38 MAPK, NF-?B p65 expression and LDH lever increases, and ?-arrestin-1, I-?B expression decreases, along with severe damage to cytoskeleton and higher PMVECs cellular permeability than they are in group NP.Conclusions 1) HPMECs grow in a good condition and have the normal cell morphology; ?-arrestin-1 shRNA expression plasmids are constructed successfully and are transfected into HPMECs.2) Effect for PHC to protect HPMECs from damaging may due to increasing of P-arrestin-1.3) PHC has a depression effect through increasingp-arrestin-1 on LPS-induced HPMECs permeability. And ?-arrestin-1 has an intimate effective relationship with HPMECs permeability transformation. |
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