Objective To investigate the regulatory effect of CsA on the expression of NK cell inhibitory receptor ILT4 and cytotoxicity of NK cells.Methods NK cells treated with CsA ( 10 mg/L) or DMSO for 12,24 and 36 h were chosen as three experimental groups and control groups respectively.RTqPCR and flow cytometry were performed to detect the alteration of ILT4 at the mRNA and protein level respectively.The expression of HLA-G in human gastric cancer cell line BGC-823 and human placental choriocarcinoma cell line JEG-3 were measured at the same time,and then the cytolytic activity of the untreated NK cells and NK cells treated with CsA for 36 h against BGC-823 and JEG-3 cells was determined with MTT.One-way analysis of variance was employed to compare the different ILT4 expression at different time points after medication; Dunnett test was performed to carry out the pairwise comparison between each mean.The difference of HLA-G expression between JEG-3 cells and BGC-823 cells,and the difference of NK cell cytolytic activity against JEG-3 cells and BGC-823 cells were analyzed by student's t-test.Results RT-qPCR assay indicated that the relative levels of ILT4 mRNA in NK cells treated with CsA for 12,24 and 36 h in turn were 0.99 ± 0.27,1.79 ± 0.29,6.79 ± 0.64,and those of their contrast groups treated with DMSO were 0.86 ±0.11,0.94 ±0.12,1.06 ±0.17.The expression of ILT4 in NK cells treated with CsA for 24 h or 36 h was higher than that in NK cells of their contrast groups respectively ( t value of 4.69,14.99,P ＜0.05,respectively),but there was no significant difference between the two groups of NK cells treated for 12 h ( t =0.78,P ＞0.05 ).Through flow cytometry,the positive rates of ILT4 protein expression in NK cells treated with CsA for 12,24 and 36 h [(5.16 ± 0.42 ) ％,( 6.23 ± 0.48 ) ％,( 23.8 ± 1.5 ) ％]were higher than those in NK cells after treatment with DMSO for 12,24 and 36 h respectively[(3.08 ±0.19)％,(3.35 ±0.12)％,(3.36 ±0.21 )％ ;t value of 7.70,10.06,20.72,P＜0.01,respectively].The expression of ILT4 in NK cells treated for 36 h was much higher than that in NK cells for 12 and 24 h at the mRNA and protein level (t value of 16.38,14.12 ;21.81,20.56,P ＜ 0.01,respectively).Meanwhile the killing rates of NK cells treated with 10∶1 effector-target ratio CsA on BGC-823 cells (low HLA-G expression) were ( 8 1.96 ± 2.80 ) ％ ( before treatment) and ( 60.23 ± 1.57 ) ％ ( after treatment),which were higher than those on JEG-3 cells (HLA-G-overexpression) [(53.46 ±2.21 )％ ( before treatment),(28.30 ± 1.85 ) ％ ( after treatment)].The changes of cytotoxicity of NK cells treated with CsA against target cells showed that CsA inhibited the killing activity of NK cells to BGC-823 and JEG-3 cells (t value of 11.74,15.16,P＜0.01,respectively),and the inhibitory rates were (26.48 ±2.42)％ and (47.10 ±1.59 ) ％ respectively.CsA had a higher killing rate inhibition on JEG-3 than on BGC-823 ( t =12.31,P ＜0.01 ).Conclusion CsA induces upregulation of ILT4 in NK cells,and the cytotoxicity of NK cells to tumor cells can be affected by interaction of ILT4 and HLA-G.

Objective:To evaluate the effects of sevoflurane on right ventricular myocardial fibrosis caused by pulmonary arterial hypertension (PAH) in rats.Methods:Eighteen SPF healthy adult male Wistar rats, weighing 260-300 g, were divided into 3 groups ( n=6 each) by a random number table method: control group (group C), group PAH and PAH plus sevoflurane group (group PS). The PAH model was established by single intraperitoneal injection of monocrotaline 60 mg/kg in group PAH and group PS, while the equal volume of normal saline was intraperitoneally injected in group C. Sevoflurane 1.5 MAC was inhaled for 1 h starting from the end of injection, twice a week for 6 weeks in total, in group PS.Echocardiography was performed at the end of 6th week to measure right ventricular end-diastolic diameter (RVEDD), right ventricular anterior wall end-diastolic thickness (RVWTd), interventricular septal end-diastolic thickness (IVSTd), pulmonary artery inner diameter (PAID) and pulmonary valve orifice maximum peak velocity (PV). At the end of 6th week, the hearts were taken to measure the weight of right ventricle, interventricular septum and left ventricle, and Fulton′s index was calculated, and the tissue of the lower lobe of the right lung was taken, the outer diameter and inner diameter of the vascular wall were measured to calculate the vascular wall thickness index (WT), and total vascular area and lumen area were measured to calculate the vascular wall area index (WA) after HE staining.The myocardial tissue of the right ventricle was obtained to observe the degree of myocardial fibrosis (with a light microscope after Masson staining) and to detect the expression of TGF-β1 (after immunofluorescence staining) and expression of TGF-β1, phosphorylated Smad3 (p-SMad3) and Smad7 (by Western blot). Results:Compared with group C, Fulton′s index, RVEDD, RVWTd, IVSTd, PAID, WT and WA were significantly increased, PV was decreased, the expression of TGF-β1 and pSmad3 in right ventricular myocardial tissues was up-regulated, the expression of Smad7 was down-regulated( P<0.01), and myocardial fibrosis occurred in group PAH.Compared with group PAH, Fulton′s index, RVEDD, RVWTd, IVSTd, PAID, WT and WA were significantly decreased, PV was increased, the expression of TGF-β1 and pSmad3 in right ventricular myocardial tissues was down-regulated, the expression of Smad7 was up-regulated ( P<0.05 or 0.01), and myocardial fibrosis was significantly improved in group PS. Conclusion:Sevoflurane can improve the myocardial fibrosis in right ventricle induced by PAH in rats, and the mechanism may be related to inhibiting activation of TGF-β1/Smad3 signaling pathway.

Objective:To evaluate the effect of sevoflurane on Ca 2+ transporter expression in cardiomyocytes during right ventricular remodeling in rats with pulmonary arterial hypertension. Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 200-250 g, were divided into 4 groups ( n=6 each) by the random number table method: control group (CM group), sevoflurane group (CS group), monocrotaline group (M group) and sevoflurane + monocrotaline group (S group). Monocrotaline 60 mg/kg was intraperitoneally injected in group M and group S, and monocrotaline lysate was intraperitoneally injected in group CM. The rats in S and CS groups inhaled 2.5% sevoflurane for 1 h, twice a week, at an interval of 3 days starting from the first day after injection of monocrotaline. Pulmonary artery acceleration time and pulmonary artery ejection time were measured by transthoracic echocardiography at 6 weeks after monocrotaline injection. The chest was exposed under 3% sevoflurane anesthesia, the heart was perfused, and the pulmonary artery branch and right ventricular myocardial tissues were retained. The wall thickness of pulmonary arterioles and cross-section area of right ventricular cardiomyocytes were observed by HE staining. The expression of Ca 2+ transporter in right ventricular cardiomyocytes was detected by Western blot. Results:Compared with CM group, the ratio of pulmonary artery acceleration time to pulmonary artery ejection time was significantly decreased, the cross-section area of right ventricular cardiomyocytes was increased, the wall thickness of pulmonary arteriole was increased, the expression of type 1 sodium-calcium exchange and inositol triphosphate receptor was up-regulated, and the expression of voltage-dependent L-type calcium channel α1C subunit, type 2 ryanodine receptor, sarcoplasmic reticulum calcium pump 2α and proteinphilin-2 was down-regulated in M group ( P<0.01). Compared with group M, the ratio of pulmonary artery acceleration time to pulmonary artery ejection time was significantly increased, the cross-section area of right ventricular cardiomyocytes was decreased, the wall thickness of pulmonary arteriole was decreased, the expression of type 1 sodium-calcium exchange and inositol triphosphate receptor was down-regulated, and the expression of voltage-dependent L-type calcium channel α1C subunit, type 2 ryanodine receptor, sarcoplasmic reticulum calcium pump 2α and proteinphilin-2 was up-regulated in group S ( P<0.01). Conclusions:The mechanism by which sevoflurane improves right ventricular remodeling is related to regulating the expression of Ca 2+ transporter in cardiomyocytes of rats with pulmonary arterial hypertension.