| Obstructive sleep apnea syndrome(OSAS)is characterized by apnea or hypopnea,episodes of upper respiratory tract obstruction,which directly causes chronic intermittent hypoxia(CIH),decreased blood oxygen saturation,and sleep fragmentation during sleep.It is a major public health problem and occurs in 3-7% men and 2-5% women.More and more researches have shown that OSAS is an important risk factor for increasing the morbidity and mortality of cardiovascular diseases,including pulmonary hypertension,diabetes,cognitive dysfunction,ischemic heart disease,stroke and other complications,OSAS is considered to be an independent risk factor for hypertension.Modern medicine for OSAS patients generally use drug intervention,oral appliance treatment,nasal continuous positive airway pressure(CPAP)treatment and surgical treatment,however,so far,a variety of treatment measures is not ideal.Chinese medicine has also carried out a positive exploration.In this study,we observed the Xiaotan Huayu Liqiao Formula treatment of rats exposed to CIH increased aortic endothelial function.Then,we further investigated to underlying the molecular mechanisms.Objective: We investigated the effects of of Xiaotan Huayu Liqiao Formula on CIH-induced endothelial dysfunction in rats by tail arterial pressure(SBP),serum NO and histopathological changes.Then we further measured expression of ET-1,e NOS,p-e NOS,Rho A,ROCK,p-MYPT-1 in rats aortic tissue using Western blotting techniques.Based on these experimental methods,we evaluated the mechanisms underlying protective effect of Xiaotan Huayu Liqiao Formula on endothelial dysfunction by CIH rats.Method: Adult(180-200 g)male Sprague-Dawley rats were provided by Hebei Medical University.Rats were randomly divided into five groups,with10 rats in each group: Normoxia,CIH,XL + CIH,XM + CIH and XH + CIH.Ten rats in each group were intragastric treatment every day.All drugs were given as follows: Normoxia group,water 1 m L/100 g;CIH group,physiological saline 1 m L/100 g;XL + CIH group,Xiaotan Huayu Liqiao Formula 24 g/kg,XM + CIH group,Xiaotan Huayu Liqiao Formula 48 g/kg,XH + CIH group,Xiaotan Huayu Liqiao Formula 96 g/kg.In addition to the Normoxia group,the other rats were exposed to CIH as previously described.Briefly,during CIH exposure,rats were placed in commercial hypoxic chambers that were flushed with 100% N2 to inspire a fraction of an inspired O2(FIO2)nadir of 9% for 1.5 min.FIO2 gradually returned to 21% over the remainder of each cycle.The exposure cycle was repeated every 3 min for 8h/day,7 days/week for 3 weeks during the animal's sleeping hours.Normoxia group rats underwent identical handling and exposure,but the chambers were flushed with room air rather than N2.At the end of modeling,the rat aorta was subjected to experimental study.SBP was measured weekly in conscious animals.At the end of the model,rats were without food and water for 12 hours,and blood was taken from the femoral artery for the examination of blood.Then,rats were sacrificed with an overdose of 25% pentobarbital sodium(i.p.).The ventral surface was sterilized with 75% ethanol before a vertical incision was made to expose the abdominal cavity and thorax.The aortic arch was located and the vessel was traced along the spine,separating it from the connective tissue prior to removal and one of it immediately placing to 4% paraformaldehyde and other tissue store in a refrigerator at-80 ?.The serum concentration of NO and ET-1 level in plasma were detected by biochemistry assay.The aortic histological changes in rats were hematoxylin eosin(HE).The protein expression of ET-1,e NOS,p-e NOS,Rho A,ROCK and p-MYPT-1 were detected by western blot.1 Effect of Xiaotan Huayu Liqiao Formula on SBP in CIH ratCIH(158 ± 3.44 mm Hg)rats showed a significantly increased tail artery SBP at the completion of the experimental period by the age of 21 days compared with Normoxia(147 ± 1.48 mm Hg)rats(P < 0.01).Treatment with different dose of Xiaotan Huayu Liqiao Formula(148 ± 2.65 mm Hg,149 ±3.43 mm Hg,149 ± 3.57 mm Hg)decreased SBP compared with CIH rats(P <0.05).2 Effect of Xiaotan Huayu Liqiao Formula on aortic histopathologyA normal tissue architecture of the endothelium was seen in the normoxia group,composed of a complete endothelial monolayer and regularly shaped and oriented endothelial cells.Aortas in the CIH group exhibited marked histopathological changes of the endothelial layer with cellular shrinkage,partial exfoliation and small hyperchromatic nucle.Compared with the CIH group,the injuries on the aortic tissues were alleviated in the different dose of Xiaotan Huayu Liqiao Formula.3 Effect of Xiaotan Huayu Liqiao Formula on NO level in serum and ET-1 level in plasmaCIH(28.9 ± 2.23 ?mol/L)rats showed a significantly decreased NO level in serum compared with Normoxia(38.1 ± 2.10 ?mol/L)rats(P < 0.05).Treatment with different dose of Xiaotan Huayu Liqiao Formula(35.7 ± 1.72,36.7 ± 1.22,38.5 ± 1.15 ?mol/L)increased NO level in serum compared with CIH rats(P < 0.05,P < 0.05,P < 0.01).CIH(97.9 ± 3.24 pg/m L)rats showed a significantly increased ET-1 level in plasma compared with Normoxia(79.4 ± 4.05 pg/m L)rats(P < 0.05).Treatment with different dose of Xiaotan Huayu Liqiao Formula(85.8 ± 1.75,83.9 ± 3.5,82.1 ± 4.4 pg/m L)decreased ET-1 level in plasma compared with CIH rats(P < 0.05,P < 0.05,P < 0.05).4 Effect of Xiaotan Huayu Liqiao Formula on ET-1 protein expression in CIH aortas.CIH(1.04 ± 0.0667)rats showed a significantly increased ET-1 protein expression compared with Normoxia(0.350 ± 0.0295)rats(P < 0.01).Results:Treatment with different dose of Xiaotan Huayu Liqiao Formula(0.831 ±0.0254,0.814 ± 0.0275,0.684 ± 0.0232)decreased ET-1 protein expression compared with CIH rats(P < 0.05,P < 0.05,P < 0.01).5 Effects of Xiaotan Huayu Liqiao Formula on e NOS and p-e NOS expression in CIH aortas.The phosphorylation of e NOS is a marker of e NOS activity.CIH rats showed a significantly decreased e NOS(0.411 ± 0.0266)and p-e NOS(0.267± 0.0128)protein expression compared with Normoxia(e NOS: 1.10 ±0.0954,p-e NOS: 0.670 ± 0.0443)rats(P < 0.05;P < 0.01).Treatment with different dose of Xiaotan Huayu Liqiao Formula increased e NOS(0.567 ±0.0444,0.602 ± 0.0223,0.736 ± 0.0250)and p-e NOS(0.324 ± 0.0120,0.424±0.0216,0.578 ± 0.0286)protein expression compared with CIH rats(P <0.05,P < 0.01,P < 0.01).6 Effects of Xiaotan Huayu Liqiao Formula on Rho A/ROCK pathway protein expression in CIH aortas.CIH rats showed a significantly increased Rho A(1.29 ± 0.0341)and ROCK(1.10 ± 0.0499)protein expression compared with Normoxia(Rho A:0.508 ± 0.0638,ROCK: 0.174 ± 0.0316)rats(P < 0.01).Treatment with different dose of Xiaotan Huayu Liqiao Formula decreased Rho A(0.955 ±0.0796,0.831 ± 0.104,0.6594 ± 0.0710)and ROCK(0.834 ± 0.0624,0.586 ±0.0395,0.310 ± 0.0652)protein expression compared with CIH rats(P < 0.05;P < 0.01).7 Effect of Xiaotan Huayu Liqiao Formula on p-MYPT-1 protein expression in CIH aortas.Because MYPT-1 is a major downstream target of Rho A/ROCK-mediated Ca2+ sensitization and a regulator of myosin light chain(MLC)activation.We tested the activation of Rho A/ROCK pathway by amount of phosphorylated MYPT-1.CIH rats showed a significantly increased p-MYPT-1(1.03 ± 0.0241)protein expression compared with Normoxia(0.339 ± 0.0656)rats(P < 0.01).Treatment with different dose of Xiaotan Huayu Liqiao Formula decreased p-MYPT-1(0.705 ± 0.0213,0.506 ± 0.0170,0.359 ± 0.0251)protein expression compared with CIH rats(P < 0.01).Conclusions:The results of these studies indicate that Xiaotan Huayu Liqiao Formula has good protective effect on reduced blood pressure and endothelial function by CIH,and endothelial protection may be related with inhibiting the Rho A/ROCK signaling pathway,adjusted expression of e NOS,p-e NOS,NO and ET-1 levels. |
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