Effects and mechanisms of Artemisia argyi essential oil on monocrotaline-induced pulmonary hypertension in rats     
YinYing1,2, YunfengSun1, HongchunLi1, BoYang1, HuiWang1, QingmeiWu1, Ping Huang3*  
1. Department of Pharmacy, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang 310012, China
2. Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, China
3. ZhejiangCancerHospital, Hangzhou,Zhejiang310022,China        
 
 
Abstract: In the present study, we aimed to investigate the effects of Artemisia argyi essential oil (AO) on pulmonary hypertension (PH)inducedbymonocrotaline(MCT)andtoexploretheunderlying mechanism.A total of 80 Sprague-Dawley ratswererandomlydividedintofourgroups as follows:controlgroup, modelgroup,bosentan(0.1g/kg)groupandAO(0.1g/kg)group.After30dofexperiment, hemodynamicparameters,lungandrightventricle hypertrophy indexweredetermined. HEand Immunohistochemistrystainingoflungs were performedto detectthe injuriesandprotein expressions. Theresultsshowedthatlevels of mPAP,mRVP,maxRVP,wW,LI,RV and RVHI as well asthe expressionsofNF-κBp65andα-SMA were increased,smallpulmonaryarterythickened, the cavity of the arteriole narrowed, and there was marked infiltration of inflammatory cells in lungs of rats receiving MCT compared with the normal group. After the administration of AO, the levels of mPAP and mRVP were significantly decreased,and the wW, LI, LV+S and pulmonary arterial remodeling were markedly improved. The expression levels of NF-κB p65 and α-SMAwerereduced by AOcomparedwiththe modelgroup.Ourresultssuggestedthat AOreduced the progressionof PH induced byMCTthroughinhibiting the expressions of NF-κB p65 and α-SMA.                  
Keywords: Artemisia argyi essential oil; Pulmonary hypertension; NF-κB; α-SMA    
CLC number: R962                Document code: A                 Article ID: 10031057(2018)318310

 
1. Introduction
Pulmonary hypertension (PH) is characterized by sustained pulmonary vascular resistance induced by a group of heterogeneity. It is caused by hypoxic pulmonary vasoconstriction, pulmonary vascular endothelial dysfunction and pulmonary vascular reconstruction induced by peripheral airway obstruction, abnormal pulmonary vascular parenchymal and chronichypoxia[1]. Current epidemiological analysis has reported that the prevalence rate of PH is 1% of the global population[2]. The clinical manifestations show increased right ventricular afterload, decreased activity toleranceor even death when right heart failure occurs. Therefore, PH is considered as a mortal threat to humans.
Calcium channel antagonists, prostacyclin, ET receptor antagonists, phosphodiesterase III inhibitors, angiotensinconverting enzyme inhibitors and adenosine are frequently used to the treatment of PH[3]. However, due to their failure of long term efficiency and side effects, it seems necessary and critical to focus on researching and discovering new drugs with high safety, and it is deemed to be a good idea to find drugs from natural plant sources.
The leaves of Artemisia argyi Levl.et Vant, a kind of perennial herb, have a long history of medical use. In pharmacological study, it has been found thatArtemisia argyi has effects of anti-bacterial, anti-viral, anti-asthmatic, expectorant, anti-allergic, anticoagulant, immunity enhancement, antipyretic and sedative, anti-tumor, inhibition of cardiac contraction and so on[4].Recently, Artemisia argyi essential oil (AO) is widely used in the therapy of chronic obstructive pulmonary disease (COPD) in traditional folk medicine of America, Indonesia and China because of its propertiesof relaxing bronchial smooth muscle. Pulmonary vascular remodeling refers to the dysfunction of cellproliferation, apoptosis, migration and adaptive changes of vascular wall structure under the stimulation of severalfactors, including vasoactive substances, growth factors and hemodynamic factors[1].
The currently used models of PH mainly include: bypass surgery between abdominal aorta and postcava induced PH[6,7]; chronic hypoxia-induced PH; monocrotaline (MCT) plus experimental lung resection induced pulmonary reconstruction[8]; and intraperitoneal or subcutaneous injection of MCT (60 mg/kg) induced PH[9].
MCT pyrrole will circulate along the bloodstream to the lung and do harm to endothelial cells in pulmonary vascular, thereby causing chronic inflammation. Inflammation plays an important role in MCT-induced PH, which fits the pathogenesis of PH.
2. Materials and methods
2.1. Reagents
MCT was purchased from BaiShikai Chemical Technology Inc. (Shanghai, China). Artemisia argyi essential oil was obtained from Zhejiang Institution of Chinese Medicine (Zhejiang, China), which contains volatile oils, flavonoids, eucalyptus terpenes, triterpenesand so on. Saline was provided by Kelun Pharmaceutical (Sichuan, China). Pentobarbital sodium was supplied by Xing Galaxy Chemical Co., Ltd. (Hubei, China). Formaldehyde solution was purchased from Mitaka Chemical Reagents Company (Zhejiang, China). Polyoxyethylene (20) sorbitanmonooleate (Tween-80) was obtained from Wenzhou Qingming Chemical Plant (Zhejiang, China). Heparin was provided by Benny Pharmaceutical and Biochemical (Changzhou, China). α-SMA monoclonal antibody was supplied by Abcam (San Francisco, CA, USA). NF-κB/p65 mAb was purchased from Cell Signaling Technology (Danvers, MA, USA). Bosentan was obtained from Allpack Group (Middlemore Lane West, United Kingdom).
2.2. Experimental animals
All animal experiments were approved by the Animal Care and Use Committee of Zhejiang University. A total of 80 Sprague-Dawley (SD) rats, including 40 males and 40 females, weighing (200±20) g were provided by the Shanghai Lab., Animal Research Center. Rats were housed under controlled conditions (temperature of 20–26 ºC, humidity of 40%–70%). Males and females were maintained separately and given free access to food and water under a 12-h light-dark cycle.
2.3. Study design
At the first day of the experiment, 60 SD rats received a single subcutaneous injection of MCT (60 mg/kg) to induce PH, while the other 20 rats received saline alone to serve as a normal control group. On day 2, the rats injected with MCT were randomly divided into three groups (n = 20/group), including model group (saline), bosentan group (0.1 g/kg) and AO group (0.1 g/kg). During the experiment, all the rats were intragastrically administrated with respective reagents once a day for 4 weeks. At the end of the experiment, the hemodynamic parameters, pathological injuries and related protein expressions in each group were analyzed.
2.4. Determination of hemodynamic parameters
Right-heart catheterization: a specially made PE50 catheter pre-filled with sodium heparin was inserted into the right ventricle (RV) through the jugular vein and the pulmonary artery after the rats were anesthetized. The systemic blood pressure (BP), mean pulmonary arterial pressure (mPAP) and right ventricular systolic pressure (RVP) were determined by a pressure transducer connected with recording system.
2.5. Determination of lung and right heart hypertrophy index
At the end of the experiment, the rats were sacrificed,and the lungs and hearts were collected. Saline was injected into the pulmonary arteries to wash out the blood. Hilars oflung was cut off, and lung wet weight (wW) was weighed.Lung index (LI) = lung wW/body weight (BW) × 100%.After the measurement, lungs were fixed with 4% paraformaldehyde for 10 h, followed by hematoxylin & eosin (HE) and immunohistochemistry (IHC) staining.
According to Monge et al.[5] and Julian et al.[6] left ventricle plus septum and right ventricle were isolated as follows: hearts were isolated, the right ventricle was carefully separated from the left ventricle plus septum (LV+S), and then the left ventricle plus septum (LV+S) was weighed. Ventricles were blotted dry and weighed separately to measure the right ventricle free wall weight (RV) in order to determine the index of right ventricle hypertrophy (RVHI). RVHI = [RV/(LV+S)] × 100%.
2.6. HE staining
Lung sections (5μm) were prepared after paraffin embedding, and then HE staining was carried out. Sections were examined under a light microscope. Morphological changes were observed, and inflammatory cells were identified according to morphology, location and staining characteristics of cells. The thickness of pulmonary artery was measured by using Leica Qwin software.
2.7. IHC staining
At the end of the experiment, 6-μm frozen sections of rat lungs were prepared and stored at –20 ºC for IHC staining. IHC staining was performed according to the instructions of HSP007/8 immunohistochemistry kit, and the specific experimental procedure was listed as follows: (1) slices were fixed with pre-chilled acetone for 10 min and washed with PBS for 2 min/times × 3 times; (2) slices were transferred into 0.01 mol/L citrate buffer (pH = 6.0) for antigen retrieval for 5 min, then cooled to room temperature, and washed with PBS for 2 min/times × 3 times; (3) the step (2) was repeated for three times, and the repaired slices were soaked into 3% H2O2–methanol solution for 10 min to remove endogenouscatalase and washed with PBS; (4) reagent A (blockingbuffer: goat serum) was added dropwisely until complete cover of the tissue sections, and then the slices were incubated at 37 ºC for 10 min; (5) each slice was added with an appropriate amount of antibodies (α-SMA 1:200, NF-κB/p65 1:800), incubated at 4 ºC overnight and then washed with PBS for 2 min/times × 3 times; (6) reagent B (biotinylated secondary antibody solution) was added to the sections until complete cover, then incubated at 37 ºC for 10 min and washed with PBS; (7) reagent C (HRP-labeled streptavidin working solution) was added to the sections until complete cover, then incubated at 37 ºC for 10 min and washed with PBS 2 min/times × 3 times; (8) DAB chromogenic reagent was added at room temperature for 5 min; (9) hematoxylin staining for 3 min, and rinsed with water to absorb excess floating color; (10) the sections were differentiated with ethanol hydrochloride and washed with water for 2 min; (11) the sections were dehydrated with alcohol (70%, 80%, 95% I, 95% II, 100% I, 100% II) and cleared with xylene (xylene, 10 min). Subsequently, the sections were mounted with neutral gum. The IHC staining was observed under a microscope to observe the staining intensity and scope of α-SMA and p65.
2.8. Statistical analysis
The data were expressed as mean±SEM. Significance between individual groups was analyzed by one-wayANOVA. A P value of <0.05 was considered as statistically significant. 
3. Results
3.1. The effect of AO on general conditions and mortality of rats
During the experiment, rats behaved normally in the mental state, food, water intake, fur outlook, action behavior, responsive action and urine amount.
The rats in the model group behaved normally in mental state, food and water intake at the early time. However, in the 3rd week, the rats became depressed, food intake was decreased with no change of water intake and responsive action, and the fur was luster but shedding when crawled. At the end of the experiment, most rats seemed shortness of breath and lost BW with dull fur and curled up with less dynamic.
Compared with the model group, rats treated with AO had normal mental state, water intake, and increasedfood intake. The fur was luster but shedding when crawled. The rats seemed to be more active and responsive with normal urine amount in the groups treated with AO. It was observed that rats in the AO group showed a transient mental apathetic and less stable gait when walking, which could be alleviated after about 20 min. No animals died accidently during the experiment.
3.2. Effects of AO on hemodynamic
Blood pressure (BP) was decreased, and the mean pulmonary arterial pressure (mPAP), mean right ventricularsystolic pressure (mRVP) and maximum right ventricular systolic pressure (max RVP) were significantly increased (P<0.05) in the model group compared with the normal control group. AO significantly reduced mPAP to 36.7±6.3 (compared with the model group, P<0.001) and mRVP to 27.6±6.1 (compared with the model group, P<0.001),while bosentan exerted the similar effects with 39.8±9.1 for mPAP and 28.2±7.9 for mRVP (P<0.05; P<0.01). In addition, AO reduced max RVP, but showed no statistical significance, while bosentan significantly lowered the max RVP (P<0.05) (Fig. 1).
 
 
Figure 1. Effects of AO on hemodynamic changes in rats (n = 20). BP (A), mPAP (B), mRVP (C) and maxRVP (D) levels in all groups. Control represents the normal group, MCT model represents the model group, and AO represents Artemisia argyi essential oil. Values are expressed as mean±SEM. #P<0.05 compared with normal group;*P<0.05, **P<0.01, ***P<0.001 compared with model group.
   
3.3. Effects of AO on lung wW and LI in rats
MCT significantly increased lung wW and LI compared with the normal control group (P<0.05). AO reduced wW (2.00±0.26 vs. 1.77±0.20) and LI (0.590±0.085 vs. 0.516±0.047) compared with the model group (P<0.01), while levels of wW and LI showed no significant change in the bosentan-treated group (Fig. 2).


Figure 2. Effects of AO on lung wW (wW) and LI in rats (n = 20).The lung wW (A) and LI (B) in all groups. LI = lung wW/BW×100%. Control represents the normal group, MCT model represents the model group, and AO represents Artemisia argyi essential oil. Values are expressed as mean±SEM. #P<0.05 compared with normal group; **P<0.01 compared with model group.
 
3.4. Effects of AO on right ventricular hypertrophy index in rats
Right ventricular free wall weight (RV) and right ventricular hypertrophy index (RVHI) were significantly higher in the model group compared with the normalcontrol group (P<0.05). AO-induced RV was improved with no significance, and the left ventricular septal weight (LV+S) declined compared with the normal control group (P<0.05). There was no significant effect of AO on RVHI compared with the model group. It showed no obvious improvement on heart damage caused by MCT in the bosentan group (Fig. 3).


Figure 3. Effects of AO on right ventricular hypertrophy index in rats (n = 20). RV (A), LV+S (B), RVHI (C) levels in all groups. Right ventricular hypertrophy index (RVHI) = [RV/(LV + S)] × 100%. Control represents the normal group, MCT model represents the model group, and AO represents Artemisia argyi essential oil. Values are expressed as mean±SEM. #P<0.05 compared with normal group; *P<0.05 compared with model group.
 
3.5. Effects of AO on lung pathological changes in rats
HE staining showed that pulmonary artery was regular and small in the normal control group. In the model group, smooth muscle cells in pulmonary artery were significantly proliferated with thickened wall of pulmonary vascular (P<0.001) (Fig. 4A). AO decreased the thickness of pulmonary vascular wall compared with the model group but showed no significance (Fig. 4B).


Figure 4.
Effects of AO on lung pathological changes in rats. HE staining of the lung (A) and the calculation of pulmonary vascular thickness (B) in all groups. Scale bar is 100 μm. Control represents the normal group, MCT model represents the model group, and AO represents Artemisia argyi essential oil. Values are expressed as mean±SEM. ###P<0.001 compared with normal group.
 
3.6. The effect of AO on the expression of NF-κB p65 in lung
IHC results revealed that p65 subunit of NF-κB was mainly expressed in the cytoplasm of bronchial epithelial cell (brown) with occasional expression in nucleus in lungs of normal rats. The expression of p65 in nuclear of bronchioles was significantly increased in the model group compared with the normal control group. AO reduced the p65 expression in bronchiolar epithelial cells compared with the model group (Fig. 5).


Figure 5.
The effect of AO on the expression of NF-κB p65 in lung.Antibody against NF-κB p65 (brown) was used in IHC staining of lung sections from MCT-induced PH, and nuclear staining was carried out by using hematoxylin staining (blue). Images were viewed at a magnification of 40× and 60×. Scale bar is 50 μm and 100 μm. Control represents the normal group, MCT model represents the model group, and AO represents Artemisia argyi essential oil.
 
3.7. The effect of AO on the expression of α-SMA in lung
IHC staining showed that α-SMA was rarely expressedin pulmonary artery in the normal control group. But its expression was significantly increased and pulmonary artery smooth muscle remodeled in the model group compared with the normal control group. AO reduced the α-SMA expression in lung artery compared with the model group (Fig. 6).


Figure 6
. The effect of AO on the expression of α-SMA in lung. Antibody against α-SMA (brown) was used in IHC staining of lung sections from MCT-induced PH, and nuclear staining was carried out by using hematoxylin staining (blue). Images were viewed at a magnification of 40× and 60×. Scale bar is 50 μm and 100 μm. Control represents the normal group, MCT model represents the model group, and AO represents Artemisia argyi essential oil. 
4. Discussion
4.1. The establishment of PH model
In our study, 4 weeks after single intraperitoneal injection of MCT, pulmonary artery thickened, luminal stenosis, perivascular infiltrated with inflammatory cells, and right ventricular hypertrophy index (RVHI) and mean pulmonary arterial pressure (mPAP) were significantly higher compared with normal rats, suggesting that PH model was successfully established.
4.2. Effects of AO and bosentan on PH in rats
Studies have shown that enhanced pulmonary vasoconstrictionis caused by endothelial dysfunction. The characteristic of endothelial dysfunction is the out of balance between vasodilator such as nitric oxide (NO) and vaso-excitor such as endothelin vasoconstrictorbetween-1 (ET-1). A variety of factors stimulate theproliferation of smooth muscle cells in pulmonary vascular (vascular smooth muscle cell, VSMC). The increased extracellular matrix components, the transformation from the non-muscular artery to muscular artery, the swelling and hypertrophy of endothelial cell eventually lead to pulmonary vascular remodeling[1013]. Bosentan, a non-selective ET receptor inhibitor, is used to treat PH in clinic. Studies have shown that elevated circulating ET-1 level is observed in PH patients, and it is positively related to increased pulmonary resistance. The inhibitor of ET-1 can improve pulmonary vascular remodeling to treat PH. In our study, the results showed that bosentan significantly improved levels of mPAP, mRVP and max RVP. Artemisia argyi is frequently used for the treatment of diseases, such as infections by fungi, bacteria and viruses, inflammation, cancer, malaria, asthma and so on. In our study, AO improved wW, LI, mPAP, mRVP, the right ventricular hypertrophy index and remodeling of pulmonary artery. Therefore, we, for the first time, reported that AO exerted beneficial effect for PH.
4.3. Effects of AO on the expressions of α-SMA and NF-κB p65 in lung
α-SMA is a protein capable of contraction and antigenicity stability, and it is a marker of vascular smooth muscle. The increased expression of α-SMA in pulmonary artery smooth muscle and increased thickness of small pulmonary arteries in the group injected with MCT indicated the proliferation of pulmonary artery smooth muscle cells and reconstructionof pulmonary artery. Therefore, AO exerted an anti-inflammatory effect, improved microcirculation and reduced vascular disease by inhibiting the expression of α-SMA. NF-κB is a dimeric complex consisting of two Rel protein family members p65 and p50, and p65 subunit presents in lung[14,15]. When rested, NF-κB and its inhibitor IκB (inhibitory κB) bind together in cytoplasm with no biological activity. However, when cells are stimulated by a variety of cytokines, viruses, lipopolysaccharide and so on, IκB is degraded and separated with NF-κB, and NF-κB is translocated into nucleus to bind at the κB sites in DNA to activate the transcription of target genes[16]. Gene transcription of cytokines, adhesion factors and chemokines plays an important role in the inflammatory response, and it is closely related to pulmonary inflammatory diseases, such as bronchial asthma and acute respiratory distress syndrome[1618]. Muraoka et al. have reported that hypoxia can stimulate the activation of NF-κB[19]. Qilin Ao et al. have reported that hypoxia activates the NF-κBin pulmonary vascular smooth muscle cells, and the activation of NF-κB inhibits apoptosis of pulmonary vascular smooth muscle cells and promotes proliferation, leading to pulmonary vascular remodeling[20]. NF-κB plays an important role in the gene transcription of ET-1 by increasing the production of ET-1 in vascular endothelial cells to cause vasoconstriction[9,2123]. Theexpression of NF-κB in MCT-induced PH in lung is also increased[9]. The results showed that the expression of NF-κB p65 was increased after MCT injection, and AO significantly inhibited their expressions. Therefore, the specific mechanisms underlying the beneficial effect of essential oil on PH might be the inhibition of the expression of NF-κB p65 and reduction of a variety of inflammatory mediators, enzymes or receptor expressions.
5. Conclusions
We identified that AO could slow the process of PH induced by MCT in rats. Further study found that AO reduced the expressions of NF-κB p65 and α-SMA in lung, which might be involved in inhibiting pulmonary vascular disease, relieving lung injury and reducing PH. Therefore, considering the complex constituent of herbal medicine, it is necessary to further explore the active ingredient, dose range, period and mechanisms of AO on PH.
Acknowledgements
This work was supported by the Natural ScienceFoundationofZhejiangProvinceofChina (Grant No. LQ14H280003), the TCM Project of Zhejiang Province(Grant No. 2011ZA006), andthe Chinese Integrative MedicineFoundation of Zhejiang Province (Grant No. 2013LYSX020), Zhejiang Provincial Program for the Cultivation of High-level Innovative Heath Talents (Ping Huang), Zhejiang 151 Elites Project, the second level (Ping Huang).
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艾叶油对肺动脉高压大鼠的干预研究
应茵1,2, 孙云峰1, 李宏春1, 羊波1, 王晖1, 吴青梅1, 黄萍3*
1. 浙江省立同德医院, 浙江 杭州 310012
2. 浙江省中医药研究院, 浙江 杭州 310007
3. 浙江省肿瘤医院, 浙江 杭州 310022  
摘要: 本研究建立野百合碱(MCT)诱导的肺动脉高压大鼠动物模型, 观察艾叶油对大鼠肺动脉高压的治疗作用。将80SD大鼠随机分为正对照组、模型组、波生坦组(0.1 g/kg)和艾叶油组(0.1 g/kg)。造模给药30天后, 测定血流动力学参数、肺指数(LI)和右心肥厚指数(RVHI)。肺组织切片进行苏木素-伊红(HE)染色, 光镜下观察肺组织形态学改变。采用免疫组化法观察大鼠肺组织相关蛋白表达的变化。结果显示, 模型组大鼠肺动脉管壁明显增厚, 管腔明显狭窄, 管周有炎性细胞明显浸润, 且模型组大鼠右心肥厚指数(RVHI)及平均肺动脉压(mPAP)较正常对照组明显升高。艾叶油对肺动脉高压大鼠平均肺动脉压、平均右心室压、右心肥厚指数、肺指数和肺动脉的重构均有不同程度的改善; 大鼠的左心室加心室间隔重量(LV+S)显著降低。在机制研究中, 行野百合碱注射后, NF-κB p65α-SMA的表达增加, 在相同时间段内模型组和药物治疗组差异有统计学意义, 表明艾叶油可以显著抑制两者的表达。研究表明艾叶油通过下调肺组织NF-κB p65α-SMA的表达, 抑制肺血管的病变、缓解肺组织损伤、发挥抗肺动脉高压作用。 
关键词: 艾叶油; 肺动脉高压; NF-κB; α-SMA
 
  
Received: 2017-11-16, Revised: 2018-01-17, Accepted: 2018-02-10.
Foundation items: TheNatural ScienceFoundationofZhejiangProvinceofChina (Grant No. LQ14H280003), the TCM Project ofZhejiang Province(Grant No. 2011ZA006), andthe Chinese Integrative MedicineFoundation of ZhejiangProvince (Grant No. 2013LYSX020), Zhejiang Provincial Program for the Cultivation of High-level Innovative Heath Talents (Ping Huang), Zhejiang 151 Elites Project, the second level (Ping Huang).
*Corresponding author. Tel.: +86-13858024909, E-mail: hangzhou_hp@163.com      
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