Effects of moxibustion at bilateral Feishu (BL13) and Xinshu (BL15)combined with benazepril on myocardial cells apoptosis index and apoptosis-related proteins cytochrome c and apoptosis-inducing factor in rats with chronic heart failure

WANG Wei,LI Qingling,MA Qiang,XIA Ran,GAO Bing,WANG Yi,WANG Jing

WANG Wei,LI Qingling,College of Chinese Medicine,Anhui University of Chinese Medicine,Hefei 230012,China

MA Qiang,XIA Ran,GAO Bing,Graduate school,Anhui University of Chinese Medicine,Hefei 230012,China

WANG Yi,College of Acupuncture and Massage,Anhui University of Chinese Medicine,Hefei 230012,China

WANG Jing,Xin’an Medical Key Laboratory;Anhui University of Chinese Medicine,Hefei 230012,China

Abstract OBJECTIVE:To observe the effects of moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril on myocardial cells apoptosis index,the expression levels of apoptosis-related proteins cytochrome c (Cyt-C) and apoptosis-inducing factor (AIF)in chronic heart failure (CHF) rats.METHODS:Sixty-five rats were randomly divided into normal group (n=10) and model-I group (n=55).After modeling,CHF rats in model-I group were divided into model group,moxibustion group,benazepril group,moxibustion plus benazepril group (abbreviated as aibei group,the same below),10 rats in each group.Echocardiogram index was examined by echocardiography. Hemodynamic indices weremeasured by rat cardiac function meter.Serum B-type brain natriuretic peptide (BNP) was detected by enzymelinked immunosorbent assay.Myocardial cells apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining.Pathological changes of myocardial tissues were observed by hematoxylin and eosin staining.The expression levels of Cyt-C and AIF in myocardial tissues were detected by Western blot.RESULTS:Compared with normal group,ejection fraction and left ventricular diameter shortening rate in model-Ⅰ group were significantly reduced,myocardial cells of rats in model group exhibited unclear transverse striations,cells swellings and vacuoles,cardiac functions were deteriorated,serum BNP level,myocardial cells apoptosis index,and the expression levels of Cyt-C and AIF were significantly increased.Compared with model group,myocardial cells of rats in moxibustion group,benazepril group,and aibei group were dyed more evenly,muscle fibers were arranged relatively neatly,cardiac functions were improved,serum BNP level,myocardial cells apoptosis index,and the expression levels of Cyt-C and AIF were significantly decreased. Compared with aibei group,cardiac functions were worsened,myocardial cells apoptosis index,and the expression levels of Cyt-C and AIF were increased.CONCLUSION:Moxibustion at bilateral Feishu (BL13)and Xinshu (BL15) combined with benazepril could improve CHF better than moxibustion at bilateral Feishu(BL13) and Xinshu (BL15) or benazepril alone.The mechanisms might be that they can inhibit the expressions of Cyt-C and AIF,and inhibit the apoptosis of cardiomyocytes.

Keywords:heart failure;moxibustion;point BL13 (Feishu);point BL15 (Xinshu);cytochromes c;apoptosis inducing factor

1.INTRODUCTION

Chronic heart failure (CHF) is a chronic progressive disease caused by ventricular systolic and diastolic dysfunction,which is common at the end of many cardiovascular diseases.1,2According to statistics,the five-year mortality rate of patients with CHF is as high as 50%-80%,and nearly 300 000 people die from CHF every year in the world.3More and more evidences indicate the importance of apoptosis in the development of CHF.Apoptosis has gradually become the"therapeutic target" of CHF.4Cytochrome c (Cyt-C) is a cytochrome oxidase that is located outside the mitochondrial inner membrane.As an apoptotic factor,Cyt-C plays an important role in the initiation of apoptosis.5,6Apoptosis-inducing factor (AIF) is a type of conservative flavoprotein that exists between the inner and outer mitochondrial membranes.In the process of apoptosis,AIF is not only a starting factor but also a direct effector,which can induce apoptosis.7At present,acupuncture and moxibustion have significant advantages in the treatment of CHF.8-10In the previous studies,we have found that moxibustion at Feishu (BL13)and Xinshu (BL15) can effectively improve cardiac function and alleviate CHF.11-14This study focused on the effects of moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril on myocardial cells apoptosis index,the apoptosis-related proteins Cyt-C and AIF in CHF rats,in order to provide an important basis for the early clinical prevention of CHF and find more effective treatments.

2.MATERIALS AND METHODS

2.1.Experimental animals

Total of 65 healthy male adult Sprague-Dawley rats[(250 ± 10) g] (three-month-old,specific pathogen-free(SPF) grade,Certificate No.SCXK [Anhui] 2017–006)were provided by the Experimental Animal Center of Anhui University of Chinese Medicine (Hefei,China),reared at Institute of Traditional Chinese Medicine and Meridians of Anhui University of Chinese Medicine(Hefei,China).Rats were maintained at (25 ± 2) ℃ and the humidity range of 55%-70%.All animals were cared for in accordance with the policies and guidelines released by the Ethic Committee for Animal Use in Anhui Science Institute of Traditional Chinese Medicine.This study was approved by experimental animal ethics committee of Anhui University of Chinese Medicine(Hefei,China).

2.2.Main instruments and reagents

EA (61 Biotechnology Co.,Ltd.,Beijing,China);Microplate reader (Thermo Technology,Massachusetts,USA);Centrifuge (Xiangyi Laboratory Instrument Development Co.,Ltd.,Changsha,Hunan,China);Philips SONOS 5500 Ultrasonic Instrument (Philips,the Netherlands);Rat cardiac function meter (Yuyan Scientific Instrument Co.,Ltd.,Shanghai,China);BX53 optical microscope (OlymPUS Co.,Ltd.,Shenzhen,Guangdong,China).

Doxorubicin hydrochloride (10 mg/branch,Fude Pharmaceutical Co.,Ltd.,Datong,Shanxi,China);Benazepril powder (5 g,Soleil Technology Co.,Ltd.,Beijing,China);Moxa stick (10 mm × 120 mm,Nanyang Wolong Hanyi Moxa Factory,Nanyang,Heinan,China) .Rabbit polyclonal antibody Cyt-C,rabbit polyclonal antibody AIF,rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH),enzyme-linked immuneosorbent assay kit (Elirat Biotechnology Co.,Ltd.,Wuhan,Hubei,China);TUNEL apoptosis detection kit,(Biyuntian Biotechnology Co.,Ltd.,Shanghai,China);X-ray film (kodak,Berlin,German).

2.3.CHF model

The CHF rat model was built by intraperitoneal injection of Doxorubicin hydrochloride (DOX) alternately every other day.15DOX injection (1 mg/mL) was prepared with 0.9% sodium chloride solution at the dose of 1 mg/kg on the 2th and 4th day,2mg/kg on the 6th and 8th day,3 mg/kg on the 10th and 12th day,4 mg/kg on the 14th and 16th day.Ejection fraction (EF) ≤ 60% and left ventricular diameter shortening rate (FS) ≤ 28.33% were taken as criteria for the success of CHF model.16

2.4.Animal grouping

Rats were randomly divided into 2 groups by random number table method:normal group (n=10) and model-I group (n=55).After modeling,three rats were randomly selected from normal group and model-I group for model identification.CHF rats were further randomly divided into 4 groups by random number table method:model group,moxibustion group,benazepril group,moxibustion plus benazepril group (abbreviated as aibei group,the same below),with 10 rats in each group.

2.5.Intervention methods

Normal group and model group:rats were fixed on rat platforms for 20 min per day without treatment.Moxibustion group:Rats were fixed on moxibustion table.According to the standard acupoints map of rats in Experimental Acupuncture Science17and the physiological structure characteristics of rats,18with the position of the highest point of spinous process in the second thoracic vertebra (T2) as the reference,it was determined the positions of Feishu (BL13) and Xinshu(BL15) in rats were 7 mm beside the third thoracic vertebra (T3) and the fifth thoracic vertebra (T5),respectively.The mild moxibustion was performed for 20 min at the location 3 cm directly above Feishu (BL13)and Xinshu (BL15) with moxa sticks.Benazepril group:Benazepril (2 mg/kg) was intragastrically administered to rats.Aibei group:Benazepril (2 mg/kg) was applied to rats by gavage,followed by mild moxibustion for 20 min.Intervention methods were taken once a day for three weeks.

2.6.Observation index

Echocardiogram indexes:rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital(0.1 mL/100 g) and fixed in the dorsal position.Ultrasonic Instrument detected EF,FS for model identification.After intervention,Ultrasonic Instrument detected EF,FS,stroke volume (SV),and heart rate (HR).Hemodynamic indices:rats were fixed in the dorsal position and given a polystyrene catheterization with heparin to the left ventricle of the heartviathe right carotid artery.Rat cardiac function meter detected cardiac output (CO),left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure (LVEDP).Enzyme-linked immunosorbent assay for detection of serum BNP:after 30 min of blood sampling,blood was centrifuged (3000 r/min,10 min) to collect serum.Levels were determined and calculated according to the kit instructions.

TUNEL staining:left ventricular myocardial tissues were immersed (10% neutral formalin solution),followed by embedding and preparation of tissue sections (4 μm-thick).Normal myocardial cells were blue,apoptotic cells were green (using 200-fold light microscope).Myocardial cells apoptosis index (%)=(number of apoptotic positive cells/total number of myocardial tissue cells) × 100%.

HE staining:left ventricular myocardial tissues were immersed (10% neutral formalin solution),followed by embedding and preparation of tissue sections (5μmthick).HE staining was used to observe the change of myocardial structure (using 400-fold light microscope).Western blot for Cyt-C and AIF in myocardial cells:total 50 mg of left ventricular myocardial tissues were dissolved.The liquid was centrifuged (12 000 r/min,10 min).Supernatant was subpackaged in a 1.5mL centrifuge tube.After electrophoresis,blocking,the antibodies against Cyt-C and AIF were diluted to 1∶2000 in a water bath of boiling water for 10 min.The secondary antibodies were diluted to 1 ∶3000 and incubated at 37 ℃ for 2 h.The gray values were analyzed after color exposure and sun drying,and the expression levels were expressed as the ratio of Cyt-C to GAPDH and the ratio of AIF to GAPDH.

2.7.Statistical analysis

All data were expressed as the mean ± standard deviation.Data from multiple samples were statistically analyzed by one-way analysis of variance,least significant difference analysis.All results were considered statistically significant atP<0.05,<0.01.Results were graphed and analyzed by IBM SPSS Statistics17.0 (IBM,Chicago,IL,USA).

3.RESULTS

3.1.Model identification

During modeling,rats lost weight,became depressed,and body hairs fell off.After modeling,rats were fed normally for one week,then identified.Compared with normal group,EF and FS in model-I group were significantly reduced (P<0.01),and the cardiac chamber was significantly enlarged,which met the criteria for CHF rat model16(Figure 1).The survived rats were detected in the final experiment:normal group (n=10),model group (n=6),moxibustion group (n=8),benazepril group (n=7) and aibei group (n=9).

Figure 1 Model identification

3.2.Echocardiography results in different groups

Compared with normal group,EF and FS in model group were significantly decreased (P<0.01),and SV and HR were significantly increased (P<0.01).Compared with model group,EF and FS in moxibustion group,benazepril group and aibei group were significantly increased (P<0.01),SV and HR were significantly decreased (P<0.01).Compared with aibei group,EF in moxibustion group and benazepril group was decreased(P<0.05),SV and HR were significantly increased (P<0.01) (Figure 2).

Figure 2 Comparisons of echocardiogram indexes in different groups

3.3.Hemodynamic indices in different groups

Compared with normal group,CO and LVSP in model group were significantly decreased (P<0.01),and LVEDP was significantly increased (P <0.01).Compared with model group,CO and LVSP in moxibustion group,benazepril group,and aibei group were significantly increased (P <0.01),LVEDP was significantly decreased (P <0.01).Compared with aibei group,CO and LVSP in moxibustion group and benazepril group were significantly decreased (P <0.01),LVEDP was significantly increased (P <0.01) (Table 1).

Table 1 Comparisons of hemodynamic indices in different groups (x ± s)

3.4.Serum BNP level in different groups

Compared with normal group [serum BNP level:(162.14±7.94) pg/mL],model group [serum BNP level:(512.22±10.37) pg/mL] was significantly increased (P <0.01).Compared with model group,moxibustion group [serum BNP level:(219.31±12.14) pg/mL],benazepril group[serum BNP level:(221.51±4.96) pg/mL],and aibei group [serum BNP level:(208.16±8.91) pg/mL] were significantly reduced (P <0.01).

3.5.Myocardial cells apoptosis in different groups

Compared with normal group,myocardial cells apoptosis index in model group was significantly increased(P <0.01).Compared with model group,myocardial cells apoptosis index in moxibustion group,benazepril group and aibei group was significantly reduced (P <0.01).Compared with aibei group,myocardial cells apoptosis index in moxibustion group and benazepril group was significantly increased (P <0.01) (Figure 3).

Figure 3 Comparisons of myocardial cells apoptosis in different groups

3.6.Pathological changes of left ventricular myocardial tissue in different groups

In normal group,the myocardial cells were in normal size,with clear edge staining and neatly arranged myocardial fibers.In model group,the myocardial cells were swollen,the nuclei were hyperchromatic,and the cytoplasms were changed into vacuoles,the myocardial fibers were broken,dissolved,and arranged disorderly.Compared with model group,the myocardial cells in moxibustion group,benazepril group and aibei group were dyed more evenly,and the myofibers were arranged relatively neatly (Figure 4).

3.7.Expressions of Cyt-C and AIF in different groups

Compared with normal group,the expression levels of Cyt-C and AIF in model group were significantly increased (P <0.01).Compared with model group,the expression levels of Cyt-C and AIF in moxibustion group,benazepril group and aibei group were significantly reduced (P <0.01).Compared with aibei group,the expression levels of Cyt-C and AIF in moxibustion group and benazepril group were increased (P <0.01,<0.05)(Figure 5).

Figure 4 Pathological changes of left ventricular myocardial tissue in different groups

Figure 5 Comparisons of the expressions of Cyt-C and AIF in different groups

4.DISCUSSION

In Traditional Chinese Medicine,it is believed that the pathological nature of CHF belongs to the deficiency in origin and excess in superficiality,and the deficiency inQi,blood,YinandYangis the root cause,with blood stasis,phlegm,water retention andQistagnation as the criteria.Therefore,tonifyingQi,warmingYang,activating blood,and draining water should run through the whole treatment process of CHF.19Modern studies have confirmed that Xinshu (BL15) can enhance myocardial contractility,improve cardiac output and further restore cardiac function.20Feishu (BL13) can obviously improve lung function and increase lung ventilation volume.The lung controlsQi,and the heart controls blood.The relationship between the lung and heart is the relationship betweenQiand blood,so Feishu(BL13) can also be used to treat cardiovascular diseases.11-14Moxibustion,as one of the important therapies in traditional Chinese medicine for diseases,has the advantages of simple operation and less side effects.Studies in China and abroad have shown that moxibustion has a good regulatory effect on the immune system,cardiovascular system and body metabolism.11-14,21Benazepril,an angiotensin converting enzyme inhibitor,has the effects of reducing cardiac load,improving cardiac function,delaying ventricular remodeling,and optimizing myocardial metabolism,thus playing an effective role in the treatment of CHF.22-24Xieet al25demonstrated benazepril could further improve cardiac function by inhibiting excessive apoptosis of cardiomyocytes and ventricular remodeling.

In recent years,apoptosis has been a hot topic in the study of CHF.Studies have shown a positive correlation between apoptosis and the severity of CHF.Apoptosis of cardiomyocytes can cause a large decrease in cardiomyocytes,leading to the loss of heart pump function.After apoptosis,cells near apoptosis rearrange to undergo myocardial remodeling and lead to decreased cardiac function.26-28Studies have shown that Cyt-C and AIF coexist in mitochondria.When the mitochondria is stimulated by apoptotic signals,Cyt-C and AIF would be released from the mitochondria to initiate apoptosis.Under the action of apoptotic stimulation signals,Cyt-C is released into the cytoplasm through the mitochondrial outer membrane and combined with apoptosis-related factor-1,cysteine family protease-9 precursor,and deoxyadenosine triphosphate to form apoptotic bodies.Furthermore,by activating cysteine family protease-3,it can inhibit deoxyribonucleic acid (DNA) repair enzyme activity and activated nuclear endonuclease,leading to apoptosis.Under the action of apoptotic stimulation signals,AIF can be released and translocated into the nucleus,causing chromosome nuclear peripheral agglutination and large fragment fragmentation of DNA,leading to apoptosis.29-33The research conducted by Nagakannanet al34demonstrated the expressions of Cyt-C and AIF could promote excessive apoptosis.However,we also found during the development and treatment of CHF,compared with normal group,the expression levels of Cyt-C and AIF in rats in model group,moxibustion group,benazepril group and aibei group were increased,indicating the excessive apoptosis of cardiomyocytes caused by Cyt-C and AIF might be the important reasons for CHF.

Because of the slow onset and long course of CHF,CHF is often accompanied by left ventricular remodeling and functional impairment,accompanied by the decrease of EF,FS,CO and LVSP,the increase of SV and LVEDP,and the acceleration of HR.35,36In addition,there will be swelling of cardiomyocytes,hyperchromatism of nuclei,alteration of cytoplasmic vacuolation,rupture and dissolution of myocardial fibers as well as disordered arrangement in myocardial tissues.36In this experiment,after treatment,EF,FS,CO and LVSP in CHF rats were increased,while SV,HR and LVEDP were decreased,myocardial cells in moxibustion group,benazepril group and aibei group were dyed evenly,and the muscle fibers were arranged neatly,indicating that CHF rat cardiac function was improved after treatment.Studies have confirmed serum BNP level is positively correlated with the severity of heart failure,which can be used to diagnose heart failure and determine the severity and prognosis of heart failure based on its level.37In this study,we found after treatment,serum BNP level in moxibustion group,benazepril group and aibei group was significantly reduced,indicating the treatment was effective.

This study showed that moxibustion at bilateral Feishu(BL13) and Xinshu (BL15) combined with benazepril in the treatment of CHF rats had a better effect on the improvement of cardiac function than moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) or benazepril alone,which might be related to the inhibition of myocardial cells apoptosis by inhibiting the expressions of apoptosis-related proteins Cyt-C and AIF in myocardial tissues.