Hui-ling Liao , Qian Ma , Ling Fu

1Affiliated Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, China, 2 Chongqing Traditional Chinese Medicine Hospital, Chongqing, China.

Background

Essential hypertension (EH) is one of the important diseases threatening human health. Although the research on the pathogenesis and antihypertensive drugs has developed rapidly, the damage of heart, brain, kidney and other organs related to hypertension has not been well controled. The study found that vascular remodeling is the pathological basis of heart, brain and other organs damage caused by hypertension. Therefore, studying the development of hypertension vascular remodeling and reversing vascular remodeling are the key factors in the prevention and treatment of hypertension. This study focused on the effects of Zhilong Huoxue Tongyu Capsule (ZLHXTY) and its decomposed formula on vascular remodeling in thoracic aorta in rats with renal hypertension, in order to further explore its anti-vascular remodeling mechanism in rats with renal hypertension.

Materials

Experimental animals

one hundred and ten male healthy wistar rats weighing 200 g ± 20 g, clean grade, 6 weeks old, provided by animal experiment center of Southwest Medical University (Chongqing, China, license No.: SYXK(Chuan) 2013-063).

Experimental drugs

Huangqi Granules (batch number: 1410072), Shuizhi Granules (batch number:1311018), Dilong Granules(batch number: 1409121), Daxueteng Granules (batch number:1408130) and Guizhi Granules (batch number:1503081) were purchased from Sichuan New Green Pharmaceutical Technology Development Co. LTD.Captopril Tablet was purchased from Harbin Pharmaceutical Group Co., Ltd General Pharm. Factory(batch number: 140241).

Common solutions

(1) 1% sodium pentobarbital: mixed with distilled water.(2) Gavage Fluid of Captopril Group: Cartopril tablet 25 mg was ground into fine powder, mixed with sterile water 96 ml, and prepared into a solution containing crude medicine (0.331g/mL); (3) Gavage solution for the Whole prescription group: 12 g Huangqi, 2 g Shuizhi, 9 g Dilong,9 g Daxueteng and 5 g Guizhi granule were added and mixed with 98 ml sterile water, then the solution containing crude medicine (0.274 g/mL) was prepared. (4)Gavage solution for the Benefiting qi Group: take 12 g Huangqi granule and add 123 ml sterile water to mix, and prepare the solution containing crude medicine(0.089g/mL); (5) Warming meridians Group: 5 g Guizhi and 9 g Daxueteng granule were taken and mixed with 121 ml sterile water to prepare a solution containing crude medicine (0.104g/mL); (6) Promoting blood circulation for removing blood stasis Group: 2 g Shuizhi and 9 g Dilong granule were added into 124 ml sterile water and mixed to form a solution containing crude medicine (0.081g/mL).

Method

Establish a rat model of renal hypertension

After 3 days of adaptive feeding to 110 male Wistar rats,basal blood pressure was measured in all rats using ZH-HX-Z non-invasive blood pressure measurement system. Each rat was measured 5-10 times and the mean value was taken. After 24 hours of fasting and water incontinence, 12 rats in the Normal group and the Sham-operation group were randomly selected by random sampling method. The rest were surgical groups. Surgical procedure: Intraperitoneal injection of 1% pentobarbital sodium was administered to anesthetized rats and the dosage was calculated as 45 mg/kg. After the rats were anesthetized and fixed, the abdomen was prepared,disinfected, and the abdominal median line was cut open.The left renal artery was obtuse, and the acupuncture needle was placed parallel to the long axis of the artery.After the surgical suture was tightened, the acupuncture needle was pulled out to form the narrowing of the left renal artery, and then the abdominal cavity was sutured. 4 x 104U of penicillin was used to prevent infection 3 days after surgery. Blood pressure was reassessed at 8-12 am at the end of the 2 weeks after surgery. According to the high blood pressure rat standard commonly adopted in foreign countries and the domestic research standard of Gu Deguan, it is successful to make model that rats with systolic pressure higher than 150 mmHg and an increase of 20mmHg compared to preoperative ones [7,8]. Only the left renal artery was isolated in the Sham-operation group, but no ligation was performed, the other conditions are the same as the Model group. In the 7th week, one rat was selected for execution by simple random sampling in Normal group, Sham-operate group and Model group. After HE staining of the thoracic aorta,no abnormal pathological changes were observed in the Normal and Sham-operated rats. The rats in the model showed thickening of the mesangial membrane, obvious proliferation of mesangial smooth muscle cells and disordered arrangement. MT, LD, MT/LD were used as criteria for model evaluation and pre-treatment of drug intervention.

Animal grouping and administration

Ten rats were randomly selected as the Normal group,and 10 rats were used as the Sham-operation group. Both groups were given distilled water 10 ml/kg×d. Sixty models of operate successful rats were screened and randomly divided into Model group, Captopril group,Whole prescription group, Benefiting qi group, Warming meridians group and Promoting blood circulation for removing blood stasis group, 10 in each group. Only 10 ml/kg×d of distilled water was given to the Model group,and 10 ml/kg×d of corresponding drugs was given to the other groups. It was administered once a day at 8 am every day for 4 weeks. The rats were weighed once a week, and the dose was calculated according to the body weight. All rats were kept in a ventilated, thermostatic room in separate cages, 5 in one cage, drank freely and eat normally.

Method of blood pressure measurement

1 Heat up rat tail: after the rat is fixed, the rat tail is heated by a hair dryer, and the skin of the rat tail is slightly red. 2 Measurement of rat blood pressure: The pressure pulse is placed at the base of the rat tail, and the pulse transducer is attached to the upper 1/3 of the side of the rat tail. After the rats were quiet, MD 3000 system was used for measurement. Adjust the amplifier so that the pulse wave amplitude is 2-3 cm, use the inflatable ball to pump air until the pulse wave disappears and continue to increase by 30mmHg, then slowly release air to reduce pressure to 0. The pressure corresponding to the first wave at this time is called the systolic pressure.

Statistical analysis

This experiment used SPSS 22.0 software for statistical analysis. All measurement data are expressed by mean ±SD. The experimental data are in normal distribution by statistical test. The data between groups are consistent with the homogeneity of variance, so one-way analysis of variance is used to evaluate the differences between groups. And the LSD test was used for the pairwise comparison. The test standard was α =0.05, means P<0.05 was statistically significant).

Results

The effect of drugs on blood pressure

Baseline blood pressureThere was no significant difference of in rats between the groups.

Blood pressure of rats 2 weeks after operationThere was no significant difference in blood pressure between the Normal group and the Sham-operation group (P >0.05). The Model group was significantly higher than the Normal group and the Sham-operation group (P < 0.05).The blood pressure values of the group all exceeded 150 mmHg and were 20 mmHg higher than the baseline blood pressure. There was no significant difference in blood pressure between the Model group and each treatment group (P > 0.05).

Blood pressure values of rats at 6 weeks after surgeryThere was no significant difference in blood pressure between the Normal group and the Sham-operation group(P > 0.05). The Model group was significantly higher than the Normal group and the Sham-operation group(P<0.05). There was no significant difference in blood pressure between the Model group and each treatment group and between the treatment groups (P > 0.05).

Blood pressure values of rats after 4 weeks of treatmentThere was no significant difference in blood pressure between the Normal group and the Sham-operation group (P > 0.05), and they were clearly lower than the other groups (P < 0.05). The Model group was obviously higher than the Normal group, the Sham-operate group and the treatment groups (P < 0.05);The decrease of Captopril group was not significant compared with the Whole prescription group (P > 0.05),and was significantly lower than that of the remaining treatment group (P < 0.05).; The decrease in the Benefiting qi group was not significant compared with the Promoting blood circulation for removing blood stasis group (P > 0.05), which was visibly lower than that in the Warming meridians group (P < 0.05). (Table 1 and Figure 1)

Table 1. The changes of blood pressure of rats before and after treatment each

Table 1. The changes of blood pressure of rats before and after treatment each

Note：▲Compared with Normal group and sham-operate group (P < 0.05); ★Compared with model group (P < 0.05);◆Compared with captopril (P < 0.05); ▼Compared with Benefiting qi group (P < 0.05); ++ Compared with Warming meridians group (P < 0.05); ●Compared with Promoting blood circulation for removing blood stasis group (P < 0.05).1 Normal group; 2 Sham-operate group; 3 Model group; 4 Captopril group; 5 Benefiting qi group; 6 Warming meridians group; 7 Promoting blood circulation for removing blood stasis group

Groups Cases(n) Baseline blood pressure(mmHg)2 weeks after surgery(mmHg)6 weeks after surgery (mmHg)4 weeks after therapy(mmHg)1 10 107 ± 9.02 106±11.90 105±9.09 103±9.12 2 10 110±7.36 108±9.23 107±8.17 107±6.70 3 10 110±11.42 165±6.70▲ 163±7.57▲ 164±7.68▲◆▼●4 10 109±4.90 168±10.75▲ 167±9.86▲ 126±7.98▲★▼●++5 10 106±10.56 165±6.35▲ 162±7.17▲ 135±10.07▲★●++6 10 108±9.88 163±8.84▲ 161±7.78▲ 144±7.66▲★◆++7 10 108±11.53 166±10.38▲ 165±10.01▲ 157±10.60▲◆▼8 10 103±9.39 166±9.49▲ 164±7.24▲ 152±8.16▲★◆

Figure 1. The changes of blood pressure of rats before and after treatment each

Effects of drugs on the expression of NF-κB protein in thoracic aorta of rats with renal hypertension(Immunohistochemistry)

Under electron microscope, brown-yellow particles were positive for NF-κB protein, mainly distributed in vascular smooth muscle. Small amounts of brown-yellow granules were observed in the Normal group and the Sham-operated group. A large number of brown-yellow granules can be seen in the Model group. After treatment,the expression of NF-κB in the Captopril group and the Whole prescription group was significantly reduced compared with the Model group, and the and temperature were significantly reduced, while that was slightly decreased in Benefiting qi group, Warming meridians group and Promoting blood circulation for removing blood stasis group compared with Model group. The Captopril group was less than the Whole prescription group. Compared with Promoting blood circulation for removing blood stasis group, the expression of NF-κB in benefiting qi group was visibly decreased, which was similar to that in Warming meridians group. (Figure 2)

Figure 2. The immunohistochemical staining images of NF-κB of rat thoracic aorta each (×400)

Comparison of average Optical Density (IOD) of NF-κB positive expression in rat thoracic aortic smooth muscle

The difference in IOD between the Normal group and the Sham-operated group was not statistically significant (P >0.05), and was significantly lower than that of the other groups (P < 0.05). The Model group was obviously higher than the treatment group (P < 0.05). Compared with the rest treatment group, Captopril group significantly decreased (P < 0.05). The Warming meridians group was higher than the Whole prescription group (P < 0.05), lower than the Promoting blood circulation for removing blood stasis group (P > 0.05),and not significantly lower than the Benefiting qi group(P > 0.05). (Table 2 and Figure 3)

Analysis of the causes of death in rats

In the experiment, there were 8 deaths rats, 2 of which died of anesthesia accident,1 died of intraoperative renal artery rupture, 1 died of an intragastric accident, 1 died of a stroke, and 1 died of suffocation when measuring blood pressure

Table 2. NF-κB expression of rat aortic smooth muscle each

Table 2. NF-κB expression of rat aortic smooth muscle each

Note：▲Compared with Normal group and sham-operate group (P < 0.05); ★Compared with model group (P < 0.05);◆Compared with captopril (P < 0.05); ▼Compared with Benefiting qi group (P < 0.05); ++ Compared with Warming meridians group (P < 0.05); ●Compared with Promoting blood circulation for removing blood stasis group (P <0.05).1 Normal group; 2 Sham-operate group; 3 Model group; 4 Captopril group; 5 Benefiting qi group; 6 Warming meridians group; 7 Promoting blood circulation for removing blood stasis group

Figure 3. NF-κB expression of rat aortic smooth muscle each

Discussion

A considerable number of studies have shown that hypertension and its complications are closely related to vascular remodeling [1,2]. The pathological mechanism of hypertensive vascular remodeling is very complex,mainly related to blood pressure, inflammation,endothelial dysfunction, extracellular matrix metalloproteinase activity, Renin-angiotensin-aldosterone System (RAAS), blood lipids, oxidative stress and other factors. Among them, inflammation and oxidative stress are the most important. NF-κB is a very important nuclear transcription factor involved in multiple signal transduction pathways associated with inflammatory process. Its activation is one of the important initiating mechanisms for the development of hypertension vascular remodeling.

It can be seen from the ancient books of Chinese medicine that the occurrence of vascular remodeling in hypertensive patients, qi deficiency is the fundamental,turbid phlegm and static blood are the standard. At present, the methods of clearing heat-toxicity, promoting blood circulation for removing blood stasis, removing phlegm and collaterals, warming Yang and nourishing kidney are often used in the study of vascular remodeling in hypertension. ZLHXTY has the functions of scavenging oxygen free radicals, resisting platelet aggregation, reducing inflammatory reaction and so on.All the drugs have the effects of invigorating qi,promoting blood circulation, removing blood stasis and promoting collaterals [3-7].

Our earlier studies have shown that ZLHXTY can effectively reduce blood pressure, anti-atherosclerosis,depress blood-lipid by intervening in the inflammatory response [3-5], but the deep studies on the mechanism of anti-hypertensive artery reconstruction research are not enough, in addition, no researches on party component of ZLHXTY. Therefore, it is necessary to clarify the effect of ZLHXTY and each component on the arterial reconstruction of hypertension.

Conclusion

ZLHXTY can effectively interfere with blood pressure remodeling, lower blood pressure and reduce NF-κB protein expression in renal hypertensive rats. Different groups can interfere with vascular remodeling from different aspects: (1) It has effect in reducing blood pressure in Benefiting qi group. (2) The effect of reducing NF-κB protein expression in Warming meridians group is significant. (3) All the mice in the promoting blood circulation for removing blood stasis group showed decreased blood pressure and NF-κB protein expression This indicates that the mechanism of vascular remodeling in ZLHXTY in rats with renal hypertension may be related to its lowering of blood pressure, anti-oxidative stress and anti-inflammatory effect. The compatibility of each group produced a significant synergistic effect,which reflected the therapeutic advantages of multiple targets of traditional Chinese medicine.

1. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. New Engl J Med 1994, 330:1431–1438.

2. Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic hypertension. Hypertension 1989,13: 968–972.

3. Qiu YA, Chen HG, Li LH, et al. Protective effects of serum contained huangqi on endothelial cell apoptosis induced by angiotensin II. Chongqing Med 2015, 44: 741 - 745.

4. Mu HO, Su XG. Pharmacological study of Dilong.China Pharmaceuticals 2007, 16: 61-62

5. Wei J, Zhang DS. Pharmacological study of Shuizhi.J China Tradi Chin Med Inf 2011, 3: 77.

6. Hu YH. Experimental study on the lipid regulation ang anti-atherosclerosis of Shuizhi. Tianjin Medical University 2014.

7. Wang KL. Effect of compound Daxueteng on the model of non-bacterial prostatitis in rats induced by neisserin. Shenzhen J Integr Med 2014, 06: 19-20.

8. Liao HL, Yin SY, Xi XL, et al. Intervention of Zhilong Huoxue Tongyu Capsule on IL-6, TNF-α and CRP of Renal Hypertensive Rats. Henan Tradi Chin Med 2011, 31: 1239-1241.

9. Yang SJ, Wang CG, Bai X, et al. Therapentic effect of Yiqi Qufeng Tongluo on the expression of NF-κB and ICAM-1 in rabbits with hypertensive carotid atherosclerosis. J Luzhou Med Coll 2014, 37: 47-51.

10. Wu Y, Yang SJ, Bai X, et al. Effect of Zhilong Huoxue Tongyu Capsule on rats with atherosclerosis and hypolipidemic. China Guide Med 2013, 11:401-402.