Effects of 1-(2-chlorine)-phenyl-9-butyl-β-carboline (DH-330) on Cystic Echinococcosis in Mice in vivo

Huijing GAO, Xin WANG, Cartiera KURBAN, Aniwanr METIKURBAN, Bei CHEN*

1. Department of Pharmacy, the First Affiliated Hospital of Xinjiang Medical University, Urmuqi 830054, China; 2.Pharmacy College of Xinjiang Medical University, Urmuqi 830011, China

Abstract [Objectives] The paper was to evaluate the in vivo treatment efficacy of harmine derivative 1-(2-chloro) phenyl-9-butyl-β-carboline (DH-330) on mice cystic echinococcosis (CE). [Methods] Kunming mice were injected intraperitoneally into the protoscoleces and infected with secondary infection for 8 months to prepare CE model. The successfully modeled mice were randomly divided into 6 groups according to their body weight: model group, control-1, -2 groups and experimental-L, -M, -H groups, with 10 mice in each group. The model group was given distilled water and control-1, -2 groups were given 50 mg/kg albendazole and harmine, respectively. The experimental-L, -M, -H groups were given 25, 50 and 100 mg/kg DH-330. After 8 weeks of intragastric administration, the mice were dissected and vesicles were taken, and the differences of cyst weight were compared. The ultrastructure changes of cysts were observed by transmission electron microscope (TEM). The histopathology of cysts and liver were observed by hematoxylin-eosin (HE) staining method. [Results] The cyst weight of model group, control-1, -2 groups and experimental-L, -M, -H groups were (34.38±4.32), (11. 38±2.37), (15.89±1.31), (16.22±2.30), (11.69±2.95) and (9.78±1.14) g, respectively. Compared between drugs group and model group, the difference was significant (all P<0.05); compared between experimental-H group and harmine group, the difference was significant (P<0.05). Except for the model group and experimental-L group, all other groups can damage the hydatid nuclei, which lead to cell lysis and nucleoli disappear. Experimental groups can improve inflammatory cells infiltration in liver and vesicle. [Conclusions] DH-330 can reduce the cysts weight of CE mice, inhibit the growth of hydatid, and improve the inflammation of the liver and vesicles, showing a good resistance against Echinococcus granulosus, or may become an effective new drug against hydatid disease.

Key words Harmine derivative, Cystic echinococcosis, Cyst weight, Transmission electron microscope, Histopathology

1 Introduction

Echinococcosis is a serious zoonotic parasitic disease caused by echinococcus parasitizing the viscera of mammals, which mainly occurs in Xinjiang, Gansu, Inner Mongolia, Qinghai and other agricultural and pastoral areas in China. 95% of echinococcosis is cystic hydatid disease caused byEchinococcusgranulosus[1]. Albendazole (ABZ) is the preferred drug for the treatment of echinococcosis recommended by the World Health Organization (WHO), but its poor water solubility and poor absorption after oral administration lead to poor clinical efficacy, and patients with echinococcosis are prone to a series of adverse drug reactions such as nausea, vomiting and hair loss after long-term adminstration. Therefore, it is urgent to find new anti-hydatid drugs[2].PeganumharmalaL. is a perennial herbaceous plant belonging toPeganum, Zygophyllaceae, widely distributed in arid regions of Xinjiang, Inner Mongolia, Shaanxi, Gansu and Ningxia, and has been used as a national medicine by Uygur, Kazak, Mongolian and other nationalities for a long time. Harmine (HM) is the main alkaloid inP.harmalaand has good anti-hydatid effect[3], but its high neurotoxicity limits its clinical application. Therefore, in order to reduce the neurotoxicity of HM, our research group modified the structure of β-carboline at 1, 7, and 9 positions with HM as the nuclear parent, and obtained a novel compound 1-(2-chlorine)-phenyl-9-butyl-β-carboline (DH-330) (Fig.1)[4]. The purpose of this study was to evaluateinvivoresistance effect of DH-330 onE.granulosus, and to lay a foundation for the development of new anti-echinococcosis drugs.

Fig.1 Chemical structure formula of harmine derivative 1-(2-chlorine)-phenyl-9-butyl-β-carboline (DH-330)

2 Materials and methods

2.1 Materials

2.1.1Animals. Kunming white mice aged from 6 to 8 weeks with body weight of (20±2) g were purchased from the experimental animal center of Xinjiang Medical University, animal license number SYXK(Xin) 2016-0004. The scheme was approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (Approval number: IACUC20170420-04).

2.1.2Drugs and reagents. Harmine (HM) control, 100 mg/bottle, content≥98%, CAS number 442-51-3, batch No.X04F6L1, produced by Shanghai Yuanye Biotechnology Co., Ltd. DH-330, 10 g/bottle, yield 45% (content ≥95%), batch No.170815, provided by Xinjiang Huashidan Pharmaceutical Co., Ltd.[4]. Albendazole, 0.2 g/tablet, approval H12020496, batch No.20130412, produced by Tianjin Smith Kline & French Laboratories Ltd. 4% Paraformaldehyde, produced by Beijing Solarbio Technology Co., Ltd.

2.1.3Instrument. JSM-1230 transmission electron microscope was manufactured by JEOL Electronics Company, Japan; EG 1150H paraffin embedding machine and ST 5020 multi-function dyeing machine were products of Germany Leica Company; Ci-L upright metallurgical microscope was produced by Nikon, Japan.

2.2 Methods

2.2.1Preparation of cystic echinococcosis (CE) mouse model[5-6]. Kunming white mice were intraperitoneally injected with 0.5 mL of 1 640 suspension containing protoscoleces (containing 2 000 protoscoleces). About 7-8 months after infection, B-mode ultrasonography showed that there was multi-ascus CE in the abdominal cavity. Several asci were found close to each other and their ascus walls were thicker than those of simple cysts. It was judged as successful modeling if the vesicle diameter was greater than 0.5 cm.

2.2.2Grouping and administration. Successfully infected mice were randomly divided into 6 groups: model group, control-1, -2 groups and experimental-L, -M, -H groups, 10 mice each group. The mice in model group were given distilled water, and those in control-1, -2 groups were given 50 mg/kg albendazole and 50 mg/kg harmine, respectively. The mice in experimental-L, -M, -H groups were intragastrically administrated with 25, 50 and 100 mg/kg DH-330 respectively for consecutive 8 weeks.

2.2.3Cyst weight and cyst inhibition rate[6]. After administration, the mice were dissected, and the cysts of mice in each group were stripped and weighed to calculate cyst weight and cyst inhibition rate. Cyst inhibition rate=(C-T)/C×100%. WhereCrepresents the cyst weight ofE.granulosusin model group and control groups;Trepresents the cyst weight ofE.granulosusin experimental groups.

2.2.4Observation of ultrastructural changes of vesicles by transmission electron microscope (TEM)[7]. Vesicles were randomly collected from each group and fixed with electron microscopic fluid. Then, they were rinsed with phosphate buffer (pH 7.2-7.4) for 3 times, fixed with 1% osmium tetroxide for 1 h, rinsed with distilled water for 3 times, dehydrated in acetone gradient, immersed and embedded. Ultra-thin sections were stained by dual electron staining of lead nitrate anduranyl acetate, and observed under TEM.

2.2.5Observation of histopathological changes of mice by hematoxylin-eosin (HE) staining[8]. Vesicles and liver tissues were randomly selected from each group, fixed with 4% paraformaldehyde, embedded, sliced, and stained with HE. The histopathological changes in each group were observed under light microscope.

3 Results and analysis

3.1 Cyst weight and cyst inhibition rateCompared with model group, all experimental groups significantly decreased the cyst weight, with statistical significance (allP<0.05). Compared with control-2 group, experimental-H group significantly reduced the cyst weight, with statistical significance (P<0.05, Table 1).

Table 1 Cyst weight and cyst inhibition rate of mouse

3.2 Ultrastructural changes of vesiclesThe ultrastructural changes of vesicles are shown in Fig.2. In model group,E.granulosusvesicles had clear structure of germinal layer, neat and abundant microtrichia, large and round parenchymal nuclei, clear nucleoli, a little heterochromatin at the nuclear membrane boundary, uniform cuticle structure, and clear lamellar structure (Fig.2A). In control-1 group (50 mg/kg albendazole),E.granulosusvesicles had reduced microtrichia, blurred boundary of parenchymal nucleus, disappeared nucleoli, destroyed cuticle texture, and blurred lamellar structure (Fig.2B). In control-2 group (50 mg/kg harmine),E.granulosusvesicles had thin germinal layer, rare and irregular microtrichia, few and dissociated parenchymal cells, completely destroyed cell structure, disappeared nucleoli, loose cuticle and disappeared lamellar structure (Fig.2C). In experimental-M and experimental-H groups (50 and 100 mg/kg DH-330),

Note: A. Model group; B. Control-1 group; C. Control-2 group; D, E, F. Experimental-L, -M, -H groups; the same below.Fig.2 Ultrastructure of vesicles of mice in 6 groups (×1 000)

E.granulosusvesicles had damaged cortical cell structure, disappeared nucleoli, loose cuticle structure with widened gap, but lamellar structure was not disappeared (Fig.2E). In experimental-L group (25 mg/kg DH-330), the vesicle structure ofE.granulosuswas not obviously damaged (Fig.2F).

3.3 Histopathological changes in experimental mice

3.3.1Histopathological changes in liver. The histopathological changes in liver are shown in Fig.3. The liver in model group had atypical hyperplasia, disappeared liver plate, and distinct infiltration of inflammatory cells around blood vessels. The liver in control-1 group (50 mg/kg albendazole) had a little flake-like necrosis in liver lobules, and there was few inflammatory cell infiltration around blood vessels. The liver in control-2 group (50 mg/kg harmine) had significantly reduced flake-like necrosis in liver lobules, and there was mild inflammatory cell infiltration in liver lobule and portal area. In experimental-M and experimental-H groups (50 and 100 mg/kg DH-330), there was less inflammatory cell infiltration around liver vessels. In experimental-L group (25 mg/kg DH-330), there were a large number of inflammatory cell infiltration around liver vessels, which was more serious.

Fig.3 Pathological changes of liver tissue in experimental mice (hematoxylin-eosin, ×200)

3.3.2Pathological changes of vesicles. The pathological changes of vesicles are shown in Fig.4. In model group, the vesicles had clear outline of germinal layer, smooth inner wall, no focal necrosis and calcification, and no foreign body granuloma. There were many corneous cells in the outer layer, which could absorb nutrients and protect the germinal layer. In control-1 group (50 mg/kg albendazole), the germinal layer structure was damaged, with unclear outline, and the outer cuticle cells were reduced without calcification. The structure and outline of germinal layer in control-2 group (50 mg/kg harmine) were damaged, with unsmooth inner wall, and there was a little calcification. In experimental-M and experimental-H groups (50 and 100 mg/kg DH-330), the vesicles had damaged structure of germinal layer, exfoliated cells, unsmooth inner wall, and more calcification. In experimental-L group (25 mg/kg DH-330), the vesicles had clear structure and outline of germinal layer.

Fig.4 Pathological changes of vesicles in mouse (hematoxylin-eosin, ×200)

4 Discussion

Cystic echinococcosis (CE) is a chronic, complex and neglected zoonotic parasitic disease caused by the larva ofE.granulosusparasitizing in humans and animals. The disease is distributed worldwide, mainly in Inner Mongolia, Sichuan, Qinghai, Tibet, Ningxia, Xinjiang and other pastoral and semi-pastoral areas[9]. A national survey on echinococcosis in China showed that about 50 million people in 9 western provinces were at risk of infection from 2012 to 2016, and nearly 170 000 people suffered from echinococcosis[10]. Echinococcosis seriously harms human health and animal husbandry development, and is one of the main causes of “poverty due to disease and return to poverty due to disease” among farmers and herdsmen in western China. It is also a parasitic disease that China focuses on prevention and control[11].

There are a large number of alkaloids inP.harmala, mainly β -carboline and quinazoline alkaloids. The seeds and whole grass ofP.harmalacontain 4%-7% β-carboline alkaloids, mainly harmine and harmaline[12]. Harmaline has a wide range of pharmacological effects, including anti-tumor, anti-hydatid, bactericidal, antiviral, bone protection and anti-angiogenesis[13]. Harmine shows goodinvitroandinvivoactivity against hydatid[3,6]. However, it is highly neurotoxic and can cause neurotoxic symptoms such as tremor, dyskinesia and vomiting in both animals and humans[14]. In order to reduce its significant neurotoxicity, DH-330 was synthesized by modifying its structure with harmine as the parent nucleus by computer simulation.

Harmine has good anti-hydatid activity, and its derivative DH-330 is speculated to have anti-hydatid activity. In order to confirm this prediction, Wangetal.[15]explored the resistance effect of DH-330 onE.granulosusfrom the perspective ofinvitro, and found that DH-330 had a good anti-hydatid activityinvitro. Compared with model group, DH-330 at low, medium, and high doses significantly reduced the cyst weight, with statistically significant differences. Compared with harmine, DH-330 in experimental-H group could significantly reduce the cyst weight, and the cyst inhibition rate was similar to that of positive drug albendazole. The results of TEM and histopathology showed that DH-330 could treat hydatid disease by attacking the nucleus of protoscolex and destroying cysts. The histopathological results of liver showed that DH-330 had slight damage to liver tissue at high dose, but had no effect on liver tissue at low dose, and different doses of derivatives could improve inflammatory cell infiltration in different degrees.

Previous studies[16]showed that DH-330 had significantly higherinvivoandinvitrosafety than harmine. The results of this study showed that DH-330 had good anti-hydatid effectinvivo. Therefore, comprehensive evaluation holds that DH-330 may be a potential anti-echinococcosis drug. More detailed mechanisms of action, pharmaceutical properties,invivoabsorption, distribution and metabolic processes of DH-330 need to be further studied, so as to evaluate the potential of the derivative as an effective new drug for the treatment of human echinococcosis and to provide a new approach for the treatment of echinococcosis.