Original Article Effects of Jiangtang Xiaozhi tablets on expression of NFκB, CYP

Original Article

Effects of

Jiangtang Xiaozhi

tablets on expression of

NFκB, CYP

E

, PPARγ and ABCA

inobese rat livers

Xiaoxia Dong1*_{, Wenting Song}2*_{, Zhengyan Ge}2_{, Long Jin}2_{, Bin Yang}1_{, Hongkun Li}1_{, Yujie Guo}2_{, Xiaohua} Hong2_{, Yanghui Wang}2_{, Jianxun Liu}2

1_{Department of Pathology,} 2_{Institute of Basic Medical Sciences, Xiyuan Hospital CACMS, Beijing, China.} *_Equal contributors and co-first authors.

Received March 7, 2016; Accepted June 5, 2016; Epub September 15, 2016; Published September 30, 2016

Abstract: To explore effects of Jiangtang Xiaozhi Tablets (JTXZ) on expressions of NF-κB, CYP₂E₁, PPARγ and ABCA₁

inobese rats’ liver. Obese rats were established by high-fat fodder feeding for 18 weeks. Then, the obesity rats were divided randomly into: control model group, metformin group (30 mg/kg), simvastatin group (4 mg/kg), and 3 JTXZdosage groups (2, 4 and 8 g/kg, crude herb mass equivalent) (n=10). Drugs were administered orally once

daily for 8 weeks. Subsequently, rats were sacrificed and bloods were collected from the abdominal aorta to deter -mineserum total cholesterol (TC) and triglyceride (TG). In addition, livers were collected to determine expressions of

NFκB, CYP₂E₁, PPARγ and ABCA₁ using immunohistochemical assays. Our results showed that after treatment with JTXZ, body weight (BWs, P<0.01) and TC (P<0.05) are obviously decreased compared with the model rats, and TG was also decreased by treatment with JTXZ at the doses of 8 and 4 g/kg (P<0.05); importantly, the JTXZ (2, 4 and 8

g/kg) decreased the expressions of NF-κB and CYP₂E₁ significantly (P<0.01), while expressions of PPARγ and ABCA₁ (P<0.01) are significantly increased compared with model rats. These results above suggested that JTXZ reduce

body weight and blood lipid, and improve the liver steatosis of obese rats. The potential mechanism might be related

to down-regulating NF-κB & CYP₂E₁ and up-regulating PPARγ & ABCA₁.

Keywords:Jiangtang Xiaozhi Tablet, obese rats, NF-κB, CYP₂E₁, PPARγ, ABCA₁

Introduction

Increasing reports have indicated that obesity have become one of the most prevalent diseas-es of the world [1, 2]. Obdiseas-esity could cause a number of metabolic diseases, and in particu-lar obesity is closely associated with Type 2 Diabetes Mellitus (T₂DM), which insulin resis-tance exists in both conditions [3, 4]. Fur- thermore, obesity is also the most direct rea-son of fatty liver disease [5]. Chinese herbal medicine and its prescriptions have been used in China for thousands years to treat various diseases [6, 7]. Jiangtang Xiaozhi tablet (JTXZ) is a standardized herbal formulation containing the following herbal medicines: Glossy Privet fruit (12 g), Astragalus membranaceus (6 g), Coptis chinensis (3 g), Semen Litchi (6 g), Euonymus alatus (4.5 g) and Curcuma longa (4.5 g). JTXZ was commonly used in clinically for treating T₂DM and metabolic syndrome. Previous studies have found that JTXZ has

obvi-ous hypoglycemic effects on diabetes animals induced by alloxan and streptozotocin [8, 9]. Further experiments have also shown that JTXZ

has significant hypoglycemic and hypolipidemic

effects in KK-Ay _{obese diabetic mice and type 2} diabetic rats, and can markedly increase the numbers of pancreas islets and reduce adipo-cytes [10]. However, the detail mechanisms of JTXZ are still not clear. Therefore, our present study is designed to explore the effects of JTXZ

on the expressions of NF-κB, CYP₂E₁, PPARγ

and ABCA₁ in livers of obese rats.

Materials and methods

Animals

animals were housed individually in an air-con-ditioned room (22-25°C; relative humidity: 45-60%) with free access of food and water within the Laboratory Animals Center of Xiyuan Hospital, China Academy of Chinese Medical Sciences, which is a Grade 2 animal breeding facility [License No.: SYXK (Beijing) 2009-0001]. Standard diet was provided by Beijing Keaoxieli Fodder Co., LTD [License No.: Beijing Fodder (ingredient) No. 238, executive standard GB14924, 3-2001]. High fat diet formula was prepared by Beijing Huafukang Biological Te- chnology Co., LTD consisting 10% of lard oil, 10% of sucrose, 1.5% of cholesterol, 0.5% of bile salt, 5% of egg yolk powder 5% and 73% of standard diet. All experiments conducted in this project were approved by the local animal eth-ics committee of our Hospital.

Chemicals and materials

Jiangtang Xiaozhi tablets (Batch number:

20100308) were provided by the Department of Pharmaceutical Chemistry, Basic Medical Institute, Xiyuan Hospital, China Academy of

Chinese Medical Sciences. Dry powder was used in the experiments; each gram of dry pow-der consists of 5.4 g raw herbs. Metformin Hydrochloride Tablets: was obtained from Bei- jing Union Pharmaceutical Factory (0.25 g/tab-let; batch number: 101205). Simvastatin ob- tained from the Hangzhou MSD Pharmaceutical Company Limited (20 mg/tablet; batch num-ber: 09547). Biochemical kits were obtained from Biosino Bio-Technology and Science Incorporation (TC batch number: 111221, TG batch number: 114751). Other immunohisto-chemical reagents used were summarized in

Table 1.

Animal models and experimental design

70 SD male rats were randomly allocated to seven experimental groups (n=10): Control rats were fed standard diets, while the obese rats were established by high-fat fodder feeding for 18 weeks. Then obese rats were divided into Model group, Metformin group, Simvastatin group and 3 doses JTXZ (2, 4 and 8 g/kg, crude herb mass equivalent). Drugs were adminis-tered orally once a day for 2 months.

Body weight and blood lipid

Rats body weight changes of before and after modeling were recorded, and blood samples of before and after modeling were recorded were taken via cardiac puncture in animals to deter-mine the serum total cholesterol (TC) and tri-glyceride (TG).

Determination of NF-κB, CYP₂E₁, PPARγ and

ABCA₁

Rats in each group were dissected for liver,

which was made into paraffin section with 4 μm thickness via fixation with 10% neutral formal -dehyde, dehydration with ethanol step by step,

hyalinization by xylene and paraffin embedding.

Then the immunohistochemical process was

Table 1. Immunohistochemical reagents used in our present study

Name Product code Batch number Provider

NF-κB ab31481 GR78568-1 Abcam Co.

CYP₂E₁ A628146 GA72736-1 Abcam Co.

NF-κB and CYP₂E₁ secondary antibody (MaxVisionTM_{) KIT-5020 1303144707E Fujian Maixin Biotech. Co.}

DAB DAB-2031 1305312031 Fujian Maixin Biotech. Co.,

PPARγ ab27649 GR82388-1 Abcam Company

ABCA₁ ab125064 GR82846-7 Abcam Company

PPARγ and ABCA₁ secondary antibody and DAB (DaKo REALTM _EnVisionTM_{) K5007 00091392 Beijing Xiya Jinqiao Biol. Tech. Co.}

Table 2. Body weight changes in rats with/with-out high-fat diets feeding

Group n Baseline (g) After 18 weeks _{modeling (g)} Control rats 10 129.74 ± 9.78 575.19 ± 46.43 High lipid rats 60 130.64 ± 6.16 635.60 ± 51.28**

Data are expressed as mean ± SD, **_{P<0.01, compared with}

the control rats.

Table 3. Blood lipid changes in rats with/with-out high-fat diets feeding

Group n TC (mmol/L) TG (mmol/L)

Control rats 10 1.45 ± 0.21 1.16 ± 0.22

High lipid rats 60 2.15 ± 0.36** _{1.31 ± 0.32}*

Data are expressed as mean ± SD, *_P<0.05, **_P<0.01,

performed by staining with each antibody (Dilution 1:100). Then, the tissue sections were observed under microscope. Integral optical density (IOD) = Average optical density (AOD) × positive area (A).

Statistical analysis

All Data are presented as mean ± S.D, and were evaluated with one-way ANOVA and two-tailed Student’s t test between different groups.

The statistical significance of differences

be-tween groups was analyzed by using SPSS soft-ware (SPSS for Windows 18.0, SPSS Inc., USA),

and the differences were considered significant

at P<0.05.

Results

Results of the model establishment

As can be seen from Table 2, there is no signifi -cant difference in body weights between the control group and high lipid group before model establishment. However, after 18 weeks’ feed-ing with high-fat fodder, the rats’ body weights

obese rats

After the obese model rats were prepared, rats were treated with JTXZ at the doses of (2, 4 and 8 g/kg, crude herb mass equivalent) for 8 weeks. Before drug treatments, there was no difference in body weights among all the test-ing groups. Interesttest-ingly, our results showed th- at similar to the metformin (0.3 g/kg), the JTXZ

at the doses of 4 and 8 g/kg could significantly

decrease the body weight of obese rats (P< 0.05), compared with the model rats (Table 4).

Effects of JTXZ on blood lipid of obese rats

We can see from the Table 5 that our results

revealed that JTXZ (4 and 8 g/kg) significantly

decreased the TC of obese rats (P<0.05), com-pared with the model rats. In addition, the TG of the obese rats could be also show a decreasing trend by treating with JTXZ.

Effect of JTXZ on expressions of NF-κB and CYP₂E₁

Our present results showed that expressions of

NF-κB and CYP₂E₁ increased significantly

(P<0.001) in the model rats, compared with the rats in control group. Interestingly, as shown in

Table 6 and Figure 1A, 1B, JTXZ (2, 4 and 8 g/

kg, crude herb mass equivalent) significantly decreased the expressions of NF-κB (P<0.01)

and CYP₂E₁ (P<0.01), compared with the model rats, with a doses-dependent manner. Our

results suggested JTXZ possesses significant inhibitory effects on NF-κB and CYP₂E₁ in livers of obese rats.

Effect of JTXZ on expression of PPARγ and

ABCA₁

In our present study [(Table 7; Figure 1C, 1D],

the expressions of PPARγ and ABCA were

sig-Table 4. Effects of JTXZ on body weight of obese rats

Groups Doses Before treatment (g) After treatment (g)

Control 576.58 ± 41.57 605.50 ± 41.39

Model 635.42 ± 62.38* _{734.20 ± 66.61}**

Metformin 0.3 g/kg 636.08 ± 58.76 667.10 ± 42.28#

Simvastatin 4 mg/kg 636.83 ± 54.41 704.90 ± 58.68

JTXZ 2 g/kg 635.58 ± 39.85 695.80 ± 53.17

4 g/kg 633.42 ± 43.69 672.00 ± 54.97#

8 g/kg 634.17 ± 57.69 641.90 ± 25.70##

Dose of JTXZ was represented as crude herb mass equivalent. Data are expressed as mean ± SD (n=10), *_P<0.05, **_{P<0.01, compared with the}

control rats; #_P<0.05, ##_{P<0.01, compared with the model rats.}

Table 5. Effects of JTXZ on blood lipid of obese rats

Groups Doses TC (mmol/L) TG (mmol/L)

Control 1.15 ± 0.21 0.43 ± 0.06

Model 1.64 ± 0.40** _{0.61 ± 0.18}**

Metformin 0.3 g/kg 1.38 ± 0.22* _{0.49 ± 0.14}

Simvastatin 4 mg/kg 1.24 ± 0.25# _{0.58 ± 0.13}**

JTXZ 2 g/kg 1.48 ± 0.24** _{0.63 ± 0.13}**

4 g/kg 1.30 ± 0.24# _{0.59 ± 0.11}**

8 g/kg 1.28 ± 0.33# _{0.47 ± 0.13}

Dose of JTXZ was represented as crude herb mass equiva-lent. Data are expressed as mean ± SD (n=10), *_P<0.05, **_{P<0.01, compared with the control rats;} #_P<0.05.

of high lipid rats were significantly

greater than that of the control rats (P<0.001) (Table 2).

Our results, presented in Table 3, showed that there was no obvious dif-ference in rats between the two group; however, after 18-week high-fat diets’ feeding, TC (P<0.01) and TG (P<0.05)

levels were significantly higher than

that of the control rats. The results above suggested that the obese model rats were successfully established.

Table 6. Grayscales of NFκB and CYP₂E₁

Groups Doses NF-κB (IOD) CYP2E1 (IOD)

Control 61688.44 ± 34057.09 9867.88 ± 3437.17

Model 167297.44 ± 45886.61** _{29349.73 ± 10955.71}**

Metformin 30 mg/kg 109302.03 ± 38047.52## _{17618.89 ± 5180.88}##

Simvastatin 4 mg/kg 108050.35 ± 35325.91## _{19917.70 ± 7366.51}##

JTXZ 2 g/kg 96104.76 ± 32033.91## _{16389.65 ± 4757.06}##

4 g/kg 135679.87 ± 55025.26## _{24203.79 ± 6827.15}##

8 g/kg 117037.68 ± 34849.37## _{19700.33 ± 5681.56}##

Integral optical density (IOD) = Average optical density (AOD) × positive area (A). Dose of JTXZ was represented as crude herb mass equivalent. Data are expressed as mean ± SD (n=10), **_{P<0.01, compared with the control rats;} #_P<0.05, ##_P<0.01,

compared with the model rats.

Figure 1. Effects of JTXZ on expressions of NF-κB (A), CYP₂E₁ (B), PPARγ (C) and ABCA₁ (D) in obese rats’ liver (×40). (a-g) represented the Control group, Model group, Metformin group, Simvastain group and JTXZ groups (2, 4 and 8 g/kg, crude herb mass equivalent), respectively.

Table 7. Grayscales of PPARγ and ABCA₁

Groups Dose PPARγ (IOD) ABCA₁ (IOD)

Control 772644.30 ± 217462.79 110327.13 ± 48590.24

Model 510345.36 ± 239765.58** _{68230.17 ± 43590.37}**

Metformin 30 mg/kg 620230.73 ± 124459.16## _{100453.50 ± 48839.81}##

Simvastatin 4 mg/kg 650431.79 ± 161080.01## _{95152.72 ± 44817.41}##

JTXZ 2 g/kg 646398.05 ± 149053.07## _{96827.80 ± 40401.22}##

4 g/kg 641288.81 ± 108764.48## _{93641.09 ± 43456.52}##

8 g/kg 754298.41 ± 126515.12## _{98970.79 ± 37725.83}##

Integral optical density (IOD) = Average optical density (AOD) × positive area (A). Dose of JTXZ was represented as crude herb mass equivalent. Data are expressed as mean ± SD (n=10), **_{P<0.01, compared with the control rats;} #_P<0.05, ##_P<0.01,

nificantly decreased (P<0.001) in the model

group compared with those of the control group. However, JTXZ treatments (2, 4 and 8 g/

kg, crude herb mass equivalent) significantly up-regulated the expression of PPARγ (P<0.01)

and ABCA₁ (P<0.01) compared with those of the model group (P<0.01), with a doses-depen-dent manner, suggesting that JRXZ has obvi-ous up-regulating effects on expressions of

PPARγ and ABCA₁ in livers of obese rats.

Discussions

Obesity is defined as excessive fat tissue accu -mulation and is one of the important risk fac-tors of T₂DM [3]. Obesity-induced nonalcoholic fatty liver disease (NAFLD) is most commonly, which reaches 50% of the morbidity in T₂DM patients and nearly 100% in the crowd with dia-betes accompanied with obesity. Fatty liver is one of the clinical pathological syndromes given prior to diffuse macro-vesicular steatosis in lobular liver cell and hepatocell [5, 11].

NF-κB is a widespread kind of eukaryotic cells

transcription factors, which can be activated by oxidative stress via inducing expression of Fas ligand on liver cell membrane surface and plays a key control role in the induction of

inflammatory factor expression [12, 13]. In

addition, CYP₂E₁ is a main source for lipid per-oxide in vivo. Its high expression could enhance oxidation reaction in the liver cells, thereby causing liver cell membrane lipid peroxidation; therefore, it decreases the protection role of antioxidant system and damage hepatocyte [14, 15], thus causing hepatocyte fatty

degen-eration and necrosis. PPAR-γ is mainly

expressed in adipose tissue and liver, which can promote fat cell differentiation. It is acti-vated by a variety of fatty acid and its

deriva-tives. The activated PPAR-γ regulates the utili -zation of saccharides in the muscle tissue through affecting the translocase in the adi-pose tissue [16]. ABCA₁ is a member of the ATP-binding cassette transporter superfamily, which is mainly involved in the reversal of the cholesterol. It undertakes an important task in cholesterol scavenging of macrophages, thus clearing the excessive cholesterol [17, 18]. The rats are fed with high fat fodder for 18 weeks in our experiment. The obese rat model is successfully established when the rat BWs,

TC and TG are significantly increased. After

8-week drugs treatment, high and medium

dose groups have significantly lower body

weight, obese symptoms, and TC and TG levels than those of the model group. In each dose

group, liver NF-κB and CYP₂E₁ expressions are

obviously decreased, while liver PPARγ and

ABCA₁ expressions are obviously increased, suggesting JTXZ can inhibit the activation of

NF-κB and reduce lipid peroxidation, thus reducing inflammation and liver steatosis.

Up-regulation of ABCA₁ and PPAR-γ expression

can also promote that JTXZ can active PPAR-γ,

inhibit expression of inflammatory cytokines,

enhance the transcription of ABCA₁, thus elimi-nating excessive cholesterol in the organization and reducing liver lesions.

In conclusion, JTXZ can significantly reduce the

body weight and blood lipid and improve the liver steatosis of obese rats. Its mechanism may be related to the down-regulation

expres-sion of liver NF-κB and CYP₂E₁, and reduction of lipid peroxidation damage; it may also be

relat-ed with up-regulation expression of PPARγ and

ABCA₁, promotion of adipocyte differentiation, increased insulin sensitivity and improvement of IR.

Acknowledgements

This work was supported by the National Major

Scientific and Technological Special Project for “Significant New Drugs Development” (No.

2010ZX09102-213, 2012ZX09103201-040) and Beijing Natural Science Foundation project (No. 7154233), and National Basic Research Program (No. 2015CB554400).

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Jianxun Liu, In- stitute of Basic Medical Sciences, Xiyuan Hospital CACMS, 1 Xiyuan Playground, Beijing 100091, Chi- na. Tel: +86-10-62835601; Fax: +86-10-62874049; E-mail: jianxliu_xh@126.com

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