Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows

Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows
Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows

Agricultural Sciences in China 2010, 9(10): 1497-1503

October 2010? 2010, CAAS. All rights reserved. Published by Elsevier Ltd.

Effects of Yimu Shenghuasan Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows

DU Jin-liang, QIN Jian-hua, CHU Jing-sheng, XU Li-na and MA Yu-zhong

College of Animal Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R.China

Abstract

In order to investigate the mode of action of Yimu Shenghuasan preparation in endometrial cells of dairy cows, the primary cultured endometrial cells in cows were isolated and the inflammatory models were made by lipopolysaccharide (LPS) induction. The inflammatory cells were treated with gradient concentration of herbal medicine preparation, Yimu Shenghuasan for 48 and 72 h. The expression of cytochrome P450 (CYP450) was detected by Western blot. The amounts of IgG and IgA in sera were also detected in the endometritis of dairy cows. The expression level of CYP450 in the endometrial cells of dairy cow was increased gradually, and the amounts of IgG, IgA were increased significantly as compared with those in the control group. The expression level of CYP450 in the inflammatory cells was increased significantly in the treatment of 2000 μg mL-1 of Yimu Shenghuasan after 48 h of treatment.

Key words:dairy cow, endometrial cell, CYP450, Yimu Shenghuasan preparation, immune function

INTRODUCTION

Endometritis is a common postpartum disease (Wathes et al. 2009; Herath et al. 2009; Donofrio et al. 2008; Lee and Kim 2007), which could cause serious eco-nomic loss in animal husbandry. In veterinary clinics, antibiotics and hormones are popularly used for the treat-ment of cow endometritis. Due to their extensive use, the drug resistance in bacteria has been developed. Further, the animal immunity has been reduced, and the residual drug residues have been found in the meat and milk, which cause hazards to human health. Therefore, the development of an efficient, stable and secure traditional Chinese medicine is the current need in animal husbandry.

There were many ways to treat this disease, such as antibiotics, hormone, biological methods etc.(Wang et al. 2008; Jia and Qi 2005). However, these methods have many disadvantages. The treatment of cow endometritis by traditional Chinese medicine in China has a long history, and a number of classi-cal preparations have been established (Li and Gu 2008).

In this paper, the mode of action for Yimu Shenghuas an preparation was investigated by in vivo and in vitro experiments. In vivo experiments were carried out with the cellular and molecular biological methods, while the immune functions were investi-gated with in vitro experiments. The successful iso-lation of the primary cultured endometrial cells in cows was achieved, and Western blot was employed to in-vestigate the mechanism of Yimu Shenghuasan prepa-ration for endometritis in cows. This will provide a theoretical basis for the herbal preparations to treat endometritis in cows.

This paper is translated from its Chinese version in Scientia Agricultura Sinica.

DU Jin-liang, Tel: +86-312-7528443, E-mail: xiaoduhaoabcd@https://www.360docs.net/doc/cd2192864.html,; Correspondence MA Y u-zhong, Professor, Tel: +86-312-7528446, E-mail: dkma@https://www.360docs.net/doc/cd2192864.html,

1498DU Jin-liang et al.

MATERIALS AND METHODS

Chemicals and reagents

The chemicals and reagents used in the present study consisted of LPS (Sigma,USA), DMEM/F-12 (Invitrogen, USA), FBS (Hangzhou Sijiqing Company, China), pre-stained protein marker (Beijing Tiangen Bio-technology Company), CYP450 antibody (ADL), alka-line phosphate enzyme marked IgG (Beijing Zhongshan Company, China), NBT, BCIP (Amresco, USA), and IgG, IgA(Shanghai Lanji Biological Company, China).

T est animals

Twenty endometritis infected cows, 10 healthy cows as control, 10 endometritis infected cows treated with Yimu Shenghuasan, and 10 endometritis cows without any treatment were used in the present experiments. The jugular venous blood samples were collected at an interval of 1, 3, 5, and 7 d after treatment, and the sera were separated at 4°C.

Isolation and culturing of endometrial cells from cows

The uterus was rinsed into D-Hank’s, endomertrium was scraped, and 1 g L-1 collagenase I was added to digest for 8-10 min at 37°C in water bath. The filtered solution was centrifuged at 1200 r/min for 10 min, and the centrifugation was repeated for 3 times. The pellet was suspended in DMEM/F-12 medium containing 15% FBS, and the viability was determined by trypan blue exclusion. The endometrial cells were then inoculated into flasks and cultured at 37°C/5% CO

2

.

Establishment of inflammatory model

The endometrial cells were treated with gradient con-centration of LPS, and incubated at 37°C/5% CO

2

for 48 h. The 20 μL of MTT at the concentration of 5 mg mL-1 MTT was added and incubated for 6 h. The 100 μL of DMSO was then added and vortex for 5 min. The absorbance was recorded to determine the optimal LPS concentration. This LPS concentration was used to treat the endometrial cells for 24-72 h, and the amount of IL-lβ and TNF-a were detected with double-anti-body sandwich ABC-ELISA method.

Treatment of the endometrial cells with Yimu Shenghuasan preparation

The Yimu Shenghuasan preparation (consisted of honeysuckle,forsythia,dandelion,tokyo violet herb5 g each, motherwort 6 g, angelica, chuanxiong, pre-pared rhizome of rehmannia, safflower,radix glycyrrhizae3 g each) was soaked in deionized water for 1 h, the crude extract was then concentrated into 4 g mL-1 solution, and made into powder by vacuum freeze-drying. The powder of Yimu Shenghuasan preparation, was dissolved into DMEM/F-12 medium, and filtered with 0.22 μm microfilter. The solutions of honeysuckle, motherwort, angelica,and radix glycyrrhizae were prepared individually with the simi-lar procedure as described above. These drugs were used to treat the cow endometrial inflammatory cells for 48, 72 h, and the cell proteins were collected and quantified.

Expression of CYP450 by Western blot analysis

The cell lysates were denatured, analysed with 12% SDS-PAGE, and transferred electrophoretically to ni-trocellulose membranes. After blocking the non-spe-cific binding sites with 5% non-fat milk, the blots were incubated with CYP450 antibody. The antibodies were subsequently localized with lgG conjugated with AP. The expression of CYP450 was detected by NBT/BCIP. RESULTS

Morphological character of endometrial cells in cow

The endometrial cells in cow obtained by digestion were grown well, and the viability of cells was about 95%. Under microscope, the most of cells were globu-lar at 72 h, and were confluent at 120 h. The epithe-lial cells were like slabstones and the stromal cells were fusiform (Fig.1).

Effects of Yimu Shenghuasan Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows 1499

Table 1 The inhibitory action of LPS on endometrial cell growth in cow (n = 6)

Concentration (ng mL -1)Absorbance value Inhibition Control group 0.1704 ± 0.0067010.1706 ± 0.00350100.1658 ± 0.00512.6500.1622 ± 0.0024 4.81000.1526 ± 0.0059*10.42000.1356 ± 0.0062**20.4500

0.1282 ± 0.0056**

24.8

*

and **, significant at P < 0.05 and P < 0.01, respectively.

Table 2 Effect of LPS on the amounts of TNF-a and IL-1b in the endometrial cells of dairy cows

Time TNF-α

IL-1β

(h)Control group LPS group Control group LPS group

2421.96 ± 4.7022.63 ± 2.29 21.08 ± 6.52 35.78 ± 7.18

4822.16 ± 4.4341.44 ± 8.74*21.54 ± 5.2190.17 ± 8.38**

72

20.20 ± 3.89

63.18 ± 6.08*23.16 ± 8.56136.77 ± 9.13**

*

and **, significant at P < 0.05 and P < 0.01, respectively.

Screening of optimum LPS concentration

It was observed that the nearest IR = 10 was 100 ng mL -1group, so the optimal LPS concentration was l00 ng mL -1(Table 1).

CYP450 expression in endometrial cells of dairy cows induced by Yimu Shenghuasan preparation

When the inflammatory cells were treated with gradi-ent concentration of the herbal medicine preparation of Yimu Shenghuasan, the expression of CYP450 in endometrial cells of cow was increased gradually at 48 h treatment. At the concentration of 5 000 μg mL -1, the expression of CYP450 was decreased, and was disappeared at the concentration of l0 000 μg mL -1. The expression of CYP450 was increased in the beginning, but was decreased later at 72 h after treatment. The optimal dose was 2 000 μg mL -1 at 48 h (Fig.2).

CYP450 expression in endometrial cells of dairy cow induced by radix glycyrrhizae

When the endometrial cells were treated with radix glycyrrhizae for 48, 72 h, the expression of CYP450had no significant change at lower concentration,but the expression was decreased at the concen-tration of l0 000 μg mL -1 for 72 h after treatment (Fig.3).

The expression of CYP450 in endometrial cells of dairy cow induced by angelica preparation

The inflammatory cells were treated with angelica preparation for 48, 72 h. It was found that the expression of CYP450 in endometrial cells was decreased with increase in the dose and time course (Fig.4).

The expression of CYP450 expression in endometrial cells of dairy cow induced by motherwort preparation

The inflammatory cells were treated with gradi-ent concentration of motherwort preparation . It was found that the expression of CYP450 was increased gradually with the increase in the con-centration of motherwort preparation. Further,the expression was more obvious with the increase in the time (Fig.5).

Fig.1

Endometrial cells in dairy cow (120 h, × 250).

Establishment of inflammatory model

During the incubation period, the secretion of IL-l β,TNF-α in the control group were very low. In LPS group, along with the time extension of LPS induction,the secretion of two cytokines was increased. The amounts of IL-l β and TNF-α at 48, 72 h in LPS groups were significantly higher than those in the control group (P < 0.01). Similarly, the amounts of IL-l β and TNF-a at 72 h group were significantly higher than those in 48 h group (P < 0.01) (Table 2).

1500DU Jin-liang et al .

The expression of CYP 450 in endometrial cells of dairy cow induced by honeysuckle

The inflammatory cells were treated with gradient concentration of honeysuckle preparation for 48 and,72 h. The expression of CYP450 increased gradu-

ally with the increase in the concentration of honey-suckle preparation. Further, the expression was more obvious with the increase in the time. The expres-sion of CYP450 was decreased significantly at the concentration of l0 000 μg mL -1 and at 72 h after treatment (Fig.6).

Yimu Shenghuasan

01020501002005001 0002 0005 00010 000- 10 μmol L -1 PCN -----------+

Fig. 2 CYP 450 expression in endometrial cells of dairy cow induced by Yimu Shenghuasan preparation. Radix glycyrrhizae 01020501002005001 0002 0005 00010 000- 10 μmol L -1

PCN -----------+

Fig. 3 CYP 450 expression in endometrial cells of dairy cow induced by radix glycyrrhizae preparation.

Angelica

01020501002005001 0002 0005 00010 000- 10 μmol L -1

PCN -----------+

Fig. 4 CYP 450 expression in endometrial cells of dairy cow induced by angelica preparation.

Honeysuckle 01020501002005001 0002 0005 00010 000- 10 μmol L -1 PCN -----------+

Fig. 6 CYP 450 expression in endometrial cells of dairy cow induced by honeysuckle preparation.

Motherwort

01020501002005001 0002 0005 00010 000- 10 μmol L -1 PCN -----------+

Fig. 5 CYP 450 expression in endometrial cells of dairy cow induced by motherwort preparation.

Effects of Yimu Shenghuasan Preparation on the Cytochrome P450 in Endometrial Cells and Immune Function of Dairy Cows 1501

DISCUSSION

Inflammatory model of cow endometrial cells induced by LPS

The LPS is reported to induce the inflammation in en-dometrial epithelial and mesenchymal cells (Leung et al .2001; Rozeboom et al. 2001). In this experiment,LPS was used to simulate the clinical endometritis pathogenesis to investigate the pathogenesis of cow endometritis. The early acute inflammatory cytokines IL-l β and TNF-α were the indicators to determine whether the inflammatory model was made successfully. The results showed that the cells were grown well at the concentration of 100 ng mL -1 LPS treatment, and the amounts of IL-l β, TNF-α after 48,72 h of treatment were significantly higher than those in control group (P < 0.01). This indicated that the endometrial cell inflammatory model was made successfully. The establishment of this model made the foundation for the treatment of cow endometritis

(Walsh et al. 2007, 2008) and the evaluation of drug efficacy.

The expression of CYP450 in endometrial cells of dairy cows induced by Yimu Shenghuasan preparation

At present, there are lot of drugs for the treatment of cow endometritis (Rutigliano et al. 2008; Drillich et al.2007; Pol and Ruegg 2007; LeBlanc et al. 2006), but the results are not satisfactory. The use of molecular biology methods to study the mode of action of drug is also limited. The expression level of CYP450 have been popularly used to screen and research new drugs in the world (Ma et al. 2004, 2005; Cabrera et al.2009; Sinz et al. 2008; Yu et al. 2009). Using this approach, the early prediction of drug pharmacoki-netic in vivo and its possible metabolites and potential toxicity could be possible. However, it could also pre-dict the effectiveness of drugs in vitro and in vivo (McKerrow et al. 2009). The efficacy and toxicity of

Table 3 Effects of herbal medicine preparation,Yimu Shenghuasan on the amount of IgG in endometritis dairy cows (g L -1, n = 10)

Group

Treatment days (d)

3

5

7

Medication group 6.4563 ± 0.32419.2362 ± 0.4119 A 9.6987 ± 0.3471 A 10.1861 ± 0.5601 A Untreated group 6.4687 ± 0.4582 6.3468 ± 0.3389 6.2865 ± 0.3962 6.4964 ± 0.4015Control group

5.7912 ± 0.5231

5.8243 ± 0.3438 B

5.8454 ± 0.2961 B

5.9243 ± 0.3565 B

Values in the same column with different letters mean significant differences (P < 0.01). The same as below.

Table 4 Effects of herbal medicine preparation,Yimu Shenghuasan on the amount of IgA in endometritis dairy cows (g L -1, n = 10)

Group

Treatment days (d)

03

5

7

Medication group 0.7247 ± 0.0423 1.2346 ± 0.2789 A 1.5789 ± 0.3721 A 2.0034 ± 0.3461 A Untreated group 0.7512 ± 0.06310.7986 ± 0.08290.7435 ± 0.02860.8203 ± 0.0231Control group

0.6652 ± 0.0216

0.6200 ± 0.0332 B

0.6531 ± 0.0255 B

0.6251 ± 0.0200 B

Measurement of IgG

Before treatment, the amount of IgG in endometritis cow sera was slightly higher than that in the control group. After treatment, the amount of IgG was in-creased significantly (P < 0.01), but there was no change in the untreated group. The Yimu Shenghuasan prepa-ration had significantly increased the amount of IgG in endometritis cow sera, and strengthened animal immu-

nity (Table 3).

Measurement of IgA

Before treatment, the amount of IgA in endometritis cow sera was slightly higher than that in the control group (Table 4). After treatment, the amount of IgA increased significantly (P < 0.01), but there was no change in untreated group (Table 4).

1502DU Jin-liang et al.

screening results in vitro, gives an initial comprehen-sive evaluation for candidate drugs, in order to nar-row the screening scope.

In this experiment the inflammatory cells were treated with the different concentrations of herbal medi-cine preparation,Yimu Shenghuasan, and the expres-sion of CYP450 which related to the drug metabolism were studied. The results showed that the expression of CYP450 in endometrial cells was increased with the increase in dose. The drug had a satisfactory effect at 2000 μg mL-1, and 48 h after treatment. When the drug concentration reached to 10000 μg mL-1, the ex-pression of CYP450 was disappeared, it showed that the concentration was too high that caused serious in-jury and even death to the endometrial cells, so the ex-pression of CYP450 was weakened or disappeared. In this study, it was interesting that when the indi-vidual preparation of radix glycyrrhizae, angelica, motherwort,or honeysuckle were used to treat the in-flammatory cells, their induction on CYP450 were different, and sometimes even contrary.

Effects of herbal medicine preparation,Yimu Shenghuasan on the amount of IgG, IgA in endometritis of dairy cow

All major subtypes of immune globulin (Ig) are existed in uterine secretions (Li et al. 2009; Sharkey et al. 2008). IgA could combine the pathogens, in order to prevent the pathogens adhere on the surface of uterus mucosa. IgG could regulate and active complement, and comple-ment could also exude to the uterine cavity by serum which induced by mast cell degranulation and eosino-phil granulocyte, to participate the immune response in the uterine cavity.

Some papers showed that Chinese herbal medicine could improve the antibody level and immunity of ani-mal body. Epimedium preparation has been reported to improve the levels of sera hemolysin in mice, immu-nized with sheep red blood cells, and increase the num-ber of antibody producing cells and antagonize anti-body formation against cyclophosphamide (Hu et al. 1999). Similarly, Duan et al. (2005) reported that Chi-nese herbal medicine could significantly increase the IgG level in chicken.

In this experiment, 3 d after treatment of Yimu Shenghuasan preparation, the sera IgG, IgA in medica-tion group were significantly increased. This showed that Yimu Shenghuasan preparation could increase the sera Igs, and enhance humoral immune function. Thus, it enhances the resistance against some diseases in dairy cows.

CONCLUSION

In this study, the endometrial cells of cow were iso-lated successfully, and the endometrial cells inflamma-tory model of dairy cows was established. The inflam-matory cells and the cows were treated with Yimu Shenghuasan preparation. It was found that the Yimu Shenghuasan preparation had a satisfactory effect on enhancing the immune function in dairy cows. The results have made a foundation for the treatment of endometritis in cow.

Acknowledgements

The project was supported by the National Technology Support Project of China (2006BAD04A10-4).

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