Xingyao Long,Yanni Pan,Xin Zhao,*

a Chongqing Collaborative Innovation Center for Functional Food,Chongqing University of Education,Chongqing 400067,PR China

b College of Food Science,Southwest University,Chongqing 400715,PR China

c School of Biological and Chemical Engineering,Chongqing University of Education,Chongqing 400067,PR China

ABSTRACT This research the preventive effect of Kudingcha polyphenols(Kp)on colitis based on animal experiments.Experimental mice divided into four groups,including normal group,model group,low-concentration Kp(LKP)group and high-concentration Kp(HKP)group,and they all smeared and given 0.15 mL 1%oxazolone solution by lavage to induce BALB/c mice colitis.DAI,colon weight/length ratio,serum levels of cytokines,related antioxidant activities of colon tissues such,and the mRNA expressions.The experimental results show that KP can significantly(p ＜0.05)reduce DAI in mice with colitis and inhibit shortening of colon caused by colitis,increase colon weight/length ratio.It can also significantly reduce(p ＜0.05)the activities of MPO,NO,MDA in mice colon tissues with colitis,increase the activity of GSH,and significantly increase(p ＜0.05)the level of IL-2 and decrease the level of IL-10 in mice serum with colitis.The results of qPCR assay show that Kp can significantly increase(p ＜0.05)the expression of Cu/Zn-SOD,Mn-SOD,GSH-Px,CAT,c-Kit,SCF and decrease the expression of IL-8,CXCR2 in mice colon tissues with colitis.Kp has a good preventive effect on colitis,and high concentration of PT has better preventive effect.

Keywords:Kudingcha polyphenols Oxazolone Antioxidant Colitis mRNA expression

1.Introduction

Ulcerative colitis (UC) is an inflammatory bowel disease with a potential risk of cancer,onset age is between 20 and 50 years old,it seriously threatens the quality of life [1].The pathogenesis and treatment of UC are the focuses of researches and an important principle to control and treat UC is to discover and maintain it without deterioration[2].In clinical treatment drugs for UC can cause side effects after long-term use,so another important aspect of UC research is to look for functional food that can alleviate UC but have no side effect[3].Oxazolone can be used to build experimental colitis,and oxazolone-induced colitis is a type of IL-4 mediated Th2 inflammation,whose histological features and inflammatory distribution are similar to human UC.The oxazolone mice colitis model can be used as a tool to study the inhibition effect and mechanism of functional food on UC[4].

Oxygen free radical(OFR)mainly includes superoxide anion radical (O2－) and hydroxyl radical (OH－).In physiological condition,there are defense systems to remove OFR in our body,including antioxidant enzymes(SOD and GSH-Px)and antioxidants(vitamin C,vitamin E and carotene).So the weakened antioxidant system or overproduced OFR can cause damage to tissues and cells[5].During UC,a large number of phagocytes are activated and oxygen consumption increases on intestinal diaphragm.A large number of O2－are produced under the effect of xanthine oxidase and arachidonic acid.Covalent binding between OFR and biological membrane unsaturated fatty acids activates a chain reaction in body,causing lipid peroxidation reaction,producing oxidative damage to OFR and decomposing MDA,which decreases membrane fluidity and increases permeability [6].Internal flow of a large number of Ca2+and the metabolism of arachidonic acids form highly-bioactive inflammatory mediators,such as prostaglandins,thrombin,leukotriene,etc.,which aggravates the injury of UC intestinal mucosa.The traditional medicines for UC are salicylic acid and hormone.Although they have many anti-inflammatory functions,they have great side effects [7].Nowadays,the focus of research is to find natural antioxidants in order to use its functional groups with reducibility in molecules to directly scavenge free radicals and lipid peroxidation free radicals,thus preventing and restraining oxygen free radical reactions and lipid peroxidation reactions to inhibit UC[8].Polyphenol in plants is a kind of active ingredient,and it has been confirmed that it can scavenge free radicals and attain lipid peroxidation free radicals in peroxide process,breaking the chain of free radical oxidation reactions and effectively removing free radicals in body,thus inhibiting inflammation[9].

Taking KP as research object,this study observe the prevention effect of KP on oxazolone-induced colitis and observe the mechanism of KP inhabiting experimental colitis by using antioxidant function,so the results will accumulate certain theoretical basis for the further development of KP,which is helpful to the development and utilization of KP.

2.Materials and methods

2.1.Kudingcha polyphenols extraction

The 100 g Kudingcha broke into powder and added into 100 mL 45%(volume ratio)ethanol solution.The mixed solution extracted under 90°C for 30 min,the extraction and the water extracts.pH of the water extract adjusted to 6.0 and added into 160 mL AlCl3(6g)and ZnCl2(12g)mixed precipitants for precipitation.The mixture centrifuged at 3000 r/min for 10 min and added 200 mL hydrochloric acid with 12% volume ratio for precipitation.The precipitate dissolved to collect supernatant,and then 200 mL ethyl acetate into the supernatant for 2 times for extraction.In the end,polyphenols extract obtained by rotary evaporation of the liquid[10].

2.2.Animal experiment

The 50 BALB/c mice randomly divided into 5 groups,including normal group,model group,SASP (sulfasalazine) group,lowconcentration KP (LKP) group and high-concentration KP (HKP)treatment group,with 10 in each group.Normal group and model group normally fed without any treatment while the other three groups respectively given SASP,LKP or HKP with the concentration of 500 mg/kg,250 mg/kg and 500 mg/kg for 2 mL by lavage per day for 26 d.On the 15thday,all mice shaved 2 cm×2 cm in abdomen.Normal group smeared with 0.2 mL ethanol(99%)while the other groups smeared with 0.2 mL oxazolone solution(3%mass ratio and 99%ethanol as solvent).On the 19thday,after fasted for 24 h,mice narcotized with 0.1 mL/10 g chloral hydrate,and then inserted into the blunt end of a silicone tube from the anus to 3.5 cm deep of mice intestine.Normal group injected 0.15 mL 50% ethanol solution while other groups injected 0.15 mL 1% oxazolone solution(50% mass ratio and ethanol as the solvent).The catheters pulled out after 20 s,meanwhile,mice inverted for 30 s[11].On the 26thday,all mice put to death to collect their plasma.Then the weight and length of the colon were measured.At the same time,disease activity index(DAI)determined by the formula DAI=(weight loss score+stool state score+hematochezia score)/3(Table1).

Table1 The standard of DAI score.

2.3.Determination of MPO,NO,GSH and MDA in colon tissue

Clean mice colon tissues mixed with saline nine times heavier than the colon.The colon tissue homogenate by using ultrasonic grinding,and the content of MPO,NO,GSH and MDA in mice colon tissue determined according to kits (Nanjing Jiancheng Bioengineering Institute,Nanjing,Jiangsu,China)instructions.

2.4.Determination of cytokine IL-2 and IL-10 in serum

The cytokine levels of IL-2 and IL-10 in mice determined by ELISA method according to kits(Abcam,Cambridge,Massachusetts,USA)instructions.

2.5.qPCR assay

The mice colon homogenate is added into RNAzol to extract total RNA of colon tissues.Then the total RNA concentration is diluted into 1 μg/μL.The 2 μL diluted total RNA extraction is in turn added into 1 μL oligodT18,RNase,dNTP,MLV enzyme and 10 μL 5×buffer(Thermo Fisher Scientific,Waltham,MA,USA)to synthesize cDNA under the condition of 37°C for 120 min,9°C for 4 min and 4°C for 3 min.The expression of mRNA of Cu/Zn-SOD,Mn-SOD,GSHPx,CAT,c-Kit,SCF,IL-8 and CXCR2 (Table2) are amplified by the method of qPCR(94°C 5 min for one time,94°C 30 s,58°C 30 s,72°C 30 s for 30 times,72°C 5 min for one time)with housekeeping gene GAPDH as internal amplification.The relative transcription levels of the mRNAs were calculated according to the 2－ΔΔCrformula[12].

2.6.Statistical analysis

The experimental determinations for three times and then average the results using SAS9.1 statistical software.Data of each group by one-way ANOVA method to see whether there significant differences between different groups whenp＜0.05.

3.Results

3.1.DAI value of mice

As shown in Table3,at the 20th,22nd and 26th in the experiment,the DAI values of mice in model were higher than mice in other group at the same time.SASP and KP could reduce the DAI values,and the HKP showed the stronger reducing effects than LKP.

Table2 Sequences of qPCR primers were used in this study.

Table3 DAI value of each group mice.

Table4 Colon length and colon weight/colon length of each group mice.

3.2.Colon length and colon weight/colon length

As shown in Table4,the colon length and colon weight/colon length of mice in normal group were largest,but model group mice showed the least colon length.The colon length and colon weight/colon length of mice in SASP group were smaller than those of mice in normal group,and larger than those of mice in LKP,HKP group.Meanwhile,HKP treated mice showed the larger colon length and colon weight/colon length than LKP treated mice.

3.3.Levels of colon tissue

As shown in Table5,the MPO,NO,MDA levels of colon tissue in mice of model group were highest,but GSH level was lowest,and the normal group mice showed the crosscurrent.After treatment with SASP and KP,the MPO,NO,MDA levels were reduced and GSH was raised,HKP treated mice had the lower MPO,NO,MDA levels than LKP treated mice,but higher than SASP treat mice; and HKP treated mice had higher GSH level than LKP treated mice,but lower than SASP treat mice.

Table5 MPO,NO,GSH and MDA colon tissue contents of each group mice.

3.4.Cytokine levels in serum

As shown in Table6,the IL-2 level in mice of normal group was highest,but IL-10 level was lowest.After inducing colitis,the IL-2 level was reduced and IL-10 was raised,SASP increased the IL-2level and decreased the IL-10 level compared to untreated colitis mice(control group).HKP also could increase the Il-2 and decrease IL-10 level compared to the control group,and these abilities were stronger than LKP but weaker than SASP.

Table6 IL-2 and IL-10 serum levels of each group mice.

3.5.Cu/Zn-SOD,Mn-SOD,GSH-Px and CAT mRNA expression in colon tissue

As shown in Fig.1,the Cu/Zn-SOD,Mn-SOD,GSH-Px and CAT mRNA expression in colon tissue of mice in control group were weakest,but these expressions in mice of normal group were strongest.After treatment with SASP and KP,the Cu/Zn-SOD,Mn-SOD,GSH-Px and CAT expression of mice in HKP group were stronger than those of mice in LKP group,but weaker than those of mice in SASP group.

3.6.c-Kit,SCF,IL-8 and CXCR2 mRNA expression in colon tissue

As shown in Fig.2,the c-Kit,SCF mRNA expressions in colon tissue of mice in control group were weakest,but the IL-8,CXCR2 expression in mice of control group were strongest.In order,the c-Kit,SCF expression of mice in normal group,SASP group,HKP group and LKP group were higher than the mice in control group,respectively;but the IL-8,CXCR2 expression were weaker than the mice in control group,respectively.

4.Discussion

Colitis can result in weight loss,diarrhea and bleeding,etc.DAI can measure the degree of colitis in terms of weight,fecal trait and hematochezia [13].DAI shows that KP can reduce the symptoms of oxazolone-induced colitis and the effect becomes more obvious with the increase of KP concentration.Colon length and colon weight/length ratio are also standards of colitis degree,because the colon length of colitis mice is shorter than that of normal mice and colon weight/length ratio is also lower[14].

The increase of MPO activity in colon shows that the concentration of neutrophils in inflammatory tissues decreases.In the process of colon inflammation,iNOS will produce NO,which aggravates the lesion of colonic tissues.Besides,as NO increases,MPO activity will also increase,thus worsening the inflammation [15].Colitis can also produce a large number of reactive oxygen(ROS)and reactive nitrogen(RNS),which can then cause oxidative stress toxicity reaction and inflammation in colon tissues[16].After inflammation,the large number of ROS will destroy the balance between oxidants and antioxidants in body and reduce the content of GSH in colon tissues[17].Large numbers of lipid peroxidation will increase the production of its end product MDA[18].

Fig.1.The mRNA expression of Cu/Zn-SOD,Mn-SOD,GSH-Px and CAT of colon tissue in mice.a–e Mean values with different letters in the same bar are significantly different(p ＜0.05) according to Duncan’s multiple-range test.SASP:sulfasalazine dose (500 mg/kg); LKP:low concentration Kudingcha polyphenols dose (250 mg/kg); HKP:high concentration Kudingcha polyphenols dose(500 mg/kg).

Fig.2.The mRNA expression of c-Kit,SCF,IL-8 and CXCR2 of colon tissue in mice.a–e Mean values with different letters in the same bar are significantly different(p ＜0.05)according to Duncan’s multiple-range test.SASP:sulfasalazine dose(500 mg/kg);LKP:low concentration Kudingcha polyphenols dose(250 mg/kg);HKP:high concentration Kudingcha polyphenols dose(500 mg/kg).

Oxazolone-induced colitis is a kind of inflammation mediated by Th2 cells and the mechanism is imbalance between Th1/Th2 immune networks [19].Cytokine IL-2 and IL-10 are respectively produced by Th1 and Th2 cells and directly related to colitis,so too low level of IL-2 and too high level of IL-10 mean the worsening of colitis[11].

Ulcerative colitis show not only diarrhea and hemafecia,but also colon power disorder.Studies have shown that ICC(interstitial cells of Cajal)are associated with colon power,thus directly involved in the process of colitis [20,21].During inflammatory intestinal diseases,SCF has a direct effect to maintain the number and function of ICC.SCF is the natural ligand of c-Kit,so if the path of SCF/Kit signal is damaged,the proliferation and differentiation of ICC will decrease,thus aggravating colitis[22].

IL-8 has inflammatory activity and chemotaxis and CXCR is the receptor of IL-8.So IL-8 and CXCR2 are both related to colon cancer and CXCR2/CXCL7 has prognosis value for colon cancer.In the liver metastasis of colon cancer,disease-free survival and overall survival for patients with high expression of CXCR2/CXCL7 will shorten[23].In addition,studies have confirmed that the expression of IL-8 and CXCR2 will increase when colitis occurs,and similar results are obtained in this study.

SOD is an important antioxidant enzyme system in body,including Cu/Zn-SOD,Mn-SOD,etc.,which can effectively eliminate oxygen free radicals,inhibit lipid peroxidation in intestinal tissues and stabilize cell membrane [24].The activity of SOD indirectly reflects the ability to remove oxygen free radicals in body.When ulcerative colitis occurs,a large number of phagocytes in the intestinal mucosa circulate from the blood into the intestinal mucosa and submucosa and part into the intestines.When they swallow foreign matters or are activated,oxygen consumption increases and a large number of O2－are produced under the influence of NADPH oxidase,NADH oxidase,xanthine oxidase and arachidonic acid.O2－can produce non-free radical hydrogen peroxide under the influence of SOD and reduce the damage of intestinal mucosa by SOD[25,26].

The activity of antioxidant enzymes,such as SOD and CAT,on colonic mucosa,submucosa and muscularis mucosae are low,while the activity of xanthine oxidase is high,which reduce OFR removal,increase OFR and aggregate UC.So regulating the increase of CAT can alleviate UC[27].GSH-Px is a kind of important enzyme to catalyze the decomposition of hydrogen peroxide,which widely exists in body and can restore harmful peroxide to harmless hydroxyl compounds,thus protecting the structure and function of cell membrane from the damage caused by oxidation of hydrogen peroxide and protecting colonic mucosa[28].

According to research about the inhibition effect of KP on oxazolone-induced colitis in mice,the experimental results show that KP can significantly reduce DAI in colitis mice,inhibit colon length shortening caused by colitis and improve colon weight/length ratio.KP can significantly reduce the content of MPO,NO,MDA and IL-10 in colon tissues of mice with colitis and increase the level of GSH and IL-2 in serum.The detection of mRNA level in colon tissues also finds that KP can up-regulate the mRNA expression of Cu/Zn-SOD,Mn-SOD,GSH-Px,CAT,c-Kit,SCF and down-regulate the expression of IL-8,CXCR2.As a result,KP can effectively prevent oxazolone-induced colitis and can be used as the raw material for functional food,so it has great value for utilization and development.

Acknowledgments

The present research was supported by the Program for Innovation Team Building at Institutions of Higher Education in Chongqing(CXTDX201601040) and the Introduction of High-level Personnel Research Start-up Fund of Chongqing University of Education(2013BSRC001),China.