The Effect of Green Tea on IL-6 and CRP level in Model of Polycystic Ovary Syndrome as an Inflammation State

Background: Having low-grade chronic inflammation state symptoms such as elevated C-reactive protein and interleukin-6 play a crucial role in polycystic ovary syndrome (PCOS). Green tea has antiinflammatory properties. This research evaluates the effect of green tea on inflammatory indexes. Adult Wistar rats were exposed to subcutaneous administration of estradiol valerate for PCOS induction. PCOS rats were divided into control and experimental groups received intraperitoneal injection green tea extract daily. Animals were anesthesia with chloroform. Ovary and serum were taken to measure the inflammatory markes using ELISA kits and Histomorphometric studies. The data were analyzed using One-Way ANOVA with significance level P<0.05. Results: The results indicated the significant reduction in inflammatory markers and significant changes follicular layers thickness in the treatment group as compared with control. Disscoution: it can be concluded that having anti-inflammatory substances, green tea is effective in symptoms of this syndrome and metabolic syndrome.

expression is basically regulated by Beta Kappa core factor (NK-kb) and interleukin IL-6 causes stimulus, regulates synthesis of acute phase protein, activates axis of hypothalamus -pituitary and disorder in signal transduction of glucose through changing activity of serine -threonine kinases.Interleukin-6 plays a key role in the pathogenesis of chronic inflammation, insulin resistance, and CV diseases [2].CRP is a member of pentraxinsfamily.As the most sensitive inflammatory indices, CRP plays an important role in immune response.In human, CRP gene lies on 1q21 to 1q23 chromosomes.CRP protein is produced by liver and it is almost the strongest inflammatory marker, as its increase is responsible for inflammation.Appearance of CRP is considered as a strong predicator for metabolic abnormality.This substance is produced in liver and intimal of vessels.Despite CRP's inflammatory role, using different mechanisms, such as producing nitric oxide (NO), increasing molecules adhesion, and changing absorption of low-density lipoprotein (LDL) by macrophages, it is able to damage vessels.The increase of this protein (as an independent predicator of CV risk) increases damages to CV system 2 -5 times.Its content is mainly regulated by some cytokines, especially 1L6 [2].PCOS is a reproduction abnormality together with a metabolic disorder.It is seen among 5-10 percent of women during the age of fertility and it is the major factor of infertility [3][4].Women with PCOS are chronically with anovulation; however, ovulation may occur spontaneously and pregnancy may sometimes occur.Irregular menstruation, which is one of the signs of PCOS syndrome, is usually continued since menarche, as the menstruation pattern never occurs [5].Biochemical disorders indicating PCOS, which are seen in patients, include increased serum concentration of the androgenic hormones such as testosterone, androstenedione, dehydroandrosterone, dehydroandrosterone sulfate, hydroxyl progesterone-17 produced in ovary [6], hirsutism, oligomenorrhoea, amenorrhea [7][8], abnormality in releasing gonadotropin together with the increase of LH secretion in proportion to FSH [5] In this syndrome, obesity and increase in body fat (accompanied by a central lipid distribution pattern, which is described as visceral obesity [9] and some disorders including sweet diabetes type-II, cardiovascular abnormalities and endometrial, breast cancer seem to be common [10].Moreover, it is followed by insulin resistance, increased insulin, and blood sugar drop [11].Green tea is a product made by leaves and sprouts of Camellia sinensis, which is almost the commonest drink after water.Green tea is a non-fermented tea [12].Flavonoids of green tea account for 36 percent of the dry weight of tea and it is the major part of theflavonoids is flavan-3-ols, which is called catechin.Two-hundred and thirty seven milliliters of green tea contains 30-130 mg epigallates.Catechins are strong in-vivo and in-vitro antioxidants.It also contains some minerals and vitamins that increase antioxidant potential of this type of tea [13].The biological effects of green tea that is based on the epigallate effects include cardiovascular diseases and some cancers, anti-hypertension effect, body weight control (weight reduction), antiviral and antibacterial activities, participation in anti-diabetic processes (through controlling saliva and intestine amylase that prevents starch breakdown).[14] Other effects include antimutagenic and anti-inflammatory properties, protecting against ultraviolet rays, increasing density of bone minerals and anti-fibrotic properties, reducing resistance against cholesterol and triglycerides, and reducing resistance against insulin [13,15].PCOS can be considered as a metabolic disorder accompanied by endocrinal disorder, as women with such syndrome suffer cardiovascular problems.Syndrome of insulin resistance was also seen in these patients.The correlation between pre-inflammatory cytokines and insulin resistance is a pathophysiological relationship and a guiding path for insulin resistance and cardiovascular illness followed by PCOS [16, 17 and 18].Moreover, prevalence of the illness among the obese people exceeds the thin people.Obese women show higher il-6-generating levels as compared with thin women.These data show that adipose tissue is a great source of IL-6 in women.In addition, polymorphism c-174g exists inside IL-6 promoter.It is shown in vitro that this gene is related to the increase of IL-6 level and hyperandrogenismin thin women.Therefore, http://www.ispacs.com/journals/ijcmb/2014/ijcmb-00014/International Scientific Publications and Consulting Services IL-6 can establish a relationship between anthropometric and syndrome of metabolic and hyperandrogenismin people with PCOS [19, 20, 21, 22 and 23].On the causes of PCOS, it can be stated that PCOS is an endocrine abnormality and many markers involve in its prevalence, including mutation in the genes encoding gonadotropin and/or mutation in inflammatory markers [24].We study the amount of inflammatory markers, as the recent studies extensively show that there is a close correlation between inflammation and chronic illnesses, especially insulin resistance and PCOS.There are many inflammatory factors involved in PCOS.The present study examines the effect of green tea extract as an inflammatory marker on il-6 and CRP, as many studies show that these factors are strong managers of inflammation system [24, 25 and 26].

Methods
This study was conducted on 96 Wistar rats with the average weight 200±20 gr.The rats were kept under the approximate temperature of 22˚C, 12:12 dark-light.Before drug administration, the rats were kept under the above conditions at least for 7 days so that they become accustomed to the environment completely.During this period, the animals consumed normal dish and sufficient water.The rats selected for induction had two successive regular periods of estrous cycle after preparing vaginal smear daily test for 12-14 days [16].Hormonal induction using estradiol valerate was applied in this project.For this purpose, 2 mg/kg estradiol valerate is injected subcutaneously.After injection, vaginal smear test is prepared daily.The test is continued as long as the changes of estrus cycle and its irregularity are appeared and we reach the stage of Persistent Vaginal Cornification (PVC), which is usually 60 days after injecting estradiol valerate.To confirm induction of syndrome, 2 ml blood sample is taken from a rat's heart.histomorphometric studies by the hematoxylin and eosin technique was performed.Ovaries were placed in bouin fixative for histological analysis; fixed samples were kept in alcohol solutions of 20 to 100% for a period of 45 min to dehydrate, and later in alcohol/xylen (50:50) and xylen (3 times) for clearing, and finally blocked in paraffin.Samples were sliced to sections of 7 micron thickness using a microtome, and these sections were placed on slides, previously coated with gelatin, prior to staining with hematoxylin-eosin for histological observations.After syndrome confirmation using the measurement of hormone levels and the histomorphometrical studies on part of an inducted group, the remaining rats were divided into five experimental groups: The PCOS rats that received no injections (PCOS control),The rats that received saline (sham), The PCOS rats that received of 50 mg/kg of green tea extract (GT-Treated-1), The rats that were received intraperitoneal injection 100 mg/kg of green tea extract (GT-Treated-2), The rats that were received intraperitoneal injection 200 mg/kg of green tea extract (GT-Treated-3) for 14 days, consequently.

Green Tea Extraction
The green tea was collected in Lahijan city located in Gilan province.The collected samples were dried in the shade and its leaves were removed from the plants' stems.Then, they were pulverized to a powder form by mechanical means.To prepare hydro-alcoholic extract, 200 gram of the powder of the dried green tea leaves was soaked with 1500 ml of ethyl alcohol for 48 hours.The obtained solution was evaporated after it was filtrated in the constant flow of the air.Then, it was completely dried.Thereafter, dried extract was collected.Then, the hydro alcoholic extract of the green tea was used to prepare the desired dose of the treatment [16].

CRP Measurement
After taking blood samples from hearts of the rats and preparing blood serum using anELISA kit (Millipore's MILLIPLEX® MAP Rat/Mouse CRP Single Plex USA), CRP contents were measured.Serological analysis was carried out to measure IL-6 serum level and hormonal changes.An ELISA kit rat IL-6 platinum ELISA/ (Bender Medsystems, Vienna, Austria) was used to diagnose IL-6 serum level as per the manufacturer's instructions.This method was iterated 3 times.The results expressed as (pg/ml) MEAN±SD were used for individual rats.Sensitivity of this method for IL-6 was 12 pg/ml.First, all the wells washed using buffer solution 1%.Then add 100 cc assay buffer to the blank wells, 50 cc assay buffer and 50 cc serum to sample wells.Then 50 cc biotin was added to all wells and put them under the room temperature (RT) for 2 hours.Next, wash the wells using buffer solution 6 times and add 100 cc HRP and leave them for 1 hour RT.After washing, add 100 cc TMB to the wells for 10 minutes RT.Then add 100 cc Stop solution to them and read the stripe using an Elisa Reader Machine at a wavelength of 450 nm.

IL-6 assay
In this study, PCOS induction led to a significant rise in IL-6 inflammatory index (p < 0.001 vs. control rats).The effect of green tea (100mg/kg) on the level of IL-6 in PCOS rats was examined for 14 days after complete induction of PCOS.As shown in Figure 1, the respective IL-6 levels in control, PCOS and Green tea-treated PCOS rats were 50, 100 and 200 pg/mL.Our study indicated that green tea reduces IL-6 content in the green tea-treated groups as compared to the control group.It also indicated that green tea with dose of 100 mg/kg BW is more effective than other doses and such effectiveness is significant as compared to 50 and 200 mg dosage (Fig1).
CRP is an exquisitely sensitive systemic marker of inflammation and tissue damage.The cute-phase response comprises the nonspecific physiological and biochemical responses of endothermic animals to most forms of tissue damage, infection and inflammation.Plasma CRP is produced only by hepatocytes, predominantly under transcriptional control by the cytokine IL-6, although other sites of local CRP synthesis and possibly secretion have been suggested.PCOS induction led to a significant raise in this systemic inflammatory index, and its reduction in rats treated with green tea was significant (Fig 2).

Histological Finding
Examining morphology of the tissue sections prepared from ovaries in the experimental group of follicles at different stages including graph showed that the follicles had thick granular layer and thin theca compared to the PCOS control group.Moreover, a significant number of corpus luteum, indicators of ovulation, was observed.However, greater number of cystic follicles was observed in the PCOS control group.They had thin granular layer and thick theca.Moreover, low number of corpus luteum was observed which indicated that injection of estradiol valerate induced polycystic ovaries reduced the number of active follicles as well as ovulation (Fig3).c,d,e), a decrease in the number of cysts and the ovarian stroma level, and the appearance of a corpus luteum can be detected in the experimental group receiving 50,100,200mg/kg of green tea respectively.X400.

Discussion
In the present study, we investigated the inflammation as the main feature of PCOS and the therapeutic effect of Green tea on PCOS.PCOS is a complex and diverse abnormality indicating one or many abnormalities within a phenotype.One spectrum of these abnormalities can be studied in the field of metabolic processes, as it is seen the patients with PCOS who are usually obese, insulin resistance, hyperinsulinemia, and diabetes type II (27,28).The following reasons indicate that they play a crucial role in creation and intensification of PCOS symptoms with the following reasons: First, CRP is one of the major proteins of inflammation, which is secreted by liver in response to the increase of inflammatory cytokines such as Interleukin (IL)-1 beta, IL-18, IL-6 [29].High concentration of CRP, which is related to the risk of CV diseases, is seen in women with PCOS.According to the study of Nabiuni et al, the rate of this protein in the polycystic rats induced by estradiol was significantly high .Second, the increase of c-174g factor expression, which is in the Il-6 gene promoter area and regulates the activity of promoter, due to the increase of il-6 level, intensifies insulin resistance, obesity, and hyperandrogenism.These are the symptoms common between PCOS and metabolic syndrome (MBS) [19, 20, 21, 22 and 23].Third, free fatty acids are the basic ligands for toll like receptors that are central regulators of innate immune receptors.By increasing lipolysis process and consequently increasing free fatty acids, androgens cause inflammation.Therefore, free fatty acids act as a direct connection between hyperandrogenism (well-known factor of PCOS) and inflammation (one of the features of metabolic syndrome) [32].Regarding the close correlation between MBS and PCOS, it seems that we may reduce fertility, CV, and inflammatory problems by controlling the factors involving creation and development of these two syndromes.The major factors involving in both syndromes include interleukin-6 and CRP.Various studies indicate that CRP and IL6 in women increase PCOS [33,34].A group realized that they increase in women with PCOS and others believe that they increase in women with PCOS suffering from obesity and insulin resistance [35].The genetic studies indicate that single nucleotide polymorphisms (SNPs) are in il-6 and CRP genes.With the plasma levels of il-6 and CRP increasing, they increase [36].
In PCOS model, blood mononuclear cells (PBMCs) migrate into a developing adipose tissue.They are then differentiated between the macrophages secreting cytokines such as TNF [37].In addition, in vascular smooth muscle cells and CRP adipocytes cells (that increase in PCOS), TNF and IL6 are produced by activating signaling path of p38 MAPK-TLR4.According to the studies of Spaczynskiet al., tumor necrosis factor alpha (TNF-α), which is produced from these two paths, causes proliferation and differentiation of single cells and increase of follicular cell layer, which is seen in PCOS [38].The IL-6, which was released from adipose tissues, increases expressions of glucose transporter GLUT4, insulin receptor substrate 1 (IRS-1), TNF-a, and suppressor of cytokine signalling-3 (SOCS3) in hepatocytes and induces insulin resistance through decreasing lipogenesis and inhibiting glycogen synthesis.Moreover, the adipose tissue itself releases Nonesterified fatty acids (NEFAs) that increases capacity of muscle, liver and pancreatic beta cells.In the presence of lipid, NEFA also increases insulin resistance.Ability to create inflammatory states by IL6 can be discussed from two perspectives: Releasing IL-6 and TNF-α and free fatty acids excites JNK and IKK-B/NF-KB path.Therefore, inflammatory mediators develop incremental adjustments.White adipose tissue (WAT) releases chemokine ligand (CCL) 5, CCL2, and CCL8 through LY6C monocytes that increase classically activated macrophages (CAMs) and inflammatory phenotype.It is interesting that item increases insulin resistance and item reduces sensitivity to insulin [39].In 1990, Watson some evidence that proved IL-6 secretion by ovary [40].Basic production of IL-6 occurs in culture of primary ovarian tumors and ovarian cancer cell.In addition, IL-6 is produced from numerous granulosa cells of many species.IL-6 production in granulosa cells was shown in various studies on rats, cows, rabbits, and humans.IL-6 production in granulosa cells is excited by FSH, IL-1, and lipopolysaccharide (LPS) [41].In 1998, Keck et al. proved that human granulosa cells produce IL-6 and have IL-6 receptors.Therefore, IL-6 is able to affect the activity of granulosa cells and specifically the steroidogenesis of these cells through the autocrine mechanism.Interfering IL-6 in regulating production of granulosa steroids in animals was proved [42].Relying on these studies, we can create a strong link between syndrome symptoms, including imbalance in releasing pituitary and ovarian hormones and changing at il6 level that result in intensification.The changes were proved by the results of this study and the studies of the earlier authors of the present research.According to the literature, it was proved that PCOS has a close relationship with metabolic syndrome and inflammation (cardiovascular disorders (CVD), cytokine increase, disorder of hormones level of hypothalamus -pituitaryovarian axis, and insulin resistance).Therefore, it is probable that treating the rats with green tea would lead to adjusting these syndromes to improve PCOS.The relationship between mortality caused by CVD and consumption of green tea was highly impressive during last decade.In this regard, reduction of consuming10 green tea cups a day in a Japanese society caused 42 percent of the mortality caused by CVD in men.This was more effective in women.In addition, a reduction of 37 percent of mortality caused by infarction and myocardial infarction was observed.The studies show that the use of green tea extracts capsules for 12 weeks reduces blood CRP in human studies [43].In the study of Sentle et al; EGCG oral consumption, the major catechin of tea, considerably reduced CRP in the rats with high-fat diet [44,45].Due to inhibiting NF-kB and ap1, epigallates inhibits TNF expression.Fellaini's study showed that after treating the transgenic rats with high expression of TNF in their lungs with green tea, TNF and interleukin reduced expression for 70 percent and 80 percent, respectively both at mRNA level and protein level [46].Oxidative Stress activates MARKs p38 and JNK, which plays a key role in regulating production of inflammatory cytokine.EGCG of green tea inhibits both JNK and p38 through inhibiting transcription factors of Ap-1 and NF-kB.It also inhibits p38 inducing interleukins and JNK activity and changes in binding affinity of AP-1 to DNA.In addition, EGCG can retard cytokines expression through affecting down-regulate MRNA [47,48,49].Epigallates inhibit proliferation of adipocytes and their differentiation in 3t3-l1 cells.It also inhibits lipid oxidation, increases glut-4 in animal models, improves energy in human, and inhibits induction of cytokine-induced beta cells.Anti-obesity effects of epigallates are due to increasing adiponectin expression.Catechin gallate decreases level of blood glucose through affecting alimentary canal and blocking glucose received from tissues; the latter partially increases glucose contents.Epigallate (the major compound of green tea) increases sensitivity to insulin and reduce hepatic glucose [50].

Conclusion
In the present study, reduction of cytokine level of the suffering rats indicates the effectiveness of epigallate on the female rats, which had a collection of a serious symptom, including hormonal imbalance and histological and metabolic abnormalities.With a view to the earlier study of the same author, whose data have not been released yet, some significant changes indicating histomorphometric and hormonal are obvious.It is expected that green tea is a method with the minimum side effects for pregnancy of those with PCOS and lifestyle-related illnesses.

Figure 3 :
Figure 3: Histological analysis showing normal (a),PCOS(b) and green tea extract -treated PCOS (c,d,e) ovaries.Ovarian sections were stained with hematoxylin and eosin.An increased in the number of cysts (arrow), lack of corpus luteum (arrow head), and increased levels of ovarian stroma (star) in b is compared to a.In part(c,d,e), a decrease in the number of cysts and the ovarian stroma level, and the appearance of a corpus luteum can be detected in the experimental group receiving 50,100,200mg/kg of green tea respectively.X400.