Effects of acupuncture and electroacupuncture on estradiol-induced inflammation and oxidative stress in health rodents 1

PURPOSE: To investigate the effects of classical acupuncture (Ac) and electroacupuncture (EAc) on estradiol-induced inflammation and oxidative stress in health rodents. METHODS: Twenty-four eight-week old female rats were treated with estradiol valerate (EV) 4.0 mg i.m. single dose and randomly assigned to four groups (n=6): G1(control), G2 (Ac), G3 (EAc 2 Hz) and G4 (EAc 100 Hz). After 60 days all rats were anesthetized with chloral hydrate 10% (0.1 ml/30 g weight of the animal) and submitted to Ac/EAc for twenty minutes. The procedures were repeated on days three, five, seven and nine of the study. The equivalent of the human right ST-36 (Zusanli) and SP-6 (Sanyinjiao) acupoints were chosen for needling and electrical stimulation. On the 10th day of the experiment, all rats were anesthetized for collection of blood and tissues (ovaries) samples for biochemical analysis and histological examination. RESULTS: Glutathione (GSH) and malonaldehyde (MDA) concentrations increased significantly in all groups (plasma and ovary) while myeloperoxidase (MPO) activity decreased significantly in all groups compared with control group (G1). CONCLUSIONS: Both classical acupuncture and electroacupuncture decrease systemic and local oxidative stress and ovary inflammation in healthy rats exposed to estrogenic stimulation. EAc enhances lipid peroxidation at systemic and local levels in female rats exposed to estrogenic stimulation.


Introduction
Reactive oxygen species (ROS) are generated in all aerobic cells and include superoxide radical (O 2-), hydrogen peroxide (H 2 O) and hydroxyl radical (OH 2 ) 1 .Mitochondria are not only responsible for energy (ATP) production, but also are a major source of reactive oxygen species (ROS) 2 .Under physiological circumstances, there is a balance between antioxidants and oxidants produced by aerobic cellular systems.Because aerobic cells possess antioxidant systems to trap and/or inactivate such oxygen intermediates, oxidative stress is the result of an imbalance between the production of ROS and their inactivation by the protective systems 1 .
There are now numerous reports showing that estrogen and estrogen-like compounds are effective in protecting against a wide variety of insults in numerous different cell types 3 .Estrogens effectively prevent pro-oxidant stress in the mitochondria 4,5 .Tugay et al. 6 studied the effects of dexamethasone on oxidant/antioxidant status in kidneys of rats administered mercuric chloride and concluded that dexamethasone injection may partially protect the rodent kidneys against oxidative reactions by preventing the increase in xanthine oxidase activity.Estrogen has been shown to exert antioxidant properties in rodent hearts exposed to ironstimulated lipid peroxidation 7 .In aging liver oxidative stress increases due to the decrease in antioxidant bio-molecules such as estrogens which can be modified by hormonal replacement therapy.Long-time estrogen supplementation prevent the increase in lipid peroxidation induced by aging 8 .
On the other hand, adverse effects of steroids have been demonstrated .Dexamethasone is known to induce free radical toxicity in humans and animals [9][10][11] .Increased ROS generation resulting in liver oxidative stress has been demonstrated in sea bass exposed to 17β estradiol 12 .Recent experimental studies have demonstrated that chronic estradiol exposure induces oxidative stress in the hypothalamus of rats 13 .Long-acting compounds such as estradiol valerate (EV) may develop polycystic ovary syndrome (PCOS) in rodents with a single intramuscular (i.m.) injection 14,15 .
Whereas steroids may induce oxidative stress and that the use of acupuncture (AC) or electroacupuncture (EAc) can attenuate the oxidative stress in different organs and tissues such as liver and kidneys 16 and random skin flaps 17 in anesthetized rats, this study aimed to investigate the possible protective effects of Ac and EAc stimulation in young female rats exposed to a single high dose of EV.The equivalent of the human right ST-36 (Zusanli) and SP-6

Approval
(Sanyinjiao) acupoints were chosen for needling and electrical stimulation.The acupoint nomenclature used follows WHO nomenclature 18 .ST-36 acupoint is located 5mm below the head of the fibula under the knee joint, and 2mm lateral to the anterior tubercle of the tibia.SP-6 acupoint is located 5mm above the medial malleolus, about an imaginary line connecting this point to another point in the depression below the medial condyle of the tibia 19 .

Experimental groups
Animals were anesthetized intraperitoneally with a fresh-prepared solution of chloral hydrate 10% (0.1ml /30g body weight).Twenty-four rats were randomly assigned to 4 equal groups (n=6) as follows: or 100 Hz (Group 4), with duration of two seconds and pause for two seconds were applied for 20 minutes.Ac / EAc applications were repeated every other day, at the same time, for a total of five applications, held on one, three, five, seven and nine days of the experiment.Group 1 rats served as positive control and did not receive any additional treatment.On the 10th day of the experiment, all rats were anesthetized as described.A midline laparotomy was performed for blood (3.0 ml) and both ovaries were harvested for biochemical analysis and histological examination.
Tissue samples were snap-frozen in liquid nitrogen and stored in glass tubes at -70º until subsequent preparation and analysis of liver and kidney homogenates.Lipid peroxidation was assayed by measuring malondialdehyde as TBA-reactive substances 20 .
In brief, H 3 PO 4 (1%, 3 mL) and aqueous TBA solution (0.6%, 3 mL) were added to the 10% homogenate (0.5 mL).The assay medium was shaken and heated on a boiling-water bath for 45 min.After cooling, 4 mL of n-butanol was added and the mixture shaken.After separation of the n-butanol layer by centrifugation at 1200g for 15 min, its optical density was determined in a spectrophotometer (Beckman DU 640 B; Beckman Instruments, now Beckman Coulter, Inc., Fullerton, CA, USA) with 535 and 520 nm as absorption wavelengths, respectively.The difference between the results of the two optical density determinations was taken as the TBA value and the amount of malondialdehyde (MDA) in the testis was calculated, comparing with MDA standards and expressed as micromoles of MDA per gram of wet tissue.GSH levels were estimated by the method of Sedlak and Lindsay 21 which is based on the reaction between thiol groups and 5-5-dithiobis-(2-nitrobenzoic acid) to produce a compound that absorbs light at 412 nm.The amount of GSH was determined from a standard curve simultaneously obtained under the same conditions with various concentrations of GSH.

MPO activity
In brief, after collection, the sample was placed in an Eppendorf tube with cold buffer (0.

Effects of VE on ovary structure
No histological findings compatible with PCOS was identified in rats treated with VE (Figure 1).

GSH assay
Plasma GSH concentrations increased significantly in all groups compared with control (Figure 2).Tissue (ovary) GSH concentrations increased significantly in rats submitted to EA 2Hz and EA 100Hz (p<0.001),compared with control (Figure 3).

MPO activity
MPO activity decreased significantly in all groups compared with G1 (Figure 6).

Discussion
Sex steroids play a very important role both in the female reproductive system as in the male, through its regulatory effect in the various sexual and reproductive endocrine functions.These steroid hormones, particularly estrogen, are important for the development of the sexual phenotype, morphology of the reproductive tract and sexual differentiation of the central nervous system.In mammals, sex steroids play a key role in regulating the hypothalamic-pituitary-ovarian, ovarian folliculogenesis, ovulation and embryo implantation 22 .Many studies have indicated that dexamethasone induces free radical toxicity in humans and animals [9][10][11] .Recent experimental studies have demonstrated that chronic estradiol exposure induces oxidative stress in the hypothalamus of rats 13 .PCOS has been experimentally induced with a single dose of EV (4 mg) applied to virgin adult cycling Sprague-Dawley rats 14 .In another study 15 8-week-old rats receiving an i.m. injection of EV 4 mg developed PCOS.Sasikala et al. 23 treated female rats with letrozole (an inhibitor of the aromatase enzyme, responsible for the conversion of androgens into estrogens) for 28 days, resulting in alterations compatible with PCOS Surprisingly, healthy rats subjected to a single high dose EV presented no significant alterations in ovarian structure in this study.The explanation for the absence of the expected development of PCOS in female rats exposed to EV, using the same doses as other authors [14][15] is not clear.
The equivalent of the human right ST-36 (Zusanli) and SP-6 (Sanyinjiao) acupoints were chosen for needling and electrical stimulation.The Zusanli and Sanyinjiao acupoints are located on the femoral quadriceps muscle (in the belly of the anterior tibialis muscle) and on the digitorum profundus flexor muscle, respectively, innervated by branches of the emerging nerve roots from L4 to S1, and is related to somatic and autonomic innervation of pelvic segments, responsible for the regulation of its various neuroendocrine and sensory functions 24 .Ai-Hui et al. 25 mapped the territories of C fibers of the superficial peroneal, sural and tibial saphenous nerves in rats, and verified that the rats hind legs are anatomically identical to the lower limbs of human beings, thus justifying the identification of the acupuncture points in the rodent, which would occupy position and action similar to points in humans.
Yu et al. 26 evaluated the role of needle stimulation of four different acupoints: GB-34 (Yanglingquan), LR-3 (Taichong), ST-36 (Zusanli) and SP-10 (Xuehai) acupoints on regulating oxidative stress in the nigrostriatal system in the 6-hydroxydopamine lesioned rat and concluded that acupuncture stimulation prevented the reduction of GSH level as well as the increase in MDA level.
In our study, the stimulation of two acupoints, ST-36 (Zusanli) and SP-6 (Sanyinjiao), using manual Ac and EAc with low (2 Hz) and high (100 Hz) frequencies promoted a significant increase in systemic (plasma) and local (ovary) GSH levels compared with G1 levels.The use of EAc has promoted an increase in GSH levels in the liver and kidneys and in random skin flaps of rats, as demonstrated in previous studies [16][17] .Lipid peroxidation was more intense in rats treated with EAc as there was a significant raise in MDA levels in G3 and G4 rats.However, the use of Ac did not Effects of acupuncture and electroacupuncture on estradiol-induced inflammation and oxidative stress in health rodents Acta Cirúrgica Brasileira -Vol.28 (8) 2013 -587 alter MDA levels.Despite the fact that is known that there is a correlation observed between the plasma levels of MDA, and the level of 17-beta estradiol where high levels of this hormone may lead to oxidative stress 27 we cannot exclude the effects of EAc on the increase in lipid peroxidation.We have reported similar results in other tissues in two previous studies [16][17] .
In this study MPO activity decreased significantly in all groups compared with G1, after five sessions of AC and EAC.
Recently we have reported similar results in random skin flaps treated with EAc 17 .
Furthermore, increased levels of MDA in rats treated with EAc shows that the electric stimulation of accupoints may have a pro-peroxidative effect.Both Ac and EAc reduced MPO activity in ovarian tissue, possibly by inhibiting neutrophil infiltration, suggesting that these methods of treatment may have a protective role in the estradiol -induced oxidative injury.

Conclusion
As evidenced by the changes in glutathione and malonaldehyde levels and myeloperoxidase activity, the results of the present study demonstrate that both acupuncture and electroacupuncture treatments attenuates systemic and local estradiol -induced oxidative damage.

Group 4 :
Electroacupuncture 100 Hz (EAc 100 Hz) After one week of acclimatization, twenty-four 8-weekold rats received an i.m. injection of EV 4.0 mg (Sigma-Aldrich Laboratory Ltda, Brazil), in 0.2 ml of olive oil, in the quadriceps muscle.Sixty days later Groups 2, 3, and 4 rats were anesthetized as described and treated as follows: Group 2 -After routine skin disinfection with 75% ethanol sterilized disposable stainless steel needles (0.25 mm × 15 mm) were inserted perpendicularly as deep as 2-3 mm at right ST-36 and SP-6 acupoints, bilaterally.Needles were kept in place for 20 minutes.Groups 3/4 -After needle insertion, as described, electrodes were connected to all needles and to the NKL electrostimulator; pulsed square waves, Burst type 1 mA, 2 Hz (Group 3)

FIGURE 1 -
FIGURE 1 -Photomicrograph of left ovary from control group (G1) exhibiting typical normal appearance with follicles and corpora lutea in different stages of development and regression (HE, x40).
for experimental use of laboratory animals was obtained from the local Ethics Committee on Animal Use (CEUA,