nitrogen hyperbaric narcosis [4] as a result of accumu lation of heat shock proteins (HSP 70) in neurons of the rat cerebral cortex [5] makes it opportune to esti.
ISSN 00124966, Doklady Biological Sciences, 2010, Vol. 430, pp. 11–13. © Pleiades Publishing, Ltd., 2010. Original Russian Text © O.S. Alekseeva, A.N. Vetosh, D.E. Korzhevskii, V.B. Kostkin, 2010, published in Doklady Akademii Nauk, 2010, Vol. 430, No. 3, pp. 421–423.
PHYSIOLOGY
Influence of Quercetin on the Progress of Nitrogen Narcosis and Accumulation of Heat Shock Proteins in Cells of the Rat Cerebral Cortex O. S. Alekseevaa, b, A. N. Vetosha, D. E. Korzhevskiib, and V. B. Kostkina Presented by Academician V.L. Sviderskii June 10, 2009 Received June 23, 2009
DOI: 10.1134/S0012496610010047
The bioflavanoid quercetin is an antioxidant sup pressing free radical processes [1]. There is experi mental evidence that quercetin injection 6 h before stress promotes an increase in heat shock protein con tent of some tissues [2, 3]. The fact that several sessions of preliminary hypoxia weaken the manifestation of nitrogen hyperbaric narcosis [4] as a result of accumu lation of heat shock proteins (HSP70) in neurons of the rat cerebral cortex [5] makes it opportune to esti mate the quercetin effect on narcosis symptoms and the HSP70 content of the cortex. Nitrogen narcosis was chosen as a strong stress factor. Literature data show that quercetin is an antioxidant [1, 6], but there are no data on the quercetin effect on the progress of nitrogen narcosis or HSP70 content of the cortex.
ended with explosive decompression (1–2 min). The rat brain was quickly extracted, fixed with an alcohol– formalin mixture containing zinc chloride, and embed in paraffin according to the standard protocol. HSP70 was detected by the immunocytochemical method in frontal brain sections [5, 8]; then, samples were exam ined under a light microscope. Cells with different intensities of immunocytochemical staining against HSP70 in both cerebral hemispheres were counted [9]. The main symptoms were the following: ataxia of the hind feet was observed at a pressure of up to 1.7 MPa; ataxia of the fore feet, at a pressure of up to 2.6 MPa; animals assumed a lateral body position at a pressure of up to 3.5–4.1 MPa. The symptoms of nitrogen narcosis were seen under a higher pressure after a preliminary (6 h before compression) injection of quercetin. Therefore, quercetin significantly improved the body state under narcosis. On the other hand, preliminary injection of quercetin 1 h before compression deteriorated the body state under narco sis, shifting the manifestation of its symptoms to lower pressures of the oxygen–nitrogen mixture. Cells of the motor cortex with different intensities of immunocytochemical staining against HSP70 are shown in the figure. The cytoplasm response was expressed in the form of small granules. These gran ules often concentrated at one cell pole. In the nuclei, the response was also expressed in the form of small granules, their location being similar to the chromatin grain distribution over karyoplasms. The nucleole and cytoplasm of leucocytes in the cortex vascular lumen displayed pronounced responses. This gives ground to the hypothesis on HPS70 transport by blood cells into tissues.Anyway, optical examination of rat brain samples confirmed this hypothesis. The count of the cells of the motor cortex with dif ferent intensities of immunocytochemical staining against HSP70 showed that cells with moderate and weak reaction were prevailing in intact animals, and only a small amount of cells showed a strong reaction
Twentyfour adult male Wistar rats obtained from the Rappolovo farm (body weight, 200–250 g) were used in the study. The animals were divided into groups. The first group was an intact control. The remaining three groups were exposed to a high nitro gen pressure. The second group was exposed to nitro gen narcosis alone. Preliminarily, 1 h before narcosis, the animals from the third group were intraperito neally injected with querctin (ICN, United States) in physiological saline at a dose of 0.035 mg/g body weight. The fourth group was intraperitoneally injected with querctin at the same dose 6 h before the exposure to a high nitrogen pressure. A decompression chamber with a volume of 107 l and oxygen–nitrogen breathing mixture with normal oxygen content were used to produce hyperbaric conditions. The method of nitrogen narcosis is described in detail elsewhere [4, 7]. Hyperbaric exposures under a pressure of 4.1 MPa
a
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, pr. Toreza 44, St. Petersburg, 194223 Russia b Institute of Experimental Medicine, Russian Academy of Medical Sciences, ul. Akademika Pavlova 12, St. Petersburg, 197376 Russia 11
12
ALEKSEEVA et al.
2 1 2
2 3
2 3 3 Immunocytochemical reaction to HSP70 in cortex motoric region of intact rat. Different intensities of the reaction in neurons: (1) strong; (2) moderate; (3) weak (400×).
(table). The number of cells with strong and moderate reaction was increased at the expense of a decrease in the number of cells with a weak reaction after reaching a pressure of 4.1 MPa. After quercetin injection 6 h before the beginning of compression and subsequent increase in nitrogen pressure to 4.1 MPa, the number of cells with strong and moderate reactions some more, and the number of cells with a weak reaction decreased significantly compared to the control group. A quercetin injection 1 h before the beginning of nitro gen compression resulted in the exactly opposite situ ation. It should be noted that stress morphological changes (not to mention postmortal ones) in neurons during the quick explosive decompression and subse quent extraction of the rat brain had no time to The average number of neurons with different intensities of immunocytochemical staining against HSP70 in the motor cortex under nitrogen compression up to 4.1 MPa after quer cetin administration 1 and 6 h prior to stress Average number of neurons, % Exposure Intact control Nitrogen compression up to 4.1 MPa Quercetin + 1 h + nitro gen compression up to 4.1 MPa Quercetin + 6 h + nitro gen compression up to 4.1 MPa
strong moderate weak response response response 2.5 6.7
36.9 42.1
60.6 47.6
1.1
20.6
78.3
13.8
62.5
23.7
develop. Explosive decompression itself led to the for mation of nitrogen bubbles in the blood and tissues of animals and, hence, to ruptures in tissues. These microdamages as a result of fugacity explosive decom pression were seen only under the maximum magnifica tion of the light microscope (with the use of immersion). They could cause changes in the cytoplasm distribution of stained granules of the immunocytochemical reac tion but did not change their number. Consequently, the intensity of immunocytochemical staining against HSP70 proteins in cortical cells could be reasonably measured after this decompression. Thus, immunocytochemical data proved our hypothesis on HSP70 accumulation in cortex neu rons as the main defense mechanism against nitrogen hyperbaric narcosis and other stress factors. One of HSP main functions in the cell is protection of molec ular complexes against damaging factors [10, 11]. HSP is much more effective as defense against weak and moderate doses of damaging factors and less effective in the case of strong of damaging factors. Morpholog ical data confirm the protecting effect of HSP after preliminary administration followed by nitrogen hyperbaric narcosis. Quercetin protection in the case of its injection 6 h prior to stress was expressed in the antioxidant effect and modulation of synthesis, accu mulation and transport of HSP70 into tissues. Nevertheless, quercetin injection 1 h prior to nitro gen compression did not lead to this protective effect. The time course of motor activity and postural reflexes of rats during the progress of hyperbaric narcosis, as well as immunocytochemical data, confirmed this conclusion. Quercetin injected to rats 1 h prior to stress resulted in an oppposite effect. Quercetin increased the number of cells with a weak reaction and decreased the number of those with strong and moder ate reactions. Behavioral responses also gave evidence for earlier manifestation of nitrogen narcosis. It would be tempting to explain the protective effect of quercetin by its antioxidant effect. In this case, quercetin blocked or decreased the production of free radicals and triggered the mechanism of HSP70 accumulation. However, the results of quercetin injec tion 1 h prior to nitrogen compression, when the HSP70 content of brain cells decreased, contradict this assumption. Therefore, the quercetin effect is related not only to its antioxidant effect, but also to the syn thesis (or inhibition of the synthesis) of these stress proteins. The nature of these relations is obscure, but the protective effect of quercetin obviously depends on the time of its administration before the beginning of stress. In summary, in should be emphasized that querce tin injected in rats 6 h prior to nitrogen compression decreases the manifestation of nitrogen narcosis and stimulates accumulation of HSP70 in cortex neu rons. The molecular mechanisms of the stimulatory effect of quercetin on HSP in brain cells are still obscure and require further studies. However, our data DOKLADY BIOLOGICAL SCIENCES
Vol. 430
2010
INFLUENCE OF QUERCETIN ON THE PROGRESS
make it possible to use the bioflavanoid quercetin as an efficient remedy for pharmacological correction of the state of humans and animals under a high nitrogen pressure. REFERENCES 1. Terao, J., Food Fact. Health Prom., 2009, vol. 61, pp. 87–94. 2. Elia, G. and Santoro, M., Biochem. J., 1994, vol. 300, pp. 201–209. 3. Pastukhov, Yu.F. and Ekimova, I.V., Neironauki, 2005, vol. 2, no. 2, pp. 3–25. 4. Vetosh, A.N., Biologicheskoe deistvie azota (The Bio logical Effects of Nitrogen), St. Petersburg, 2003. 5. Vetosh, A.N., Alekseeva, O.S., Kostkin, V.B., and Korzhevskii, D.E., Fiziol. Zh., 2008, vol. 54, no. 2, pp. 33–40.
DOKLADY BIOLOGICAL SCIENCES
Vol. 430
2010
13
6. Crespo, I., GarciaMediavilla, M.V., Almar, M., et al., Food Chem. Toxicol., 2008, vol. 46, no. 5, pp. 1555– 1569. 7. Vetosh, A.N., Popov, A.A., Alekseeva, O.S., and Pozhidaev, V.A., Dokl. Biol. Sci., 1999, vol. 365, no. 2, pp. 114–116 [Dokl. Akad. Nauk, 1999, vol. 365, no. 2, pp. 276–278]. 8. Korzhevskii, D.E., Kratkoe izlozhenie osnov gistolo gicheskoi tekhniki dlya vrachei i laborantovgistologov (Concise Histological Manual for Medical Practitio ners and Laboratory Assistants/Histologists), St. Petersburg, 2005. 9. Vetosh, A.N., Alekseeva, O.S., Kostkin, V.B., and Korzhevskii, D.E., Patogenez, 2008, vol. 6, no. 3, pp. 49–50. 10. Welch, W., Physiol. Rev., 1992, vol. 72, no. 4, pp. 1063– 1081. 11. Meerson, F.Z. and Malyshev, I.Yu., Fenomen adapta tsionnoi stabilizatsii struktur i zashchita serdtsa (The Phenomenon of Adaptive Stabilization of Structures and Heart Protection), Moscow: Nauka, 1993.
