[Abstract] Objective observed the cancer hot Ning suppository antipyretic mechanism. Methods 2,4  dinitrophenol induced fever in rabbits model, changes in body temperature was measured at different times after the administration, and the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) detection by serum levels of TNF-α, IL-1β content changes using immunohistochemical methods to analyze the relationship between the hypothalamus protein kinase A (PKA) and changes in body temperature. Results cancer hot Ning suppository obvious antipyretic effect (P <0.05) in the low-dose group non-infectious fever in rabbits, which is high, the middle dose group compared with the model group difference was statistically significant (P < 0.01). Can significantly reduce serum TNF  α, IL  1β content. The correlation analysis showed that the temperature change of the five groups of rabbits were positively correlated with hypothalamic PKA content. Conclusions cancer hot Ning suppository antipyretic effect of non-infectious fever caused by 2,4  II nitrophenol animal model. Can inhibit the serum TNF  α, IL  1β content increased, at the same time, reduce the level of the hypothalamus cAMP  PKA may the the cancer hot Ning suppositories antipyretic one of the mechanisms.

Key words】 cancer hot Ning suppositories; a noninfectious fever; tumor necrosis factor  α; white interleukin  β; Protein kinase A

   Effects of Cancer Rening Suppository on Temperature, Levels of TNF  α, IL  1β in Serum and Hypothalamus PKA Content of Feverish Rabbits Induced by Noninfection

    JIA Ying  jie, LI Xiao  jiang, SUN Yi  yu, ZHANG Ying, CHEN Jun, ZHANG Yun  chao

    Department of Oncology, The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China Abstract: Objective To adverse reaction of Cancer Rening suppository in the treatment of fever cancer.Methods The models of noninfected fever induced by 2,4  binitro  phenol was used to observe the changes of temperature in different time, TNF  α and IL  1β in the serum of rabbit were determined by double antibodies sandwich Enzymelinked Immurosorbent Assay.Immuno  histochemical assay was used to detect the expression between PKA activities of hypothalamus and the changes of temperature.Results Different doses of Cancer Rening suppository (13.5g/kg, 4.5g/kg, 1.5g/kg) reduced the febrile responses of rabbit induced by noninfected fever.In the febrifuge experiment of rabbit, both the high and middle group have the febrifuge effect obviously.Changes of TNF  α, IL  1β in serum and PKA in hypothalamus during noninfected fever induced by 2,4  binitro  phenol in rabbits are significantly increased.Cancer Rening suppository has a protective effect on noninfected fever induced by 2,4  binitro  phenol in rabbits, decreasing the expression of TNF  α, IL  1β in serum and PKA in hypothalamus obviously.Correlation analysis indicated that the changes of the rabbit body temperature were positively correlated with the contents change of PKA in the hypothalamus of the five rabbit groups.Conclusion The antipyretic mechanisms of Cancer Rening suppository may be related to its decreasing the contents of TNF  α, IL  1β in serum and PKA in hypothalamus.It may be an important antipyretic mechanism of Cancer Rening suppository.

    Key words: Cancer Rening suppository; Noninfectious fever; TNF  α; IL  1β; PKA

The cancer hot Ning suppository According to TCM theory, science developed from traditional Chinese medicine. Qingrejiedu, Quyu Liangxue effect. Mainly used in clinical cancer febrile or non-infectious fever. The preliminary toxicological experiment results show that the safety of the clinical use of this product. About its antipyretic mechanism has not been reported. For the study of the antipyretic effect and mechanism of this product, we use 2,4  dinitrophenol preparation of rabbit fever animal model of non-infectious, by following Zoological trial, and thus provide a basis for the clinical use of drugs.

    1 Materials and methods

    1.1 Materials

    1.1.1 animal long-eared rabbits were 30, half male and half female, weighing 2.5 ± 0.5kg provided by the Experimental Animal Center of Tianjin University of TCM, license number: Tianjin moving Xu Zi (1996) No. 001.

    1.1.2 the drug cancer hot Ning suppositories, silver Bupleurum, Digupi diffusa, Polygonum cuspidatum drugs through clinical trials to determine Prescription, suppositories prepared by the hospital pharmaceutical the 6g / tablets. Herbal extract, the boiled alcohol precipitation, animal rectally per milliliter containing crude drugs 3g.

    1.1.3 Reagents 2,4  dinitrophenol, Size: 25g, lot number CNAB035  Q (Tianjin Wing Kee Trading Company). Anhydrous ethanol (alcohol analysis) 500ml, lot number 9610355 (Tianjin Wang Lan Chuang Chemical Reagent). Formaldehyde (analysis of alcohol) 500ml, lot number 20040803 (Tianjin Dengfeng Chemical Reagent). PKAγ cat (C  20) an anti-sc  905 (Santa Cruz biotechnology company).

    1.1.4 Instrument MGA  Ⅲ 219 electronic thermometer; IL-1β ELISA enzyme-linked immunosorbent assay kit provided from Beijing Shang Bosheng Biomedical Technology Co., Ltd.; TNF  α ELISA enzyme-linked immunosorbent assay kit by Beijing still Bo Biomedical Technology Co., Ltd.; enzyme-linked immunosorbent assay, 3550, the United States BIO  RAD; SG  800 low-speed desktop centrifuge.

    1.2 Methods

    1.2.1 Rabbits choice before the experiment MGA  Ⅲ 219 electronic thermometer, daily rectal temperature measured rabbits once, for three consecutive days, the average value as the basis of temperature. Select the temperature range of 38 ℃ ~ 39.5 ℃, and temperature fluctuations in less than 0.5 ℃, used in experiments [1].

    1.2.2 2,4  dinitrophenol induced heat is determined according to the previous reports [2], this experiment is determined by the pre-test After 2,4  dinitrophenol induced heat to 30mg/kg, before the test with anhydrous ethanol configuration, 2 ℃ ~ 5 ℃.

    1.2.3 modeling packet 2,4  dinitrophenol stimulation in rabbits aseptic inflammation, to simulate clinical noninfectious the fever model [3]. Experimental day, take above qualified rabbits, after determination of basal body temperature, were randomly divided into five groups according to body temperature, in addition to the group not treated as blank control group, the other four groups were subcutaneously injected 2,4  dinitro phenol-ethanol solution, dose 1.5ml/kg 2h induced increase in body temperature of more than 1 ℃ Fever, fever animals were randomly divided into model control group, cancer hot Ning the suppository is high, and in the low-dose group.

    1.2.4 administered cancer hot Ning the suppository is high, in the low-dose group, respectively, through the rectum to 9/3/1 / only different doses of the drug, that is containing raw dose 13.5g/kg, 4.5 g / kg, 1.5g/kg, administered group after modeling 3h I rectal mucosa contact administration.

    1.2.5 OUTCOME MEASURES

    (1) the impact on the body temperature of animals in each group rectal temperature changes observed modeling 0.5,1,2,3,4,5,6 h tracings average body temperature response curve (MTRC) [4], the statistical average maximum temperature reaction height (ΔT) and average 3h body temperature reaction index (TRI3), TRI calculated with reference to the Milton method [5], the fever curve and the area between the body temperature baseline TRI calculated according to the following method.

    Body temperature response index (TRI) calculated the temperature value of the vertical coordinate, 5cm = 1.0 ° C; abscissa time value, 1cm = 10min. Abscissa is the temperature baseline average of three times body temperature before injection (take 0.1 ℃ integer) as the intersection of the two coordinates, to rise in value is positive, the decline in value is negative. Each time point temperature change values ​​in the graph paper portrayed as changes in body temperature curves were fever (fever curve). The area between the body temperature curve temperature baseline is the temperature response index. Fever, that is called the heat index, reflects a good indicator of the strength of the heating effect.

    (2) serum IL  1β, TNF  α in the content of each group rabbits in modeling, modeling 3h (fever reached its peak), and 6h after modeling from the marginal ear vein blood collection 2ml stand for centrifugal serum was separated and stored at, -70 ℃ under test operation according to kit instructions.

    (3) the impact of the end of the experiment hypothalamic PKA content immediately decapitated and animals, quickly remove the whole brain and remove the hypothalamic tissue between the optic chiasm and gray nodules treated, a comprehensive analysis of entire sections of positive cells the number and intensity of staining.

    1.2.6 statistical methods using SPSS 11.5 statistical software packet processing.

    2 Results

    2.1 rabbits signs

    Injection of 2,4  II nitrophenol 30min rabbit body temperature begins to rise and to appear towering hair, shivering, curled up; 2h, as the body temperature was significantly higher in rabbits towering hair, shivering, curled up gradually disappear, but the breathing was significantly breath and rapid heartbeat. Due to systemic burning, rabbit auricle redness, fever, conjunctival hyperemia, but conscious. Their signs with the cancer hot Ning suppository treatment significantly reduced.

    2.2 on the model animal body temperature

    Modeling 0.5h after onset of fever, and to about 3h gradually reached fever peak temperature of the model group was significantly higher than the control group. The cancer hot Ning suppository high body temperature changes in the low-dose group (P <0.05) lower than the model group, including cancer hot Ning suppository, middle dose group compared with the model control group, a statistically significant difference (P <0.01), with a blank 0.05)。">compared the difference was not statistically significant (P> 0.05). The cancer hot Ning suppository ΔT, TRI3 compared with the model group was significantly lower (P <0.05). The cancer hot Ning suppository suppress fever and antipyretic role model animal, completely against the heating effect caused by 2,4  dinitrophenol, as shown in Table 1, 2.

    2.3 model animal serum IL  1β, TNF  α level

    2h modeling, modeling animal serum IL  1β, TNF  α content was significantly elevated after administration of the the cancer hot Ning suppository each group compared with the model group decreased significantly (P <0.05), suggesting that cancer hot Ning suppository model animal serum IL  1β, TNF  α increased effect inhibition are shown in Table 3 and 4. Table 1 modeling before and after the body temperature in the animal body temperature changes with this group before modeling compare *: <0.05, **: P <0.01; compared with the model group, ▲: P <0.05, ▲ ▲: P <0.01 Table model control group the 2 Cancer heat Ning the suppository animal models ΔT and TRI3 group dose the number of cases (only) ΔT (° C) TRI3 normal control group (cm2) -60.05 ± 0.081.17 ± 8.67 -62.12 ± 0.88 * 119.5 ± 36.5 * the cancer hot rather low dose group 1.5g/kg60.75 ± 0.15 * ▲ 74.3 ± 14.7 * the ▲ cancer hot Ningzhong dose group 4.5g/kg60.47 ± 0.14 ▲ 89.1 ± 14.3 * ▲ cancer hot rather high dose group 13.5 g/kg60.08 ± 0.20 ▲ 90.6 ± 15.7 * ▲

    Compared with the normal control group, *: P <0.05; compared with the model group, ▲: P <0.05 Table 3 cancer hot Ning rabbit fever peak serum with the group before modeling comparison, *: P <0.05; and comparison group modeling ▲: P <0.05; compared with the model group, ★: P <0.052.4 on animal models hypothalamic PKA content

    Blank group and high-dose groups PKA expression was weak, as shown in Figure 1,5; PKA expression of model group showed diffuse, mainly expressed in the cytoplasm of neurons and endothelial cells, as shown in Figure 2 and 3; the cancer hot Ning suppository high dose group was significantly lower than the model group, see Figure 4,5,2. Prompted the cancer hot Ning suppository 2,4  dinitrophenol rabbits induced hypothalamic PKA content increased significantly inhibited. Table 4 the cancer hot Ning rabbit fever peak serum with the group compared with the model *: P <0.05; in this group compared to the modeling, ▲: P <0.05; compared with the model group, ★: P <0.05

    3 Discussion

    The fever is mainly used for in vivo monocyte macrophages exogenous pyrogenic original to produce and release endogenous pyrogens (endogenous pyrogen, EP) Erzhi. In recent years, the study found that EP is more than one, including interleukin (IL  1), tumor necrosis factor (TNF  α), interferon (IFN), by macrophage inflammatory protein-1 (MIP  1). As fever information can be direct or indirect role in body temperature regulation center, leading to hypothalamic heating medium [6] such as cAMP, corticotropin-releasing hormone (CRH) release, so that the set point shift, increased heat production, heat reduce the role of pyrogenic. Recently, with the rapid development of molecular biology techniques and in-depth study of the pathogenesis of the fever, there are experiments confirmed [7] cAMP / PKA pathway involved in the regulation of body temperature, cAMP  PKA signal transduction mechanisms in cells [8] occupies a very important position. Current, PKA and thermoregulation widespread attention.

    TNF  α is a single nuclear  macrophage multifunctional monocytes factor TNF  α release in the disease process can directly cause cell damage by stimulating other pro-inflammatory mediators, such as IL  1 IL  6 release to expand the inflammatory cascade [9].

    IL  1 is important pyrogenic cytokine gene encoding divided into two types of IL  1α and IL  1β, IL  1β pyrogenic. Confirmed [10] involved in signal transduction of IL  1β phospholipid enzymes including phospholipase A2 (PLA2). Whose activation allows the arachidonic acid (AA) release from the phospholipase SN  2 bits, and therefore become the rate-limiting enzyme of the phospholipids inflammatory mediators. Many studies have shown that IL  1 can stimulate different cells PLA2 gene expression and protein synthesis, and increased its activity, thus splitting membrane phospholipids to produce AA, led to the production and release of PGE2 [11, 12 and by inducing the activation of COX  Ⅱ ]. The latter addition to directly affect cell function as a biologically active substance can also be the activation of adenylate cyclase (AC) so that an increase in intracellular cAMP, cAMP is an important intracellular second messenger, plays stimulation of extracellular signals into cells the role of the media within a variety of physiological activities, it can be combined with the binding subunit of cAMP-dependent protein kinase A (PKA) and make it active. Activated PKA can either make a variety of protein phosphorylation in cells and play a biological role can also be the role of cAMP response element binding protein, to play a biological role in regulating gene expression. The main process for PLA2 → AA → PGE2 → cAMP → PKA → to cause fever biological effects.

    The results of this study show that the cancer hot Ning suppository can significantly reduce by serum levels of TNF-α, IL  1β activity; by blocking cAMP intracellular PKA combine, to varying degrees, inhibition of of rabbit hypothalamic nerve cells due to 2,4  dinitramide yl phenol stimulation resulting from elevated levels of cAMP / PKA. Therefore show that the cancer hot Ning suppository antipyretic effect may be related to the regulation of cAMP / PKA signaling pathway has a certain relationship.