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9Effect of Tonifying Kidney and Eliminating Dampness on Expression of VEGF
in Acute Gouty Arthritis Rats
De-You JIANG, Shuang Liu, Ying Xie, Jie-Ru Han, Fei Chen, Yu Qiao, Jia-Yi Chang, Fu-Zhen Li
Department of JinGuiYaoLue, Institute for Chinese Medicine, Heilongjiang University of Chinese
Medicine, Harbin 150040, China
[Abstract] Objective To observe the effect of tonifying kidney and eliminating dampness on expression of vascular endothelial growth factor(VEGF) in acute gouty arthritis rats and to explore the mechanism of tonifying kidney and eliminating dampness decoction in the treatment of GA. Methods 70 rats were randomly divided into seven groups: Blank control group, model group, herbal control group, western medicine control group, high dosage herbal group, medium dosage herbal group and low dosage herbal group, each group of ten only. The uric acid sodium and oxygen polybenzoxazines potassium were jointly used to induce rat model of acute gouty arthritis. ELISA was used to detect the concentration of serum VEGF in rats and analyse the data. Histopathologic examination of synovial tissue of each rat was also performed. Results The differences of VEGF level between 7 groups were significant (P<0.01). The expression of VEGF in model group is higher than that in blank group, and the difference was significant (P<0.01). The expression of VEGF in model group is also apparently higher than that in herbal control group, western medicine control group, high dosage herbal group, medium dosage herbal group and low dosage herbal group (P<0.01), especially the effect of western medicine control group and medium dosage herbal group was more obvious. Conclusion Tonifying kidney and eliminating dampness can inhibit the expression of VEGF in acute gouty arthritis rats. It has certain curative effect on the prevention and treatment of gouty arthritis.
[Key words] Gouty Arthritis, Tonifying Kidney and Eliminating Dampness, VEGF, Experimental Studies
Introduction
Gout is a type of arthritis in which there is a build up of crystals from uric acid in the joints of the body. In gout, the body either makes too much uric acid or it is not able to flush out excess uric acid from the body fast enough. When it builds up, the uric acid forms into crystals and can deposit in joints (especially in the big toe). [1]In recent years, with the changes of people's living habits and diet, the incidence of gouty arthritis in our country has a clear upward trend. Acute gouty arthritis(AGA) [2], as a typical representative form of GA, may occur suddenly and trigger an inflammatory response which is caused by urate crystals. The inflammation leads to swelling and redness of the joint, and makes it painful to move or even to touch. Western medicine usually adopts Nonsteroidal anti?inflammatory drugs (NSAIDs), corticosteroids or colchicine to decrease the pain and swelling. However, they have obvious side-effects and the recurrence rate is very high. At present, there is still no specific remedy to treat AGA. [3]-[4] Therefore, it is a trend to seek a safe and effective traditional Chinese medicine treatment of GA in the future. This research adopts the model[5] that the uric acid sodium and oxygen polybenzoxazines potassium were jointly used to induce rat model of AGA. Colchicine, Tongfengshu Pian and Tonifying kidney and eliminating dampness were divided to different groups of rats for an intervention and then observe the concentration of serum VEGF in rats. Inquiry into the mechanism of Tonifying kidney and eliminating dampness of the prevention and cure of AGA.
Materials and methods
Experimental Animals
70 healthy male adult wistar rats (10 weeks old and weighing 180g±20g) were purchased from the Experimental Animal Center in Heilongjiang University of Chinese Medicine. During the study period, the rats were kept in an animal room under under the temperature of 20?±2? and relative humidity of 30%?35%. The 70 Wistar rats were divided into 7 groups: Blank control group,
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model group, herbal control group, western medicine control group, high dosage herbal group, medium dosage herbal group and low dosage herbal group at random, 10 rats in each group. The guidance suggestions for care of laboratory animals was followed according to the guidelines for caring for experimental animals published by the Ministry of Science and Technology of the People's Republic of China. Care was taken to minimize discomfort, distress, and pain to the animals. [6]
Test Substances
Tonifying kidney and eliminating dampness[7] is made up of cultivated land, alisma, dogberry, achyranthes bidentata, bixie which were from the Traditional Chinese Medicine(TCM) pharmacy in the First Affiliated Hospital of Heilongjiang University of Chinese Medicine. Colchicin Tablets (Cat#110807)was from Xishuangbanna Pharmaceutical Co., Ltd. Tongfengshu Pian(Cat#110601) was from Lujinzi Pharmaceutical Co., Ltd in Hubei Province. Tween80(Cat#101020) was from Tianjin Funing Fine Chemical corporation Co., Ltd. Oxygen polybenzoxazines potassium (Cat# CAS2207-75-2)was from Sigma companies in the United States. Monosodium urate crystals( Cat# CAS1198-77-2) was from Sigma companies in the United States. VEGF ELISA kits(Cat#20101108) was from Nanjing Biology Engineering Institute. Water Bath (Shanghai Yiheng Instruments Co.,Ltd), High-speed centrifuge (Beckman Coulter), Synergy H1 Hybrid Microplate Reader ( BioTek), Thermostatic oscillator (Hangzhou Allsheng Instrusment Co.Ltd).
Reagent Preparation
Preparation of MSU crystals suspended: The 1g of sterile MSU crystals were suspended in 9ml of sterile saline solution, and then added 1ml of Tween80. The solution was mixed, heated, allowed to cool to room temperature ,and finally forming 10ml of MSU crystals suspended.
Preparation of Tongfengshu Pian solution: Put the Tongfengshu Pian, which was grinded into fine powder, to sterile saline solution and then allow formation of 0.1gml-1 Tongfengshu Pian solution.
Preparation of colchicine mixed suspension: Put the colchicine, which was grinded into fine powder, to sterile saline solution and then allow formation of 0.1gml-1 colchicine suspension.
Preparation for the decoction of Tonifying kidney and eliminating dampness: Put cultivated land, alisma, dogberry, achyranthes bidentata and bixie into a marmite, add 4 times amount of water to immerse for 30min, extracting 2 times and every time with 30min, and then remove medicine residue through the filter. Finally, the solution was condensed in the rates of 0.8g/ml?0.4g/ml?0.2g/ml in 60? water bath, and then storing the obtained solutions into a refrigerator ,controlling the temperature at4?.
Preparation for the solution of oxygen polybenzoxazines potassium: Dissolve the oxygen polybenzoxazines potassium crystals in the sterile saline solution and then allow formation of 3? the solution of oxygen polybenzoxazines potassium.
Rats were given normal saline in blank and mode groups, herbal control group was given Tongfengshu Pian solution, western medicine control group was given colchicine mixed suspension, and the high, moderate and low dosage groups of Tonifying kidney and eliminating dampness decoction were studied through respective dose lavage(2g/kg?42g/kg?82g/kg).Following the above methods , the stomach feeding test on the rats is continued once a day, every day for a week, and then establish models at the late of medication
Model preparation Mode[5]
Arthritis was induced in ether anesthetised rats by injecting 0.2ml of MSU crystals suspension into the ankle joint. Ankle joint urate arthritis has advantages over other models of arthritis for therapeutic testing in that in a short time it affects a single joint in rats, and it produces responses which can be assessed by simple, sensitive measures. Mice in the blank group were injected with 0.2ml of saline into the ankle joint.
Effects of the concentration of the serum VEGF
Drawing the blood from eyeball of rats 24 hours later after modeling the mode and the concentration of the serum VEGF was determined by ELISA in accordance with the manufacturer's instruction.
Histopathological examination
Took synovial tissue of the affected joint, fixed with 10% dampen formaldehyde solution, paraffin embed, routine HE stained and morphology observed by microscope.
Statistical treatment
The measurement data were expressed as mean±SEM, and the results were analyzed by ANOVA followed by LSD-t test. The level information is analyzed by Kruskal-Wallis H followed by Rank transformation analysis. P<0.05 was considered significant.
Results and discussion
Effects of the concentration of the serum VEGF and Histopathological examination
Compared with the blank group, the level of VEGF increased significantly in rats model (P<0.01), which suggests successful mode. Compared with the model group, VEGF levels of herbal control group, western medicine control group, low dosage herbal group, medium dosage herbal group and high dosage herbal group declined significantly (P<0.01), which revealed that the agents in the above groups had effects on reducing the level of VEGF, and especially the western medicine control group and medium dosage herbal group had more obvious effects. Compared with the western medicine control group, Herbal control group, and Medium dosage herbal group had no statistical significance (P>0.05).There is statistical significance of low dosage herbal group and high dosage herbal group (P<0.05). Compared with herbal control group, there was no statistical significance of low dosage herbal group and high dosage herbal group (P>0.05), and the difference was statistical significance in medium dosage herbal group (P<0.05). (Table 1)
Table 1. Groups of rats serum VEGF level in comparison ?±s?n=10?
Table 1. Groups of rats serum VEGF level in comparison (X ±s, n=10)
Group Dose (g kg-1) VEGF level (ng ml-
Blank control group - 0.34±0.01
Model group - L21±0.211}
Herbal control group 1 0.83±0.171) 2)
Western medicine control group 5x10"4 0.67±0.091J 2)
Low dosage herbal group 2 0.89±0.111) 2) 3)
Medium dosage herbal group 4 0.69±0.041J 2) 4)
High dosage herbal group 8 0.90±0.091J 2) 3)
Note : Compared with the blank group, 1) P<0.01 ; Compared with the model
group, 2) P<0.01 ; Compared with the western medicine control group, 3) P<0.05 ; Compared
with the herbal control group, 4) P<0.05
Blank group rats joint synovial cell morphology was normal and arranged regularly(Figure 1A). The change of the model group's histomorphology was clear: the infiltration of inflammatory cells, interstitial edema and synovial cell necrosis falls off(Figure 1B). There was numerous neutrophils infiltration and synovial cell necrosis in herbal control group(Figure 1C).Western medicine control group had inflammation effusion cells, but the basic structure integrity(Figure 1D). Inflammatory cells infiltration and exudation of fibrin were found in high dosage herbal group and synovial cell edge is not neat(Figure 1E). Medium dosage herbal group had synovial tissue edema, but in which there were no seepage and inflammatory cells infiltration(Figure 1F). There were inflammatory cells and serum fiber seep in low dosage herbal group(Figure 1G).
v.. ^ 12 flMLT' -r JKSitt v : : A B C
D
■ V' v1
Fig.1. Effects of Tonifying kidney and eliminating dampness on Histopathological tissues of the ankle synovium in each group(HE, x400 )• A. Blank control group, B. Model group, C. Herbal control group, D. Western medicine control group, E. Low dosage herbal group F. Medium dosage herbal group, G. High dosage herbal group,
Studies have shown that VEGF was widely involved in diseases, such as cancer and inflammation ,in the process of angiogenesis. [8]VEGF has two important physiological function[9]:VEGF can encourage vascular endothelial cell to divide, propagate, migrate, and add vasopermeability, facilitate producing vascellum sustentaculum, to profit blood supply in ischemic region. ? VEGF can increases vascular permeability and inflammation. In 1999, Maeno, etc. discovered that the inflammatory reaction of the affected joint was positively related to the level of serum VEGF. So the serum VEGF can be used as an important indicator of evaluating the prognosis of GA. [10]
The multi-target, multi-channel overall treatment of Chinese medicine in GA is a combination of strengths and at the same time the herbal medicine has less side effects. Tonifying kidney and eliminating dampness[7] were made in long-period clinical practice and pharmacological research and consist of cultivated land, dogberry,alisma, achyranthes bidentata, bixie. Using familiar cultivated land nourishing kidney, square, born in essence, the gentleman medicine for the party. The dogberry is the basis for protecting liver and kidney.It is often used in combination with cultivated land and dogberry by the theory homogeny of liver and kidney for the root of the disease. Alisma discharge and can prevent kidney benefit too tired. Pharmacological studies indicate that alisma has obvious diuretic effect, can improve the excretion of urea and sodium chloride, and also inhibit the formation of urinary calculi. Pharmacological studies indicate that achyranthes bidentata has clear anti-inflammatory analgesic effects and obtains the ability to cure GA. Bixie is used to disperse bruised blood, for temporary relief of sprains and pains. All the reasonable side group played a total tonifying kidney and eliminating dampness effects to achieve the effects of both temporary and permanent cures.
Conclusions
In summary, this study suggested that the decoction of Tonifying kidney and eliminating dampness can improve the ankle swelling of rats' models obviously and reduce the ankle pains of rats in model group.A herbal medicine, may relieve inflammation by inhibiting the growth of VEGF. The decoction of Tonifying kidney and eliminating dampness exhibits effects similar to colchicine and has a good protective effect against GA and also provide experiment evidence for clinic.As to the other pharmacological mechanism of the herbal medicine still need to be further clarified.
References
[]Mikuls T R, Saag K G. Gout treatment: what is evidence-based and how do we determine and promote optimized clinical care?[J]. Current Rheumatology Reports, 2005, 7 (3): 242-249.
[2]Xue YM, Li CZ. The diagnosis and treatment of gout [M]. The second version.Beijing: People's Military Medical Publishing House., 2006, P2231.
[3]Lao ZY. Diagnosis and medication of primary gouty arthritis [J]. Chinese Journal of New Drugs and Clinical Remedies, 2004, 23 (9): 632-634.
[4]Cocco G, Chu D C, Pandolfi S. Colchicine in clinical medicine. A guide for internists[J]. European Journal of Internal Medicine, 2010, 21 (6): 503-508.
[5]YAO li, LIU Shumin, YU Shuyi. Improvement of Animal Model of Gouty Arthritis [J].Acta Laboratorium Animalis Scientia Sinica, 2009,17( 3):210-212.
[6] The Ministry of Science and Technology of the People's Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30
[7]WANG Junxia, HAN Jieru, ZHOU Xueming.Professor Jiang Deyou's experience in treating gout from kidney [J].Shanghai Journal of Traditional Chinese Medicine, 2010, 44 (2): 16-17.
[8]Zhu B, Liu JL, Zhu QR. Study on the serum level of VEGF and TNF-a in different stages of rheumatoid arthritis[J]. Modern Preventive Medicine, 2012,39(22):5938-5939.
[9]Liang QH, He JH, Li XL, Zhou JH, Zhang HX, Chen J, Tan Yong. Effect of bizhongxiao decoction on the expression of VEGF in thesynovial membrane of C II-induced rheumatoid arthritis in rats [J]. BULL HUNAN MEDICAL UINIVERSITY, 2002, 27(6):491-494.
[0] Maeno N, Takei S, Imanaka H. Increased circulating vascular endothelial growth factor is correlated aith desease activity in polyarticular juvenile rheumatoid arthritis[J].J Rheumatol, 1999, 26:2244-2248.
Pharmacokinetic studies ofBorago officinalis in rabbit
Ma Ying-nan, Yu ya-nan
DepartmentofPharmacology,Heilongjianguniversity of Traditional Chinese Medicine,Heilongiang
Harbin, China
Abstract:Medicine worked in the form of constituents in blood. In order to found the matters of Borago affect body directly, An UPLC-TOF/MS method has been used to analyze the metabolites and the active constituent in blood of the rabbit after fed by Borago. On the assay result, five compositions were found in urine, and one of them appears a strong ionic strength in the chromatogram of the blood.
Key word: Pharmacokinetic; Borago; UPLC-TOF/MS; active constituent
1. Introduction
Borage (Borago officinalis L.) is a large hairy annualherb that is a member of Boraginaceae 1 2 family. 1 Borageseed has a high content of c-linolenic acid and is used as adietary supplement.. 2
Borage juice and tea are used to treatinfluenza, colds, injuries, and ulcers.3-5 Borage istraditionally
used to treat respiratory, cardiovascular, andgastrointestinal diseases.6 In recent studies, it is show
that Borage can be the treatment of jaundice, rheumatism, skin inflammation, can also be used as a
7-9
diaphoretic, diuretic antitumorand analgesic. - In order to clarify theactive composition and acting mechanism of Borage, An UPLC-TOF/MS method has been used to analyze its metabolites and blood in biological samples. UPLC-TOF/MS is a recently developed technology. It can provide a higherpeak capacity, greater resolution, increased sensitivity, and higherspeed of analysis.
2. Materials and methods
