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The histologic effects of the Uyghur Medicine Xipayi KuiJiean on ulcerative colitis in rats.

INTRODUCTION

Animal experimentation is an important discipline and a vital component of experimental study in the life sciences. It plays a significant role in the development of traditional medicines as well as of modern medication. Animal experimentation can help to enhance human biological understanding by allowing internal and detailed visualizations that would otherwise be difficult to achieve--and so provide more concrete and accurate results. An ideal animal model is the key element to the success of such medical experiments.

Previously we created a rat model of UC using 2.4-dinitrochlorobenzene (DNCB) composite acid. The effects of DNCB can be explained by hapten induction of the normal immune system. The resulting symptoms, organization and pathological changes in rats correlate with the UC index, supporting its use as an ideal UC model.

Ulcerative Colitis (UC), also known as chronic nonspecific ulcerative colitis, is a type of unspecified chronic intestinal inflammation. The observed pathological changes mainly involve the hypoblast: primarily involving ulceration and erosion of the mucosa; mostly affecting the rectum and the distal colon with possible extension to the proximal colon or the entire colon (1). The main clinical signs are diarrhoea, abdominal pain, and mucoid bloody stool with pus (2, 3). Other signs include fever, weight loss, anemia, joint pain, damage to mucus membranes, renal calculi and osteoporosis (2, 4). The clinical course is long with a variety of disease manifestations, including active periods and remissions with repeated attacks. In age, young adults are more likely suffer from the disease. No obvious evidence exists that there is a difference in disease incidence rates between males and females. The disease is regarded as a significant risk factor for colon cancer, of which the incidence rate has been growing in recent years (5). It is considered one of the most challenging diseases to manage by the World Health Organization6 and is universally recognized as a clinically important entity in this country and abroad.

MATERIALS AND METHODS

Materials We used male Wistar rats with SPF health levels, weighing 200-250g. They were provided by the Experimental Animal Center of Xinjiang Medical University. Trizol, an RNA extraction reagent was purchased from Invitrogen Company. Isopropyl alcohol and ethyl alcohol was obtained from MERCK Germany. We used domestically-produced, spectroscopically pure chloroform. Our duplication reagent box was from Sangon Biotech (Shanghai) Co.,Ltd. The DNCB, acetone, and ethanoic acid for this experiment were domestically-produced and analytically pure. The Uyghur medicine, Xipayi KuiJiean, was provided by the Xinjiang Cicon Habo Uyghur Medicine Co., Ltd.

Evaluation Standards (1) Disease activity index (DAI) according to reference (7&2). CMDI evaluation standard according to reference (7).

Method: Preparation of our UC experimental animal model (steps for our improved compound method of creating an ulcerative colitis rat model): a. Rats were fed for one week to establish a baseline. b. The rats' necks and backs were depilated with 12%Na2S. c. 0.25 ml (approximately 5 drops) of 2% DNCB Acetone fluid was dripped onto the rats each day for 14 days. d. Following application of the DNCB on day 14, the rats were made to fast (allowing for free water drinking) for 24 hours. e. On day 15, 0.25 ml of a 0.1% DNCB ethyl alcohol solution was injected into the rat colons via 3mm-diameter catheters inserted 8cm past the anus. f. On day 16, the same procedure was used to inject 2 ml of 6% ethanoic acid. The solution was allowed to have an effect for 15 seconds, after which, 5 ml of physiologic saline solution was used to immediately flush the area.

After an observation period of seven days, the intervention group rats were given intravenous injections (in their tails) of a short-chain fatty acid solution with 10% glucose in a 1:1 intermixture. [The purpose of this intermixture was to prevent excessive damage from the DNCB]. These rats received 3 ml of this intermixture IV per day for three days while fasting with free water. They were returned to a conventional diet on the fourth day (following the observation period).

Experimental animal grouping: The 109 Wistar rats were stochastically divided into the following groups: normal group (7 rats); an ulcerative colitis model group (21 rats with 5 deaths); a physiologic saline/negative control group (21 rats, 10 deaths); the Xipayi KuiJiean large dose intervention group (19 rats, 7 deaths); the medium dose intervention group (20 rats, 5 deaths); and the small dose intervention group (21 rats, 5 deaths). The groups were in separate cages.

Experimental animal processing: The experimental groups (i.e., all the rats except for those in the normal and UC model groups) received interventions after 7 days of observation. 3 mm-diameter catheters were inserted in their colons for 5~8 cm to inject the following substances: physiologic saline 22.5 mL/(kg.d) to the physiologic saline/negative control group; XKJ 300 mg/kg.dto the Xipayi KuiJiean large dose intervention group; XKJ 150 mg/kg.dto the Xipayi KuiJiean medium dose intervention group, and XKJ 75 mg/kg.dto the Xipayi KuiJiean small dose intervention group.

After 20 days, the rats were made to fast overnight. The following day, blood samples were taken under ether anesthesia and rapidly placed in heparinized tubes. The blood and anticoagulant were mixed by gentle shaking of the tubes to prevent coagulation. Then, the blood plasma was separated into packets and placed in liquid nitrogen. The samples were stored at 80 until being examined by nuclear magnetic resonance.

Sections (5-8 cm) of colons were resected (away from the anus and cut along the mesentery edge). Physiologic saline was used to gently wash the residuum off the intestines. The degree of colonic mucus membrane damage, graded according to a point scale.

Samples were taken from the affected areas for histopathological evaluation. Selected tissues were fixed in 40 g/L neutral formaldehyde solution for 24 hours and then embedded in conventional paraffin wax and dyed with H&E stain.

Small portions of affected tissues were put into glutaric dialdehyde solution for preservation in order to carry out ultrastructure tissue cell observations. These tissue slices were read by specialists in a blinded fashion. Finally, additional samples of colon tissue with pathologic changes were kept at--80[??] for preservation.

Statistical processing: SPSS 15.0 statistical software was employed to process the data. When group comparison variance was uneven, T-tests were performed with a significance standard [alpha]= 0. 05.

RESULTS

Symptoms of the rats and changes in symptomatology: The rats from the normal group had glossy hair, good levels of activity and normal stool. In comparison, the model group, after being prepared, manifested decreased levels of activity, low spirits, weight loss, cheimophobia (fear of cold), and their hair was dull and sandy-colored. The model group had mucoid bloody stool with pus. The correlation of the model group's symptoms and signs with the disease activity index (DAI) demonstrated the success of the ulcerative colitis model.

Changes in the organization of the colon mucus membrane: rats from the normal group had intact colon mucous membranes without inflammatory cell infiltration or ulceration. Their colonic mucosal epithelium was complete and continuous; the gland arrangement was regular with clear structure. The lamina propria and muscularis mucosa of the inner mucus membrane was normal, and the vascular endothelium was regular.

On the other hand, intestines from the rats in the model group displayed coarsened epithelium, substantial dilation, thinning of intestinal walls, hyperemia, edema, friable mucus membranes with hemorrhage, inflammatory exudates, and their intestinal vasculature was disorganized. Examination also showed either white or colorless degenerating exudates adherent to the mucous membranes. In more severe cases, the exudates had the character ofpus. Finally, shallow, polymorphic ulcerations (of various sizes) of the mucus membranes were seen.

After 20-day applications of XKJ, colonic samples showed alleviations of the hyperemia, edema, and the quantities of ulcerations. Repair of the intestinal walls was measured at a grade of 0 on the Colon Mucus Membrane Tissue Damage Index (CMDI)--with some measurements tending towards grade 1. (Figure 2) P < 0.05

[FIGURE 2 OMITTED]

Colon mucous membrane morphological changes after intervention with XKJ: after the administration of Uyghur medicine Xipayi KuiJiean, tissue samples showed the following results: rat colons in the normal group showed complete mucous membranes without inflammatory infiltration or ulcerations. Their mucous epithelia were complete and continuous. Their glandular arrangement was regular with distinct structures. The lamina propria and muscularis mucosa of the inner mucous membranes were normal, and the vascular endothelia were regular.

But rats from the model group displayed tissue separation due to ulceration of the colon mucous membranes, massive granular cell infiltration of the submucosa and muscularis mucosa, and severe ulcerations to the depth of the submucosal vasculature.

Microscopically, inflammatory cell infiltration was reduced after exposure to XKJ. We observed a discontinuous, pale blue web of mucinous material overlaying the intestinal mucous membranes with reduced ulcerations as well as restoration of glands and epithelia. (Figure 3, 4, 5 and Table 2, 3, 4)

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

DISCUSSION

The use of a DNCB-Ethanoic acid compound to induce inflammation in the colons of rats resulted in test subjects whose symptoms, CMDI score and tissue pathological changes suggest the successful creation of an animal model of ulcerative colitis (according to a hapten induction mechanism with normal immune systems). The reaction between DNCB--as a hapten--and tissue protein stimulates a T-lymphocyte-dependent immune response (8).

Rats that were prepared in this way, as animal models of UC, showed reduced physical activity, were dispirited and lost weight. They were cheimophobic and their hair lost its gloss becoming sandy-colored. On the second day of ethanoic acid enemas, we visually observed bloody mucoid stool with pus in these rats--symptoms similar to what would be expected with genuine UC. Our animal models displayed adhesion of colon mucous membrane tissue to nearby tissues. Also, there was thickening and stiffening of the intestinal walls; hyperemia and edema of mucus membranes; erosions, and the formation of ulcers. The most severe ulcers' diameters were > 1 cm. Mucous membrane necrosis and pus formation occurred, and some rats even expired.

Tissue slices from the UC model rats showed tissue separation due to ulceration of colon mucous membranes, and massive granular cell infiltration of the submucosa and muscularis mucosa. Severe ulcerations occurred to the depth of the submucosal vasculature. The ultrastructure of colonic tissues showed obvious broadening of intercellular spaces, scattering and disorganization of the villi, and disordered cell arrangements.

These observations suggest that our ulcerative colitis rat model of rats was successful and may present as novel specimens for further experimentation on the etiology and pharmacologic treatment of this significant disease condition.

Uyghur medicine is an ethnic discipline with its own characteristic therapeutic system and illustrious history. Many folk remedies and proven traditional prescriptions have been shown to have unique curative effects on many diseases. Uyghur medicine theory emphasizes the idea that each organism is a unified whole. In Uyghur medicine, health is maintained as a balance between four hilit or "vital fluids": safra, kan, belhem and sewda (sometimes also Latinized as savda). According to this view, most diseases--such as ulcerative colitis, vitiligo, etc.--are due to an unbalancing of this organic wholeness. Uyghur medicine believes that the pathogenesis of ulcerative colitis is due to an imbalance of safra. The proposed pathogenesis involves the dilution of safra due to mixing with large amounts of unusual belhem or "vital fluid". This dilution results in a decrease in safra's "energy" function (characterized as fire in Uyghur medicine). This decreases the normal fluid functions of safra, which are: as an aid to digestion, to resist poisons, the expulsion of toxins, the normalization of kan, belhem and sewda "fluids".

Furthermore, the excessive wetness of belhem can result in a relaxation of the organization of organs and an increase in the permeability of blood vessel walls that results in additional mixing of fluid with safra leading to greater dilution. Inflowing of safra to colonic tissues results in inflammation over the long term9.

For the treatment ofulcerative colitis, Uyghur medicine advocates equal attention to be paid to recuperation and treatment, and simultaneously using targeted and whole body medication. Uyghur medicine Xipayi KuiJiean, according to traditional prescribing practice, is given as an enema preparation to adjust safra (10). It is a light yellowish-brown liquid with a delicate fragrance and an astringent but slightly sweet taste. These characteristics have "dry" and "cold" attributes in Uyghur Medicine. The drug is used to regulate safra, clean blood, stop pain, heal wounds, and cure dysentery (11). It contains Turkishgalls. Since ancient times, Turkishgalls has been regarded as one of the best substances for curing all types of chronic inflammatory disease. It is used as a disinfectant, as an astringent, to eliminate dampness, to stanch bleeding, and to reduce pain. It also has anti-immunity and anti diarrhoeal properties (12, 13, 14). Non-medicinally, it is primarily used as a tanning substance.

Previous research has confirmed that Uyghur medicine Xipayi KuiJiean has very strong anti-SARS, antioxidant, immune system modulation, anti-thrombotic, and antibacterial functions, as well as others (15-18). In the treatment of intestinal tract inflammation, there has been evidence of its curative effects8.

The gross changes of ulcerative colitis are visible via endoscopy. At the time of diagnosis, inflammation usually affects the rectum and sigmoid colon. The pathology of UC shows inflammation that is usually continuous from the rectum and may gradually spread to the proximal colon. It may involve the descending colon--which is often the case in this country--and, in more severe cases, it may involve the entire colon as pancolitis--which is rarely seen domestically. Toxic megacolon is a severe consequence of the disease.

Early UC changes to the colonic lining include: hyperemia, edema and hemorrhage at minor contact with the mucus membrane. Afterwards, necrosis of the mucus membrane and ulcerations appear. Ulcers grow gradually and form irregular, confluent ulcers that develop along the longitudinal or circumferential axes of the colon. The irregular ulcers create segments of tissue which extend into the enteric cavity because of the inflammation and edema. These form pseudopolyps. Occasionally, patches of mucus membrane can be observed hanging suspended over ulcers.

The regression in tissue pathology shows that Uyghur medicine Xipayi KuiJiean was an effective treatment of rat colitis. After administration of the medication, we discovered improvements in the health of mucous membrane tissues and in the gland structure of the large and medium dose intervention groups. The effects of the treatment include fewer glandular derangements and reduced infiltration by inflammatory cells; edema is improved and tissues showed signs of rapid healing. Even the findings from the small dose intervention group showed signs of obvious alleviation, albeit with a relatively slow rate of tissue repair. After administration of the medicine, the general types of colonic tissue histological changes were similar between the large/middle dose groups compared with the small dose group. There were no statistically significant differences in the therapeutic effects of the large versus middle dose XKJ intervention groups (P-30.05). However, there was a statistically significant difference between in therapeutic effects of both the large and middle dose XKJ intervention groups compared to the small dose intervention group.

The Uyghur medicine Xipayi KuiJiean is administered as an enema, so its usage corresponds directly to the location of pathological changes in UC. Increases in the drug concentration appear to improve the surface of intestinal tract ulcers and circulation, resulting in quick relief of of inflammation and healing of ulcers. Administration by this route allows preservation of the medication's effectiveness by avoiding degradation by digestive fluids or via digestive fermentation. According to western medical theory, it restores an organism's nutritional status; it protects and enhances an organism's defensive systems.

REFERENCES

(1.) Liszka L, Woszezyk D, Pajak J. Histopathological diagnosis of microscopic colitisJJ]. Gastroenterol Hepatol, 2006, 21(5):792~797.

(2.) Wang-Xing Peng. Current Gastroenterology[M]. Shanghai Scientific & Technical Publishers

(3.) Ou-Yang Qin, Pan-Guo Zong, Wen-Zhong Hui, et al. Recommendation of diagnosis and treatment standard of inflammatory bowel diseaseJJ]. Chinese Journal of Gastroenterology 2001, (6): 56-59.

(4.) Du-Zheng Guang. A clinical analysis of treating ulcerative colitis on 183 cases. Shandong Medical Journal, 2006, 46(22): 35.

(5.) Ou-Yang Qin, Liang-Hong Liang. Ulcerative Colitis[J]. Continuing Medical Education, 2006, 20(3): 30-34.

(6.) Zhang-zhi Ming. Clinical observation on BFGF retention enema in the treatment of chronic non-specific ulcerative colitis [J]. Chinese Journal of Gastroenterology, 2004, 24(7):17~19.

(7.) Li Lin,Wang-Zhu Li, Ke-Jian Ting, et al. Study of the experimental animal model of Ulcerative Colitis [J]. World Chinese Journal of Digestology, 2001, 9 :584-585.

(8.) Click ME, Falchuk IM. Dinitrochlorobenzene induced Colitis in the Guinea pig: Studies of Colonic Lamina Propria LymphocytesJJ]. Gut, 1981, 22: 120-125.

(9.) Halmurat Upur, Theory of Mizaj And Hilit in Uighur Medicine And Modern Study[M]. XinJiang Scientific & Technical Publishers, 2003, 7:39~40.

(10.) Halmurat Upur, Theory of Mizaj And Hilit in Uighur Medicine And Modern Study[M]. XinJiang Scientific & Technical Publishers, 2003, 7:50.

(11.) Xiang-Ying Zheng, Lei-Han Min et al. The function of herba geranium tannins in anti-inflammation, immunization and abirritation[J]. Medical Journal of National Defending Forces in Northwest China, 1998, 18(3): 172-174.

(12.) Wang-Li Min, Lu-Chun Feng, et al. Antidiarrhea effect of herba geranium tanninsDJDD Heilongjiang Medicine And Pharmacy, 2003, 26(5):28~29.

(13.) Li-Xiu Ping,Li-Chun Yuan, Qu Gui Rong. Study of Galla chinensis[J]. Acta Chinese Medicine and Pharmacology, 2002, 30(3): 72-74.

(14.) Fang-yan Zhang, MAO Xin-min, Hong-Li Wu et al. Immunological study on rat model of ulcerative colitis with the TCM syndrome of "liver depression and spleen deficiency" [J]. Journal of Xinjiang Medical University, 2004, 27(4): 367-369.

(15.) Mao Xin-ming, Halmurat Upur et al. Effects of kuijiean on cruor in experimental ulcerative colitis rats Which induced By TNBS[J]. Journal of Xinjiang Medical University, 2004, 27(5): 377-379.

(16.) Mao Xin-ming, Halmurat Upur et al. Effects of kuijiean on immunologic system in experimental ulcerative colitis rats Which induced By TNBS[D]. PhD. Dissertation by Mao Xin-ming, 2004

(17.) Hong-Il Wu, Mao Xin-ming, Effects of kuijiean on platelet agglomeration in rabbits and ulcerative colitis rats [J]. Journal of Xinjiang Medical University, 2005, l(28):28~30.

(18.) Li Haixia, Wang Zhao, Liu Yanze. Review in the Studies on Tannins Activity of Cancer Prevention and Anticancer[J]. Journal of Chinese Medicinal Materials, 2003, 6(26): 444-447.

The editor of JATMS understands that the animal experimentation referred to in the following article may not be consistent with the ethics and sensibilities of many natural therapists. Readers who find animal experimentation upsetting may want to proceed directly to the discussion of the results, which appear to support the efficacy of a traditional Uyghur medicine in the treatment of a common complaint and which explains the Uyghur philosophy of healing. Readers are encouraged to express their views on the suitability and value of such procedures to the practice of natural therapies and to their publication in JATMS.

Halmurat Upur, PhD; Kurexi Yunus, PhD; Yasen Mijiti, MPH; Huang Jing Jing, MPH; He Jie, MPH; Lian Jing Jing, MPH
Table 1 (CMDI) the index comparison of rats colon mucous membrane
damage from normal group and the model group.

 Mean
group n CMDI Rank Z P

 0 1 2 3 4

 level

normal 7 7 0 0 0 0 4.00 -3.589 0.000
group

model 10 0 0 2 4 4 12.50
group

Table 2 Pathological tissue comparison between normal and
model groups

group n pathological mean z p
 tissue rank
 comparison

 1 2 3

 level

normal group 7 7 0 0 4.00 -3.688 0.000

model group 10 0 3 7 12.50

P < 0.05

Table 3 Tissue pathological grading for different doses of XKJ

 Tissue pathological grading

group 1 2 3 Total
 level

Physiologic saline,
negative control 1 5 6 11
group

XKJ large dose group 7 4 1 12

XKJ medium dose 9 4 2 15
group

XKJ small dose group 2 5 9 16

Table 4 Comparison of tissue pathology at different doses of XKJ

group I n groupll Z P

Physiologic 11 XKJ large dose group -3.140 0.002
saline, negative XKJ medium does group -3.11- 0.002
control group XKJ small dose group -0.196 0.845

XKJ large dose 12 Physiologic saline, -3.140 0.002
group negative control group
 XKJ medium dose group -0.028 0/978
 XKJ small dose group -2.929 0.00

XKJ medium dose 15 Physiologic saline, -3.110 0.002
group negative control group
 XKJ large dose group -0.028 0.978
 XKJ small dose group -2.938 0.003

XKJ small dose 16 Physiologic saline, -0.196 0.845
group negative control group
 XKJ large dose group -2.929 0.003
 XKJ medium dose group -2.938 0.003
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Author:Upur, Halmurat; Yunus, Kurexi; Mijiti, Yasen; Jing, Jing; Jie, He; Jing, Lian Jing
Publication:Journal of the Australian Traditional-Medicine Society
Article Type:Report
Date:Dec 1, 2011
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