【摘要】：目的：建立慢性疲勞大鼠模型，觀察龜鹿益神配方顆粒對慢性疲勞大鼠一般情況和行為學指標（力竭游泳時間、鼠尾懸吊靜止時間）的影響，電鏡下觀察慢性疲勞大鼠骨骼肌線粒體微觀結(jié)構(gòu)的變化，檢測血清琥珀酸脫氫酶活性的變化，探討慢性疲勞綜合征的發(fā)病機理；觀察龜鹿益神配方顆粒對慢性疲勞大鼠骨骼肌線粒體微觀結(jié)構(gòu)和血清SDH的影響，探討其作用機制，為龜鹿益神配方顆粒治療CFS的臨床應用提供實驗依據(jù)和理論支持。 方法：雄性SPF級SD大鼠40只，體重200±20克，適應性飼養(yǎng)1周后，隨機抽取16只不進行造模，其余24只進行造模。采用強制力竭游泳，束縛和夾尾復合因素刺激，構(gòu)建慢性疲勞大鼠模型。造模成功后，未造模者隨機分為兩組，分別為正常組和正常對照組；造模者隨機分為3組，分別為模型組、蓯蓉益腎組、龜鹿益神組。從造模結(jié)束次日起，在正常飼養(yǎng)的基礎上，正常組、模型組給予生理鹽水灌胃；正常對照組、龜鹿益神組給予龜鹿益神配方顆�；鞈乙汗辔�；蓯蓉益腎組給予蓯蓉益腎顆�；鞈乙汗辔�。各組大鼠均給藥1次/d，連續(xù)14天。造模前、造模后及末次給藥后均測一次大鼠體重、力竭游泳時間及鼠尾懸吊靜止時間。上述實驗結(jié)束后，大鼠經(jīng)腹腔麻醉后，取各組大鼠右后肢骨骼肌組織一塊，電鏡下觀察線粒體結(jié)構(gòu)。經(jīng)腹主動脈采血分離上清液，采用ELISA法測定各組大鼠血清SDH活性。采用IBM SPSS19.0統(tǒng)計軟件對數(shù)據(jù)進行處理。 結(jié)果：（1）造模結(jié)束后，各組造模大鼠體重增長緩慢甚至減輕，飲食量、飲水量減少，瞇眼懶動，便溏，毛發(fā)暗淡失去光澤。與正常組比較，正常對照組體重、力竭游泳時間、鼠尾懸吊靜止時間無顯著性差異(P0.05)；模型組、龜鹿益神組、蓯蓉益腎組大鼠體重減輕、力竭游泳時間縮短、鼠尾懸吊靜止時間延長均有顯著性差異(P 0.05)。給藥結(jié)束后，與正常組相比，正常對照組力竭游泳時間延長有顯著性差異，龜鹿益神組無顯著性差異，兩組大鼠體重、鼠尾懸吊靜止時間均無顯著性差異(P0.05)；蓯蓉益腎組體重減輕有顯著性差異（P0.05），力竭游泳時間和鼠尾懸吊靜止時間均縮短無顯著性差異(P0.05)；模型組大鼠體重減輕、力竭游泳時間變短、鼠尾懸吊靜止時間延長均有顯著性差異（P0.05）。與模型組相比，龜鹿益神組、蓯蓉益腎組大鼠體重明顯增加、力竭游泳時間明顯延長、鼠尾懸吊靜止時間縮短均有顯著性差異（P0.05）。與蓯蓉益腎組相比，龜鹿益神組大鼠體重增加、鼠尾懸吊靜止時間縮短均無顯著性差異（P0.05），力竭游泳時間延長有顯著性差異（P0.05）。 （2）模型組大鼠骨骼肌肌原纖維排列紊亂，Z線、M線模糊且排列紊亂，線粒體形狀不規(guī)則，體積較正常組明顯偏小，多數(shù)線粒體出現(xiàn)空泡，膜溶解并相互連接。與模型組相比，龜鹿益神組和蓯蓉益腎組大鼠骨骼肌肌原纖維排列較整齊，Z線、M線清晰且整齊，線粒體體積大，空泡樣變少。與正常組對比，正常對照組大鼠骨骼肌排列更整齊，Z線、M線清晰且排列更有規(guī)則，線粒體結(jié)構(gòu)更完整，且體積大。 （3）與正常組相比，正常對照組血清SDH活性明顯降低，有顯著差異（P0.05）；龜鹿益神組大鼠血清SDH活性輕度升高，無顯著性差異（P0.05）；蓯蓉益腎組、模型組大鼠血清SDH活性升高，有顯著性差異（P0.05）。與模型組比較，蓯蓉益腎組、龜鹿益神組血清SDH活性降低，有顯著性差異（P0.05）。與蓯蓉益腎組相比，龜鹿益神組血清SDH活性降低，有顯著性差異（P0.05）。 結(jié)論：（1）本實驗采用非病毒感染的慢性應激刺激方法，即強制力竭游泳，束縛和夾尾激怒的復合因素刺激，構(gòu)建慢性疲勞大鼠模型。此方法操作性強，能夠模擬導致人類疲勞的內(nèi)外環(huán)境，可復制出人體的慢性疲勞狀態(tài)。 （2）龜鹿益神配方顆�？梢匝娱L慢性疲勞大鼠力竭游泳時間，縮短鼠尾懸吊靜止時間，保護線立體結(jié)構(gòu)的完整性，降低血清SDH的活性。 （3）本實驗表明，慢性疲勞大鼠骨骼肌及其線粒體結(jié)構(gòu)易損傷，且不易恢復，血清SDH活性明顯升高。龜鹿益神配方顆粒可增強骨骼肌及其線粒體膜的穩(wěn)定性，促進其損傷的恢復，，增強線粒體功能，降低血清SDH活性。提示龜鹿益神配方顆�？赏ㄟ^保護線粒體結(jié)構(gòu)、增強其功能從而達到抗疲勞的作用，進一步闡釋了慢性疲勞綜合征的發(fā)病機制及龜鹿益神配方顆粒治療該病的作用機理，為臨床應用龜鹿益神配方顆粒治療慢性疲勞綜合征提供了實驗依據(jù)。 （4）龜鹿益神配方顆�？勺鳛镃FS的預防性用藥。
[Abstract]:Objective: To establish a chronic fatigue rat model and observe the effects of Guilu Yishen Formula Granules on the general condition and behavioral indexes (exhaustive swimming time, resting time of rat tail suspension) of chronic fatigue rats. To explore the pathogenesis of chronic fatigue syndrome, observe the effect of Guilu Yishen Formula Granule on the microstructure of skeletal muscle mitochondria and serum SDH in rats with chronic fatigue, and explore its mechanism of action, so as to provide experimental basis and theoretical support for the clinical application of Guilu Yishen Formula Granule in treating CFS.
METHODS: Forty male SPF SD rats weighing 200 65 The control group was randomly divided into three groups: model group, Cistanche deserticola Yishen group and Guilu Yishen group. On the basis of normal feeding, normal group was given normal saline by gastric lavage; normal control group, Guilu Yishen formula suspension was given by gastric lavage; Cistanche deserticola Yishen group was given Cistanche deserticola Yishen formula suspension. The rats in each group were given the suspension once a day for 14 consecutive days. The body weight, exhaustive swimming time and tail suspension rest time were measured before modeling, after modeling and after the last administration. The supernatant of abdominal aorta was collected and the SDH activity was determined by ELISA. The data were processed by IBM SPSS 19.0 statistical software.
Results: (1) After modeling, the body weight of rats in each group increased slowly or even decreased, diet, water intake decreased, squint lazy, stool, hair dull loss of luster. Compared with the normal group, there was no significant difference in body weight, exhaustive swimming time, tail suspension rest time (P 0.05); model group, Guilu Yishen group, cirong Yishen group There were significant differences in weight loss, exhaustive swimming time and tail suspension rest time between the two groups (P 0.05). There were significant differences in weight loss (P 0.05), exhaustive swimming time and tail suspension rest time (P 0.05), body weight loss, exhaustive swimming time shortened and tail suspension rest time prolonged in model group (P 0.05). The weight of rats in Yishen group was significantly increased, the time of exhaustive swimming was significantly prolonged, and the rest time of tail suspension was significantly shortened (P 0.05).
(2) The myofibrils of skeletal muscle in model group were arranged disorderly, Z-line, M-line were blurred and arranged disorderly, the shape of mitochondria was irregular, and the volume of mitochondria was smaller than that of normal group. Most of mitochondria were vacuole, membrane dissolved and connected with each other. Compared with the normal group, the skeletal muscles of the normal control group were arranged more orderly, Z-line and M-line were clear and arranged more regularly, and the mitochondrial structure was more complete and larger.
(3) Compared with the normal group, the serum SDH activity of the normal control group decreased significantly (P 0.05); the serum SDH activity of the Guilu Yishen group increased slightly (P 0.05); the serum SDH activity of the Cistanche Yishen group increased significantly (P 0.05). Compared with the model group, the serum SDH activity of the Cistanche Yishen group and the Guilu Yishen group increased slightly (P 0.05). The activity of serum SDH in Guilu Yishen group was significantly lower than that in Cistanche deserticola Yishen group (P 0.05).
CONCLUSIONS: (1) A rat model of chronic fatigue was established by means of non-viral chronic stress stimulation, i.e. forced exhaustive swimming, restraint and tail-clip irritation. This method can simulate the internal and external environment of human fatigue and replicate the chronic fatigue state of human body.
(2) Guilu Yishen Formula Granule can prolong the exhaustive swimming time of rats with chronic fatigue, shorten the resting time of rat tail suspension, protect the integrity of the three-dimensional structure of the line, and reduce the activity of serum SDH.
(3) The results showed that the structure of skeletal muscle and its mitochondria was easy to be damaged, and it was not easy to recover, and the activity of serum SDH was obviously increased. Guilu Yishen formula granule could enhance the stability of skeletal muscle and its mitochondrial membrane, promote the recovery of injury, enhance the function of mitochondria and reduce the activity of serum SDH. The mechanism of chronic fatigue syndrome and the mechanism of Guilu Yishen Formula Granule in treating chronic fatigue syndrome were further elucidated, which provided experimental basis for clinical application of Guilu Yishen Formula Granule in treating chronic fatigue syndrome.
(4) Gui Lu Yi Shen formula granule can be used as a preventive drug for CFS.
【學位授予單位】：河南中醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R285.5

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