| As one of traditional acupuncture methods in Traditional Chinese Medicine, bloodletting therapy refers to discharge certain amount of blood by pricking the blood vessels with a three-edged needle or other sharp instruments in order to treat illness, with the functions of invigorating the blood and opening the collaterals, removing blood stasis and generating new blood, dispelling blockages and coagulations, and clearing heat while relieving toxins, etc. In clincal practice, the tutor of author has found that the bloodletting therapy can effectively prevent the progression of chronic liver disease to liver cancer. The preliminary experimental results showed that bloodletting therapy can reverse hepatic fibrosis. On the basis of this study, the therapeutic effect and mechanism of bloodletting therapy on the prevention of hepatic cellular cancer (HCC) were deeply discussed. Combined with the exiting research, the research group designed a experiment to discuss the effects of blood pricking on HCC rat model induced by diethylnitrosamine (DEN), by observing the pathological morphology change of the liver and the expression of tumor markers and tumor blood vessels generation promoting factors. Finally explore the preventive mechanism of blood pricking on HCC rats. We hope to provide a new method for early clinical intervention of hepatocellular carcinoma.ObjectiveThis experiment establish a HCC rat model by DEN intraperitoneal injection. At the same time of establishing the model, rats in the bloodletting preventing group were pricked on the superficial vein of Taichong acupoint, Zusanli acupoint and Yanglingquan acupoint. After the creation of the model, observed the liver tissue pathological morphology, the expression of the liver tumor markers AFP-L3, AFU and tumor blood vessels generation promoting factors VEGF, TGF-β1. At last, the mechanism of blood pricking in prevention of HCC rats were investigated.MethodsA total of 40 male Wistar rats were randomly divided into three groups, that is, the normal group(10 rats), the model group(15 rats), the bloodletting preventing group(15 rats). Rats in the model group and bloodletting preventing group were given a DEN intraperitoneal injection with twice a week to establish a HCC model. After four weeks, change the frequency to once a week until the thirteenth week. From the beginning of establishing the model, rats in the bloodletting preventing group were pricked for bloodletting 0.3-0.5ml each time and twice a week on the superficial vein of Taichong acupoint, Zusanli acupoint, Yanglingquan acupoint until the thirteenth week. After finishing the establishment of the model, the rats were made anesthesia with 10% of chloral hydrate, the liver tissues and blood were taken for detection. After cutting out the whole liver, weighted the liver, then calculated the liver index; The liver with hepatic lobule were removed for HE staining so that the pathological morphological changes of liver tissues were observed; Using the method of ELISA detected the expression of serum AFP-L3, AFU, VEGF, TGF-β1 and the liver tissues VEGF, TGF-β1.Results1. The pathological morphology change of the liver tissue:HE staining showed that the hepatic trabecular and the hepatic sinus were arranged properly in the normal group, and the hepatic cells were normal; In the model group, there were lots of cancer cells with hyperchromatic, pleomorphic nuclear, dysplastic nodules, rich interstitial blood sinus, part of the visible necrosis, which indicated the model was successful; In the bloodletting preventing group, the number of cancer cells was significantly reduced, cell staining was relatively uniform, but only a small amount of nodule formation, which showed the blood pricking was effective.2. The change of rat weight, live weight, liver index:Compared with the normal group, the weight of model group rats was sharply reduced (P<0.01), liver weight and liver index were significantly increased (P<0.01), the weight of bloodletting prevention group rats was reduced (P<0.01), liver weight and liver index were slightly increased (P>0.05,P<0.01); Compared with the model group, the body weight was obviously raised (P<0.05), the liver weight and liver index were clearly debased in the bloodletting prevention group (P<0.05, P<0.01).3. The expression of serum AFP-L3, AFU:Compared with the normal group, the content of serum AFP-L3 and AFU increased significantly in the model group (P<0.01), serum AFP-L3 and AFU content slightly increased in the bloodletting prevention group (P>0.05); The bloodletting prevention group compared with the model group, serum AFP-L3 and AFU content decreased obviously (P<0.01, P<0.05).4. The expression of serum VEGF, TGF-β1:Compared with the normal group, the level of VEGF and TGF-β1 content were significantly increased in the model group (P<0.01), slightly increased in the bloodletting prevention group (P>0.05); Compared with the model group, the level of VEGF and TGF-β1 content in the bloodletting preventing group were clearly decreased. The expression of VEGF was regulated down mostly in the liver (P<0.01), and the expression of TGF-β1 was regulated down fastly in the serum (.P<0.01).Conclusion1. In the study, we used the DEN method to induce the establishment of HCC rat model, and the method was reliable.2. Bloodletting therapy can improve the liver pathological morphology, reduce the degree of cell carcinoma, and have a positive regulation trend.3. Bloodletting therapy can significantly decrease the content of serum AFP-L3 and AFU, as well as the expression of VEGF and TGF-(31 in the serum and liver tissues.4. Bloodletting therapy has the effect of preventing or delaying the formation of HCC, which may be related to the reduction of tumor vessel angiogenesis factors VEGF and TGF-β1, and to inhibit tumor vessel angiogenesis. |
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