| Objective: Through observing the expression of VEGF, bFGF, and NGF in endothelium of sciatic nerve and surrounding tissues, acknowledging the effect by using Wen Run Tong Luo Fang, we are going to investigate the mechanism of DPN and the protective effect of the specific prescription mentioned above in order to provide more clinical evidences to the prescription which enriches and develops the theory of traditional Chinese medicine cause of disease pathogenesis and therapeutics of TCM.Method: Choosing 70 SD male rats weighing 200~220g as the subjects, and the rats which were free of eating and drinking were raised in the ordinary lab. The rats were fed adaptively for 1 week, and then 60 of them were fasted for 12 hours. After that they had one-time raperitoneal injection of plain citric acid of chain urea assistant bacterium sodium- citric acid slows liquid down (Citric acid sodium-citric acid slows liquid down 0.1mmol/L, Ph:4.2-4.5),72 hours later, they were taken some blood from the tip of the tails to test the glucose index. The rats whose glucose index were beyond 16.7mmol/L had been enrolled in the experiment. The rats which are successfully experimented are divided into 5 groups at random: model Group, group of High-dose Wen Run Tong Luo Fang, group of Low-dose Wen Run Tong Luo Fang, group of Xue Fu Zhu Yu Tang and methycobal group. Additionally, 10 rats were arranged as the normal group which were injected raperitoneally with the same volume of Citric acid sodium- citric acid slows liquid down. The rats in the Wen Run Tong Luo Fang of lower dose group were administered 17g/kg·d (according to the ten times of the dose as much as it is applied to the human on clinical setting); The rats in Wen Run Tong Luo Fang of higher dose group were administer 34 g/kg·d (according to the twenty times of the dose as much as it is applied to the human on clinical setting); Rats in Xue Fu Zhu Yu Tang were administered 12.8g/kg·d (according to the ten times of the dose as much as it is applied to the human whose weight are 60kg on clinical setting); Rats in methycobal group were administered 250μg/kg·d (according to the ten times of the dose as much as it is applied to the human whose weight are 60kg on clinical setting), and the rats in this group were also received trace element supplementation and the medication was administered according to the volume as 1ml/100g. The ordinary state of the rats was observed including the color of the skin, looks, weight, diet, drink, urination, movement and so on. 8 weeks after they were received trace element supplementation and were r killed and taken the blood. We separated blood serum and plasma to test blood glucose by the method of glucose phenylhydrazone. Then they were taken sciatic nerve and the tissues around it (about 0.5cm×0.5cm×0.5cm), and it was fixed in 4% paraformal-dehyde and using HE, after that the change of form of nervous tissues of sciatic nerve was observed by light microscope. Meanwhile we used IHCA to observe the expression of VEGF, bFGF, NGF.Results:1 General conditionsBefore modeled, animals in each group reacted smartly , had glossy hair and normal color, active moments. Three days after the rats were modeled by injection of STZ, there were the states of muti-urination, muti-drink, muti-diet, they seemed to lose weight, appear depressed and their skin were fade and exiguous. There was also deformed defecation, retard movements and inactive reaction. The whole state was not good. Given medication for 8 weeks continuously, rats in normal group seemed in good state of mood, weight , movement, skin. There was no significant upset. Rats in model group had the syndrome of increasing of quantity of drink, urination and diet, but they had fade and exiguous hair and they were easily upset and obvious loss of weight. Rats in Xue Fu Zhu Yu Tang and methycobal group similarly seemed to have increasing of drink, urination and diet, and fade and exiguous hair and they were easily upset and obvious loss of weight. The rats in the Wen Run Tong Luo Fang of lower and higher dose have released the symptoms of the increasing of drink, urination and diet. And they also had increasing of weight, smooth hair and formed defecation and active movements. The situation in Wen Run Tong Luo Fang of higher dose was better than that in lower dose group.2 Changes in weightThe weights of rats in model group were decreasing more evidently than those in normal group (P<0.01);The weights of rats in other groups were obviously increasing which have differences from the rats in model group(P<0.01); Rats in Wen Run Tong Luo Fang of higher dose group had obvious increasing weight compared to the lower dose group, Xue Fu Zhu Yu Tang and methycobal group(P<0.01); Though there is weight increasing in Wen Run Tong Luo Fang of lower dose group, but there is no statistic significance in comparing with the Xue Fu Zhu Yu Tang and methycobal group. 3 Changes in blood glucoseThe blod glucose of rats in model group had evident difference compared with that in normal group (P<0.01);And the blood glucose in Wen Run Tong Luo Fang group of higher or lower dose group has no significant changes compared with model group(P<0.01), Xue Fu Zhu Yu Tang and methycobal group had no significant changes compared with model group (P>0.05);Wen Run Tong Luo Fang group of higher dose group had obvious differences from the lower group, and so did the Xue Fu Zhu Yu Tang and methycobal group(P<0.01);There were significant differences among the Wen Run Tong Luo Fang of lower dose group, the Xue Fu Zhu Yu Tang and methycobal group(P<0.01).4 Morphologic changes in sciatic nerve tissuesSciatic nerve tissues of the rats in normal group indicates that Amyelic nerve fiber are arranged closely, medullary sheath tissues are normal. Schwann cells are scattering on the edge of medullary sheath, axons could be seen clearly in medullary sheath. The Sciatic nerve tissues of rats in model group indicated that nerve fibers are swollen, medullary sheath are decreasing, capillary vessels of epineurium have dilatation and congestion, inflammatory cells infiltrate, parts of axons disappear. Sciatic nerve tissues of the rats in methycobal group appeared to have swollen of nerve fibers, decreasing of sheath cells, most parts of medullary sheath dissolved. The Sciatic nerve tissues of rats in Wen Run Tong Luo Fang group of lower dose group and Xue Fu Zhu Yu Tang group indicated that nerve fibers were swollen, axons disappeared. In Wen Run Tong Luo Fang group of higher dose group a few normal medullary sheath could be seen, more sheath cells and normal axons in medullary sheath.5 The expression of VEGF, bFGF, NGF of sciatic nerve and surrounding tissues.The mean gray level of expression of VEGF, bFGF, NGF sciatic nerves of rats in model group seemed obviously higher than that in normal group (P<0.01);After medication, the expression of VEGF, bFGF, NGF of sciatic nerves in the other three groups seemed to have a lower mean gray level than that in model group(P<0.01); the expression of VEGF, bFGF, NGF of the surrounding tissues were lower in the mean gray level than that in model group(P<0.01). In addition, the mean gray value expression of VEGF, bFGF, NGF in the sciatic nerves of rats in Wen Run Tong Luo Fang of lower does compared with those in Xue Fu Zhu Yu Tang group has no statistic significance(P>0.05); The mean gray value expressions of VEGF, bFGF, NGF in the sciatic nerves of rats in Wen Run Tong Luo Fang of lower dose group compared with that in methycobal group were higher (P<0.05); The mean gray value expressions of VEGF, bFGF, NGF in the sciatic nerves of rats in Wen Run Tong Luo Fang of higher dose compared with those in Xue Fu Zhu Yu Tang and Wen Run Tong Luo Fang of lower dose group were lower(P<0.01); The mean gray value expressions of VEGF, bFGF, NGF in the sciatic nerves of rats in Wen Run Tong Luo Fang of higher dose compared with those in methycobal group, there was no statistic significance (P>0.05).The mean gray value expressions of VEGF, bFGF of surrounding tissues of sciatic nerves of rats in model group were much higher than those in normal group(P<0.01);The mean gray value expressions of bFGF of surrounding tissues of sciatic nerves of rats in model group was higher than that normal group(P<0.05);After medication, all of the groups have the lower mean gray value of VEGF, bFGF, NGF compared with those in model group(P<0.01). And there is no statistic significance in the comparison between the mean gray value of VEGF, bFGF, NGF surrounding tissues of sciatic nerves of rats in Wen Run Tong Luo Fang of lower group and Xue Fu Zhu Yu Tang group (P>0.05);The mean gray value expressions of VEGF, bFGF, NGF of surrounding tissues of sciatic nerves of rats in Wen Run Tong Luo Fang of lower dose were higher than those in methycobal group(P<0.05);The mean gray value expressions of VEGF, NGF of surrounding tissues of sciatic nerves of rats in Wen Run Tong Luo Fang of higher dose group were lower than those in Xue Fu Zhu Yu Tang group and warm and mild prescription of lower dose group(P<0.01);The mean gray value expressions of bFGF of surrounding tissues of sciatic nerves of rats in Wen Run Tong Luo Fang of higher dose group were lower than those in Xue Fu Zhu Yu Tang group and warm and mild prescription of lower dose group(P<0.05);there is no statistic significance in the comparison between the mean gray value of VEGF, bFGF, NGF surrounding tissues of sciatic nerves of rats in Wen Run Tong Luo Fang of higher group and methycobal group(P>0.05).Conclusions:1 Wen Run Tong Luo Fang could improve the symptoms and signs of the modeled rats.2 Wen Run Tong Luo Fang could evidently use the pathomorphism of sciatic nerves of modeled rats.3 Wen Run Tong Luo Fang could heighten the expressions of VEGF, bFGF, NGF of sciatic nerves and the surrounding tiss- use of modeled rats in order to protect the peripheral nerve to treat DNP.
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