| Obj ective:Through the study of toxicological and pharmacological effect of Tangzu granules on experimental animal, Combined with the basic theories of Traditional Chinese Medicine(TCM), evaluate the pharmacodynamics of the TCM compound preparation systematically and comprehen-sively,which for the further development and utilization, provide scientific experimental data into clinical application and safety application for high-risk diabetic foot treatment.Method:Long term toxicity test:60 SD rats, male and female, randomly divided into three groups:Tangzu granules high dose group, Tangzu granules low dose group and the control group, with 20 rats in each group, high dose group and low dose group were administered 5.10 mg·kg-1 1.02 mg·kg-1. Weigh once every week, adjusted dosage according to the changes of the weight. Daily administration for 3 consecutive months. Observe the rats'general independent activities (such as diet, stools and urine, activities, death etc) after drug administration.12h after the last drug administration, collect ten the blood samples from the abdominal aorta in each group, examine the haematology indexes (WBC,N, etc.) and blood biochemical indexes (electrolyte, liver and kidney function), observed major organs(The heart, liver, spleen, etc.), and calculate organ coefficient. Taking each piece of tissue for histopathologic analysis, compared between groups.90 days after the end of administration, each group remain male and female 10 rats were observed for 2 week recovery period without drug. Examine the same index after 14 days to understand the degree of reversible toxicity and possible delayed toxicity.Diabetic foot animal model experiment:70 SD rats, male and female,60 rats were randomly selected. Intraperitoneal injection of STZ40 mg kg-1 within the abdominal cavity rapidly, monitor blood glucose daily, if random blood glucose level?16.7 mmol·L-1, consider as successful model. Intraperitoneal injection of intermediate-acting insulin four units per day is given for rats with random blood glucose> 25 mmol·L-1. The successful model of the 45 rats were randomly divided into Tangzu granules high dose group(LA), middle dose group(ME), low dose group(SL), model positive control group(MY), Model control group(MK), and the control group (ZK). With the normal control group, N=9. Tangzu granules high, medium and low dose group daily dose is 4.08 mg·kg-1,2.04 mg·kg-1,1.02 mg·kg-1. According to the change of body weight with given Tangzu granules. Model positive control group were given the same amount of warm distilled water. Model control group and normal control group without any treatment. Daily administration for 1 consecutive months. Random blood glucose is taken every two days in each group from tail blood. Weigh weekly. Observe the rats general independent activities after administration (such as diet, stools and urine, activities, death etc). After the last Administration, intraperitoneal injection of 10% chloral hydrate(300mg/kg) for anesthetic management. Take skin samples from the back, both sides of the spine,2cm above tail for HE staining to observe the effects of epidermal thickness, the number of fibroblasts and dermal appendages(sebaceous gland, hair follicle, sweat gland). The above indexes were determined by using semi quantitative analysis in Mias 3.0 graphic analysis system. Masson staining to observe dermal collagen fibers, and semi quantitative analysis of collagen fiber area and integral optical density. Immunohistochemical staining to observe epidermal growth factor(EGF), substance P(SP).and the positive material area and integral optical density of semi quantitative analysis. Compared between groups.Result:Long term toxicity test:no significant difference between the experimental groups in general independent activities and weight (P>0.05). After 3 mouths of administration, The routine blood test of rats in high dose group had significant difference compared with the blank control group(P< 0.05), while the medium dose had no significant difference (P>0.05). The recovery period of 14 days, no significant difference between the experimental groups in routine blood test (P>0.05).During the whole experiment, no significant difference in blood biochemical,organ coefficient and histopathology (P>0.05). The main organs with naked eyes had no obvious abnormalities(no hyperemia, edema, necrosis and degeneration).Diabetic foot animal model experiment:no significant difference between the experimental groups in general independent activities (P>0.05). Compared with ZK, statistically significant difference in weight between MK in 1,2 and 3 weeks (P<0.05). no significant difference between other pairwise comparison (P>0.05); HE staining:Compared with ZK and MK,The thickness of epidermis, the fibroblast cell nuclear area and the number of skin appendages of LA, ME and SL increased significantly(P<0.05), with statistical difference. The thickness of epidermis and fibroblast nuclei area increased significantly between LA and ME, while the number of dermal appendages in SL(P<0.01). Masson staining:Positive results:the collagen was green and blue. Compared with ZK and MK, The green and blue regions in the drug group increased and the area of collagen fibers increased obviously(P< 0.01), statistically significant difference. The average integral optical density increased and the increase in the number of collagen fibers(P<0.05), The difference was statistically significant. Compared with MY, the difference was statistically significant between other pairwise comparison in the drug group(P<0.05). Immunohistochemical staining:Positive material:brown yellow, yellow, light yellow, filaments, fine granular. and distribution in dermal appendages cells and dermal fibrous tissue. Measurement of EGF positive material area and integral optical density and found that the experimental group compared with the MK, ZK and MY group, the difference was not statistically significant(P> 0.05); Measurement of SP positive material area, Compared with the ZK, group MK and MY positive area and integral optical density decreased, the integral optical density in SL, ME and LA group reduced in turn, P<0.05, the difference was statistically significant. Compared with the ZK and MK, the positive substance area increased significantly in the drug group, The difference was statistically significant(P<0.01). The comparison between groups was not statistically significant.Compared with the MY, LA and ME group in the area and integral optical density were statistically significant(P< 0.05).Conclusion:Tangzu granules safety experiment:External use dosage for Tangzu granules is a safe dosage, no toxicity, delayed toxicity and accumulative toxicity if used long-term.Diabetic foot animal model experiment:(1) Tangzu granules can enhance the barrier function of the skin, improve water changes on high-risk diabetic foot in rats, regulate skin temperature, moisten and nourish the skin, relieve the symptoms of DF effectively; (2) By using Tangzu granules, the EGF of experimental group had little difference, we speculated that EGF had no effect on intact skin, it may work through damaged skin; (3) With the extension of the duration of DM, SP in the skin had a decreasing trend. Tangzu granules may increase the SP content in the skin to increase cell proliferation and wound repair function, And then delay or prevent diabetic foot ulcer and gangrene. |
PDF Full Text Request