| Objective:Through animal experiments,study Tangzu granules toxicology and pharmacology, a comprehensive evaluation of the efficacy of traditional Chinese medicine kinetics and drug safety, for large-scale investment in the clinical use of Tangzu granules preparation.Method:Acute toxicity:64 SD rats, male and female, divided into groups intact skin and damaged skin group, two large groups they were divided into four groups, the Tangzu granules high dose group, the Tangzu granules low dose group, positive control group, the control group. N= 8. High Tangzu granules, low-dose group were administered 2.04 mg·kg-1,1.02 mg·kg-1; positive control group was treated with warm distilled water.24h washed after contact. Observed immediately after administration to rats with systemic reactions and deaths within 14 days, to be administered on days 3、7、10 and 14 weighed again. On time, within a group, were compared between groups.Irritation test:16 rabbits, male and female, divided into groups intact skin and damaged skin group, two large groups they were divided into two groups, the Tangzu granules high dose group, Tangzu granules low dose group. N=4. Each rabbit spine on the left side of the experimental side, the right side as a positive control. High Tangzu granules, low-dose group were administered 25.71 mg·kg-1,12.86 mg·kg-1; positive control side to give warm distilled water.24h washed after contact. After the removal of Tangzu granules 1、24、48、72h skin reactions, according to the technical guidelines medicine research in natural medicine irritating skin irritation score stimulation strength evaluation. On time, within a group, were compared between groups.Neuropathic diabetic foot animal model experiments:SD rats were 60, male, divided into non-surgical group (A) and the surgical group, surgery group was divided into blank control (BK) group, a positive control (BY) group, high dose (BG) group, the middle dose (BM) group, low dose (BD) group. N= 10. Disposable surgical group rapid injection STZ 0.2 ml/100g within the abdominal cavity. Respectively, after injection STZ 3、5、7 d fasting blood glucose≥13.9 mmol·L-1 were as successful model. Then every other day using the tails assay random blood glucose of rats given glucose> 25 mmol·L-1 in rats plus insulin injections, and about four intraperitoneal injection of insulin units per day. Weight tested once a week. Clamp sciatic nerve to produce an animal model of diabetic foot. After reflection rat paw test modeling show disappeared for successful modeling. 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. Positive control group given the same amount of the same method of isothermal distilled water. Daily administration 1h, for a month.2 and 4 weeks, respectively, after the first detection of rat sciatic functional index (SFI), after four weeks, the rats were sacrificed, and the operated side and the normal side gastrocnemius wet weight compared to the calculated recovery rate; surgery lateral gastrocnemius slice, producer, light microscopy of muscle cell growth, with a diameter of Mias 3.0 graphical analysis system of muscle cells; nerve regeneration cut about 10 mm, producer, light microscopy longitudinal section of the sciatic nerve myelinated axon regeneration and sheath formation. Mias 3.0 uses graphical analysis system axon area, myelin area of semi-quantitative analysis. Compare conducted between groups.Ischemic diabetic foot animal model experiment:experimental animals, grouping, STZ modeling with neuropathic diabetic foot animal model experiments. Ligation of the femoral artery to produce an animal model of ischemic diabetic foot. Administration with neuropathic diabetic foot animal model experiments. After four weeks, the rats were killed, the femoral artery blood law, under the conditions of 4℃ centrifugation,3000 rpm,10 min, the supernatant was collected using a pipette, measuring the surrounding plasma IL-6 using ELISA. CRP, e-NOS, ET, using nitrate reductase assay surrounding plasma NO. Compare conducted between groups.Result:Acute toxicity:high-dose group, and a complete set of breakage group mean weight increase with little difference between the control group (P>0.05), low-dose group, and a complete set of breakage group mean weight increase with little difference between the control group (P>0.05), the positive control group, the mean body weight increased with little difference between the control group (P>0.05).3,7,10,14 days after the first treatment, changes in body weight of rats showed a steady rise, and the similar rise each time period, no significant difference (P>0.05).Irritation test:whether it is intact and damaged skin skin group group were shown, enough sugar granules within 72 hours to one hour after the skin stimulation (P>0.05).Neuropathic diabetic foot animal models:rat sciatic functional index measurement showed enough sugar granule treatment group and the control group, the effect is no different, still no therapeutic advantage four weeks later, SFI BG, BM, BD group were compared with the BY group the difference was not statistically significant (P>0.01); comparison between BG, BM, BD group, no significant difference (P>0.01); each group gastrocnemius wet weight recovery rate, BG, BM, recovery rate in BD group BY, BK group (P<0.01); BG group was higher than BM group and the BD group (P<0.01); gastrocnemius muscle cell diameter, surgical model in each group with the A group, significantly reduced the diameter of the muscle cells, the difference was statistically significance (P<0.01), sugar granules foot surgery treatment group compared with the control group, myocyte diameter increased, the difference was statistically significant (P<0.01), BG group compared with BM, BD, a significant increase in muscle cell diameter, the difference was statistically significant (P<0.01); sciatic nerve myelin, the axon area, compared to the total area of myelin model significantly reduces surgical groups with group A, the difference was statistically significant (P<0.01), axonal area the sum of the sizes, the difference was not statistically significant, (P>0.01), enough sugar granules treated with surgical control group, significantly reduced the total area of the myelin sheath, the difference was statistically significant (P<0.01), axonal area the sum is insignificant, no statistical significance (P>0.01). BG group and BM, BD, myelin area, axon area is insignificant, no statistical significance (P>0.01).Ischemic diabetic foot animal model experiments:each group gastrocnemius wet weight recovery rate, recovery rate BG, BM, BD group and BY, BK little difference between groups (P>0.01), BG Group recovery rate and BM, BD group relatively little difference(P>0.05); the serum IL-6, CRP, NO, e-NOS, ET level, BK, BY, BG, BM, BD group compared with group A, the difference was statistically significant (P<0.01),mBY, BG, BM, BD group respectively BK group, the difference was statistically significant (P<0.01), BG, BM, BD BY group were compared with the group, in addition to IL-6 levels, BD group no significant difference between the BY group (P>0.01), the other factor levels were significantly (P<0.01),BG group, BD group compared with BM, the difference was statistically significant (P<0.01).Conclusion:(1) Tangzu granules safety studies:In a certain period, the rats no significant acute toxicity; nor rabbits in each group had mild erythema, edema, mild skin irritation reactions. Statistical analysis showed that:Tangzu granules toxicity weak groups; no significant acute toxicity and irritation reactions. Therefore, we believe that preliminary clinical topical dose of Tangzu granules safe dose.(2) After treatment enough Tangzu granules, neuropathic diabetic foot rat toe color, shape has improved, increasing the gastrocnemius muscle cell diameter, shape recovery significantly, and high-dose treatment group was better than in the low dose group good; but in the sciatic nerve sciatic functional and histological observation, no significant difference in Tangzu granules treatment group and the control group recovery.(3) After treatment enough Tangzu granules, ischemic diabetic foot rat serum CRP and IL-6 in the amount of decline, increased expression of e-NOS, NO synthesis, reduce the level of ET, endothelin balance system is adjusted, vascular endothelial cells obtained protection, microcirculation reduce and prevent foot ulcers and gangrene sugar occurs. |
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