| ObjectiveThrough experimental research, we testify the "Cited Drug Up" theory of large amount of borneol so as to provide Traditional Chinese Medicine (TCM) a new thinking and method of Acute Cerebrovascular Disease treatment. By inMethods1.Animal Experimental StudyIn 70 New Zealand white rabbits as the experimental object of study, 10 rabbits in each group and they were divided into:A. High dose Borneol and Ligustrazine Group; B. Borneol in dose and Ligustrazine Group; C. Low dose Borneol and Ligustrazine Group; D. Solvent and Ligustrazine Group; E. Physiological Saline Group and Ligustrazine Group; F. Physiological Saline Group; G. Intravenous Group; E. Reagent Group. Among them, High, middle and low concentration of borneol are respectively 20,10 and 5mg/ml. Each group of Ligustrazine concentration are all 50 mg/ml. According to 0.4ml of each rabbit, lumbar anesthesia catheter insert into one side of the rabbit nose, precisely and quantitatively drip into the reagent. After administration of 0.5,1,3,5,10 and 30min, we quantitatively and continuously collect cerebrospinal fluid through transcranial drilling windowing tube method. Using high performance liquid chromatography to compare cerebrospinal fluid concentration of ligustrazine.2.Cell Experimental StudyWe put Human airway mucous epithelial cancer cells NCI-H292 as in vitro model to study the characteristics of goblet cells. When cells grow to logarithmic gat is, A. High Dose Borneol Group B. Dose of Borneol Group C. Low Dose Borneol Group D. Solvent Group E. Physiological Saline Group F. Blank Group. Then we add the corresponding drugs (among them the concentration of the borneol are 0.2,0.1 and 0.05mg/ml). CCK8 assay was applied to detect the each group cell proliferation activity in 24,48 and 72h after administration.After treated with 1,5,10 and 15min, the expression of mucin cells were detected by ELISA method. After each group respectively add the corresponding drugs (among them the concentration of the borneol are 0.2, 0.1 and 0.05mg/ml) 1,5,10,15min later, we collect cells and extract total RNA of each group cells by TRI REAGENT. According to RevertAidTM instruction, synthesize the first strand cDNA cells in order to amplified fragment. According to the literature, we synthesize the upstream and downstream primer MUC5AC. MUC5AC 5’Primer:TCCGGCCTCATCTTCTCC,3’Primer: ACTTGGGCACTGGTGCTG.94℃ degeneration for 30s. Annealed at 60℃ for 50s. 72℃ extension for 50s.35 cycles. Refer to GAPDH for internal. Using the analysis software of image measurement for MUC5AC in each strip so that we can come to the expression in each group at different time points of the mucin quantity.Results:(1) Animal experimental studyIn addition to the group G within 1 min after intravenous administration of peak, group there were significant differences between different time points (p< 0.05), ABC three groups are about 3-5 min of cerebrospinal fluid after the treatment, the concentration of ligustrazine in peak (p< 0.05), A, B, C composite between borneol and D, and E group, the average content of ligustrazine in cerebrospinal fluid in there was no statistically significant difference, D, E average content of ligustrazine differences between groups have no statistical significance. Group G intravenous drug (group E reagent) after 1 min, the concentration of the peak of ligustrazine in cerebrospinal fluid in the peak of 9.6 mu (G/ml). ABCDE comparison between group and G group, group G in dosing concentration after 1 min started falling fast, and in addition to the group F, ligustrazine concentration in cerebrospinal fluid in the other groups decrease obviously after dosing for 10 min.ConclusionIn the high, middle and low dose borneol group, there is no obvious role by intranasal administration of Ligustrazine. The peak time of intranasal absorption is 3-5min. The preparation of borneol solvent had no significant effect on the experimental results. Intravenous administration absorb rapidly,1 min reached the peak. However, CSF drug concentration metabolism speedy and the efficacy cannot endure for a long time. It concludes that Ligustrazine can directly through the blood brain barrier into the cerebrospinal fluid. (2) Cell Experimental Study2.1 Detection method of CCK8 on cell proliferation of Borneol StudyResultsAfter 24h and 72h administration, we compared with the blank group and there is no significant influence on cell proliferation between the groups. After 48h administration, we compared with the blank group. High Dose Borneol Group (0.2mg/ml) is obviously promote the proliferation of cells. Dose of Borneol Group (0.1mg/ml) and Low Dose Borneol Group(0.05mg/ml)had no significant effect on cell proliferation. Solvent Group and Physiological Saline Group promote cell proliferation. Compared with Solvent Group, High Dose Borneol Group (0.2mg/ml) has no statistical significance between the two groups. There was statistical significance between Solvent Group and Physiological Saline Group (p<0.05).ConclusionHigh Dose Borneol Group promotes the proliferation of cells. Dose of Borneol Group and Low Dose Borneol Group had no significant effect on cell proliferation. Solvent DMSO has an effect on cell proliferation.2.2 Enzyme-linked immunosorbent assay (ELISA)for the detection of mucin expression (MUC5AC) StudyResultsThere is no statistically significant differences between the groups at different time points. There were statistically significant differences in High Dose Borneol Group, Dose of Borneol Group and Low Dose Borneol Group at different time points (p<0.05). There is no statistically significant difference between Blank Group, Solvent Group and Physiological Saline Group at each time point. Among High Dose Borneol Group, Dose of Borneol Group and Low Dose Borneol Group, High Dose Borneol Group inhibited protein MUC5AC prominently in the 10min. What’s more, Dose of Borneol Group inhibited protein MUC5AC obviously in the 5min and Low Dose Borneol Group manifested inhibitory ability of MUC5AC protein in the 15min.ConclusionThe expression of MUC5AC had no obvious effect of borneol. It probably because of the packet using drug dose is not enough so that the difference between groups was not obvious.2.3 Fluorescence quantitative PCR detection method of mucin (MUC5AC) mRNA StudyResultsAfter 1 min drug administration, compared with Blank Group, there was significant difference among the groups High Dose Borneol Group, Dose of Borneol Group and Low Dose Borneol Group (p<0.05). There is no statistically significant difference between Physiological Saline Group and Blank Group. Compared with Solvent Group, Dose of Borneol Group differences between the two groups had no statistical significance. There is no statistically significant difference between High Dose Borneol Group and Low Dose Borneol Group. After 5 mins drug administration, compared with Blank Group, there was significant difference among the groups High Dose Borneol Group, Dose of Borneol Group and Low Dose Borneol Group (p<0.05) and Solvent Group and Saline Group had no statistically significant difference between the three groups. Compared with Solvent Group, there is differences between the three groups High, Middle Dose of Borneol Group had statistical significance (p<0.05). Low Dose Borneol Group differences between the two groups had no statistical significance. After 10 mins drug administration, compared with Blank Group, High, Low Dose Borneol group differences between the three groups had statistical significance(p<0.05). Compared with Solvent Group, High Dose Borneol Group between the two groups have statistical significance (p<0.05). There was statistically significant difference between the two groups High Dose Borneol Group and Low Dose Borneol Group. After 15 mins drug administration, compared with Blank Group, High, middle Dose Borneol Group differences between the three groups had statistical significance (p<0.05). Solvent Group and Saline Group had no statistically significant difference between the three groups. Compared with Solvent Group, High, middle Dose Borneol Group differences between the three groups had statistical significance (p<0.05). There is no statistically significant difference between the two groups High Dose Borneol Group and Dose of Borneol Group. In the comparison of high, middle and low dose borneol group three groups in different time point, there were statistically significant differences in 1 min of High Dose Borneol Group and Low Dose of Borneol Group(p<0.05). There were statistically significant differences in Dose of Borneol Group in 5 mins(p<0.05).ConclusionAfter lmin drug administration, different concentrations of borneol and DMSO have varying degrees of reduction effect on gene expression MUC5AC. The downward function of Low Dose Borneol Group regulated genes MUC5AC in the 5 mins expression weakened. However, the downward function of Dose of Borneol Group regulated genes MUC5AC in the 5 mins expression was obvious. When it comes to 15 mins, the downward function of Low Dose Borneol Group and DMSO regulated genes MUC5AC in the expression weakened. |
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