| ObjectiveThrough establishment blood tumor barrier (BTB)model in vitro, to research the molecular mechanisms of MAPKs signal transduction pathway and regulation of the tight junction proteins and genes. To clarify effects and mechanism of natural borneol regulating blood tumor barrier and to pen a new way forclinical treatment of intracranial diseases such as glioma.Methods1. The establishment of BTB model in vitro.Through non-contact co-culturing of C6 rat glioma cells and human umbilical vein endothelial cells(HUVECs) to establish cell BTB model in vitro. Using endothelial cell membrane impedance(TEER) and HRP flow to evaluate its performance.2. Effect of permeability about natural borneol on BTB model.The experiment was divided into 4 groups:control group, borneol low dose group (25μg/mL), borneol middle dose group(50μg/mL), borneol high dose group (100μg/mL), each group has three wells. TEER assays and HRP flow to determine the permeability of different doses of natural borneol on BTB mode lin vitro.3. Effect of expression of MAPKs signal transduction pathway related kinase about natural borneol on BTB modle.The experiment was divided into 4 groups:control group, borneol low dose group(25μg/mL), borneol middle dose group(50μg/mL), borneol high dose group (100μg/mL), each group has three wells. Western blot to determine the expression of ERK, JNK, p38, p-ERK,p-JNK, p-p38 with different doses of natural borneol on BTB model.4. Effect of permeability of blood tumor barrier, expression of ERK, p-ERK, tight junction related protein and gene about combination of natural borneol and ERK inhibitor U0126 on the BTB modle.The experiment was divided into 8 groups:control group, natural borneol low dose group (25μg/mL), natural borneol middle dose group (50μg/mL), natural borneol high dose group (100μg/mL), U0126 group, U0126 and low dose natural borneol combination group, U0126 and middle dose natural borneol combination group, U0126 and high dose natural borneol combined groups, each group has three wells. TEER assays and HRP flow to determine the permeability of BTB model of different group in vitro. Western blot to determine the expression of protein ERK, pHERK, Claudin-5, F-actin and ZO-1 in different group. Real Time-PCR to detect the expression of TJs associated genes, Claudin-5, F-actin and ZO-1, in different group5. Effect of permeability of blood tumor barrier, expression of Rafl, p-Rafl, tight junction related protein and gene about combination of natural borneol and Rafl inhibitor Sorafenib Tosylate(ST) on the BTB modle.The experiment was divided into 8 groups:control group, natural borneol low dose group (25μg/mL), natural borneol middle dose group (50μg/mL), natural borneol high dose group (100μg/mL), ST group, ST and low dose natural borneol combination group, ST and middle dose natural borneol combination group, ST and high dose natural borneol combined groups, each group has three wells. TEER assays and HRP flow to determine the permeability of BTB model of different group in vitro. Western blot to determine the expression of protein ERK, p-ERK, Claudin-5, F-actin and ZO-1 in different group. Real Time-PCR to detect the expression of TJs associated genes, Claudin-5, F-actin and ZO-1, in different group. Results1. The successfully established BTB cell model in vitro, can be used to simulate in vivo environment, and for the study of drugs across the blood tumor barrier.2. TEER assays and HRP flow to determine the permeability of BTB model of different doses of natural borneol in vitro. Results show that compared with the control group, TEER value firstly dose-dependent decrease and then dose-dependent increase gradually, restored to the level before the medicine, respectively, in the natural borneol low dose group(25μg/mL), middle dose group (50μg/mL) and high dose group (100μg/mL). And in the 30min TEER value was the lowest(P<0.01). HRP flow Results:permeation rate of BTB modle in different natrual borneol group dose-dependent increased more obviously than the control group from 10min to 240min after natural bornel administration (p<0.01); From the transmittance of adjacent time point, low middle high dose groups are in 10-30min,30-60min, enhancement of HRP is most obvious, upgrade rate gradually decreased at 60-120min, returned to normal levels at 120-240min.3. Western blot results showed that:There was no significant difference exist at all time points expression of ERK, p38, p-p38, JNK and p-JNK (The expression of p-JNK at 180min,240min were excluded) (P> 0.05). The expression of p-ERK(30min,60min) and p-JNK (180min,240min) obviously increased in the low-dose group, with the efficacy superior to the the control group(P<0.05). The expression of p-ERK (10min,30min,60min,120min,180min) and p-JNK (180min, 240min) increased to various degrees in the middle-dose group when compared with the low-dosel group(P<0.05). The expression of p-ERK(10min,30min,60min, 120min,180min,240min) and p-JNK (180min,240min) increased more obviously in the high-dose group than in the middle-dose(P<0.05).4. TEER assays to determine the permeability of BTB model of different group in vitro, the results showed:Compared with the control group, the cell resistance was significantly increased, decreased in the U0126 group, cell resistance was significantly decreased in the borneol group, and there have a statistically significant difference (p<0.01); compared with borneol group, the resistance of the cell in the combination group was significantly increased, with a statistically significant difference (p<0.05). HRP flow to determine the permeability of BTB model of different group in vitro. The results showed: Compared with the control group, the permeation rate in U0126 group was significantly decreased, permeation rate in borneol group increased significantly(p< 0.01); the permeation rate of the combination group was significantly lower than borneol group, with a statistically significant difference (p<0.05). Western blot to determine the expression of protein ERK, p-ERK, Claudin-5, F-actin and ZO-1 in different group. The determination of kinase ERK result that there are no significant changes in the amount of protein expression on different groups, suggests that natural borneol low dose group, middle dose group, high dose group are no significant (P>0.05) within 4h. Compared with the control group, in U0126 group, of expression p-ERK and TJs associated protein was significantly reduced and increased respectivily, expression of p-ERK and TJs associated protein in borneol group was dose-dependently increased and decreased respectivily(p<0.01); compared with borneol group, expression of p-ERK and TJs associated protein in the combination group was significantly reduced and added respectivily, with a significant difference (p<0.05). Real Time-PCR to detect the expression of TJs associated genes, Claudin-5, F-actin and ZO-1, in different group. RT-PCR results showed that:Compared with the control group, in U0126 group, the expression of gene Claudin-5, Occludin, ZO-1, F-actin was significantly increased; in borneol group expression of genes Claudin-5, Occludin, ZO-1, F-actin was significantly reduced (p<0.01); compared to the borneol group, expression of genes Claudin-5, Occludin, ZO-1, F-actin was significantly increased in combined group, with a statistically significant difference (p <0.05). Its trend is consistent with the results of the protein determination.5. TEER assays to determine the permeability of BTB model of different group in vitro, the results showed:Compared with the control group, the cell resistance was significantly increased, decreased in the Sorafenib Tosylate(ST) group, cell resistance was significantly decreased in the borneol group, and there have a statistically significant difference (p<0.01); compared with borneol group, the resistance of the cell in the combination group was significantly increased, with a statistically significant difference (p<0.05). HRP flow to determine the permeability of BTB model of different group in vitro. The results showed:Compared with the control group, the permeation rate in ST group was significantly decreased.permeation rate in borneol group increased significantly(p< 0.01); the permeation rate of the combination group was significantly lower than borneol group, with a statistically significant difference (p<0.05). Western blot to determine the expression of protein ERK, p-ERK, Claudin-5, F-actin and ZO-1 in different group. Compared with the control group, in ST group, of expression Rafl and p-ftaf 1 was significantly reduced respectivily, expressionof in borneol group was dose-dependently increased respectivily(p<0.01); compared with borneol group, expression of Rafl and p-Raf 1 in the combination group was significantly reduced drespectivily, with a significant difference (p<0.05). Compared with the control group, in ST group, of expression TJs associated protein was significantly increased, expression of TJs associated protein in borneol group was dose-dependently decreased (p<0.01); compared with borneol group, expression of TJs associated protein in the combination group was significantly added, with a significant difference (p<0.05). Real Time-PCR to detect the expression of TJs associated genes, Claudin-5, F-actin and Z0-1, in different group. RT-PCR results showed that:Compared with the control group, in ST group, the expression of gene Claudin-5, Occludin, Z0-1, F-actin was significantly increased; in borneol group expression of genes Claudin-5, Occludin, Z0-1, F-actin was significantly reduced (p<0.01); compared to the borneol group, expression of genes Claudin-5, Occludin, Z0-1, F-actin was significantly increased in combined group, with a statistically significant difference (p<0.05). Its trend is consistent with the results of the protein determination. ConclusionBorneol could actived RAF/MEK/ERK signaling pathway, thereby regulated expression of downstream tight junction related genes Claudin-5, ZO-1, F-actin and expression and distribution of downstream tight junction related protein Claudin-5, Z0-1, F-actin. All of there was required for the reversibility open and increase in BTB permeability. |
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