| Objective: To explore the effect of Chromofungin(CHR), a chromogranin A(CGA) derived peptide on the increased permeability of blood brain barrier in septic mice.Methods:(1) septic mice model was induced by 10mg/kg LPS intraperitoneal injection. Seventy two male C57BL/6 mice were divided into LPS treated groups(1 h, 3 h, 6 h, 12 h, 24 h groups after LPS injection) and normal group. The normal group mice were administrated with the same dose of saline. Six mice in each group were executed directly to calculate the cerebral water contents, and another six mice were administrated Evans blue(EB) dye intravenously at the dose of 4 ml/kg circulating for 1 h before brain tissues being harvested. The permeability of blood brain barrier was detected by cerebral water contents and EB dye extravasations. The expression of ZO-1 and claudin-5 was determined by western blot.(2) Forty eight mice were randomly divided into 4 groups: normal saline group(group NS), sepsis model group(group LPS), low-dose CHR(15.5 μg/kg) pretreated group(group CL+LPS) and high-dose CHR(77.5 μg/kg) pretreated group(group CH+LPS). Six hours after LPS challenge, mice were executed. To measure the integrity of blood brain barrier, water contents, EB concentration and EB immune fluorescence in brain tissue were detected. Brain pathology was observed by hematein-eosin(HE) staining. The expression of tight junction proteins, ZO-1 and claudin-5 was also assayed by western blot.Results:(1) The water contents of brain tissue in LPS challenge mice at 3 h, 6 h were significantly higher than normal group [(80.75±1.27)%,(80.14±0.49)% vs.(78.24±0.50)%, all P<0.01], then it decreased gradually. EB concentration in brain tissues significantly increased at 6 h, 12 h, 24 h after LPS challenge(μg/g: 19.39±3.09, 20.53±6.45, 31.79±4.61 vs. 7.75±1.23, all P<0.01). The expression of ZO-1 and claudin-5 decreased at 6 h after LPS administration, and it became lower with extension of 24 h.(2) The contents of water and EB in brain tissue in LPS group significantly increased as compared with NS group [water content:(79.77±0.62)% vs.(78.28±0.44)%, P<0.01; EB content(μg/g): 13.87±4.50 vs. 7.13±1.76, P<0.05]. CHR pretreatment with either of two dosage could reverse the increase in water and EB contents in brain tissue induced by LPS, and the effect was more significant in CH+LPS group [water content:(78.15±0.73)% vs.(79.77±0.62)%; EB(μg/g): 7.09±2.59 vs. 13.87±4.50, both P<0.05]. It was shown by EB fluorescence observation that the fluorescence signal displayed only in the meninge in NS group, and EB fluorescence distributed widely in brain parenchyma in LPS group, indicating that there was more EB leakage in LPS group than that in NS group. In CHR pretreatment groups, EB fluorescence reduced in brain parenchyma, indicating that EB leakage was significantly less than that in LPS group, and it performed a dose-dependent effect. The result of HE staining showed that cerebral blood vessel structure was intact in NS group, and the gap around blood vessel was not significantly distinct. Brain structure in LPS group appeared loose, with widening space and obvious edema around the small blood vessels. Brain edema in CHR pretreatment groups was improved as compared with that in LPS group, and it was more apparent in high dose CHR group. Compared with LPS group, the expression of ZO-1 and claudin-5 in CHR pretreated groups was up-regulated, and the tight junction protein expression in high dose CHR group was higher than in low dose CHR group.Conclusion: LPS induced high permeability of blood brain barrier in mice in a time-dependent manner. Exogenous CGA derived peptide CHR could inhibit hyper-permeability of blood brain barrier in septic mice model induced by LPS, thus relieved brain edema, and protected the brain tissue. The inhibitory effect of CHR on blood brain barrier hyper-permeability was more obvious with a high dose of CHR(77.5 μg/kg). It might be related to the regulatory role of CHR on the expression of tight junction protein ZO-1 and claudin-5. |
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