ABSTRACT
Objective To investigate the effect of plateau hypoxia on the blood-brain barrier after subarachnoid hemorrhage (SAH) in rats. Methods Adult male SD rats (n = 78) were randomly divided into 4 groups: sham group (sham), SAH model group (SAH), plateau hypoxia sham group (Hp sham) and plateau hypoxia SAH model group (Hp SAH). The rat model of plateau hypoxia was established through low-pressure simulation chamber (altitude 5000 m), and the SAH model was established by endovascular perforation method. At 24 hours after SAH, neurobehavior score and SAH grade were assessed. The morphological changes of neurons and apoptosis of nerve cells in the CA1 region of hippocampal were observed by the staining of Nissl and TUNEL. The expression of phosphorylated PI3K (p-PI3K), PI3K, phosphorylated Akt (p-Akt), Akt, phosphorylated nuclear factor κB (p-NF-κB), NF-κB, matrix metalloproteinase-9 (MMP-9), occludin and claudin-5 in hippocampal were detected by the method of Western blotting. The expression of occludin and claudin-5 proteins in the CA1 region of hippocampal were observed by immunofluorescent staining. Results At 24 hours after SAH, the neurobehavior score decreased significantly and SAH grade increased significantly in the SAH and Hp SAH group (P< 0.05). Neurobehavior score decreased significantly in the Hp SAH group compared with the SAH group (P < 0.05). In the SAH group, neurons in the CA1 region of hippocampus were atrophied and deformed, the arrangement were disordered, the number of neurons decreased significantly, and the apoptosis of nerve cells increased significantly(P< 0.05). Plateau hypoxia could aggravate the morphological damage of neurons and apoptosis of nerve cells. The expression of p-PI3K/PI3K, p-Akt/Akt, occludin and claudin-5 proteins decreased significantly, while the expression of p-NF-κB/NF-κB and MMP-9 proteins increased significantly in the SAH and Hp SAH group (P< 0.05). The expression of p-PI3K/PI3K and MMP-9 proteins increased significantly in Hp SAH group compared with the SAH group. The expression of claudin-5 protein increased significantly in Hp sham group compared with the sham group (P < 0.05). Immunofluorescent staining showed that the expression of occludin and claudin-5 proteins in the CA1 region of hippocampus decreased in the SAH group. Plateau hypoxia could further decreased the expression of occludin and claudin-5 proteins. Conclusion Plateau hypoxia aggravates blood-brain barrier disruption after subarachnoid hemorrhage in rats through inhibiting PI3K/Akt/NF-κB pathway.
ABSTRACT
Objective To explore the molecular pathological mechanism of gout, and to explore the mechanism of how interleukin (IL)-37 influencing PDZK1 protein in gout through nuclear factor κB (NF-κB) pathway. Methods HK-2 cells were stimulated with inflammatory signal IL-37. The expression of PDZK1 and its subcellular localization were detected by real-time fluorescence quantitative polymerase chain reaction (real-time PCR) at different concentrations of IL-37 (defined as group A), PDTC+IL-37 (defined as group B), Wortmannin+IL-37 (defined as group C), respectively.The changes of PDZK1 protein expression in HK-2 cells were detected by adding inhibitor PDTC (NF-κB pathway inhibitor) or Wortmannin (PI3K pathway inhibitor) and inflammatory signal stimulating protein imprinting method. The comparative t test was used for statistical analysis between groups A, B and A and C. Results The average levels of PDZK1 mRNA were as follows:(group A: 28.71 ±0.35, 28.57 ±0.31, 28.78 ±0.28, 28.63 ±0.29, 28.62 ±0.19; group B: 28.71 ±0.31, 28.83 ±0.27, 28.58±0.26, 28.73±0.36, 28.68±0.35;group C:28.81±0.32, 28.91±0.29, 28.72±0.24, 28.59±0.18, 28.58±0.22). There was no significant change in PDZK1 mRNA level in group A and B. The same IL-37 was found in group A and B. The values of T were 5.73, 4.72, 4.69, 5.86 and 6.79, respectively. The P values were all greater than 0.05, and there was no significant difference between the two groups. The values of T were 6.78, 7.13, 7.47, 6.38 and 5.98 in the same IL-37 concentration groups of A and C, respectively, and the P values were all greater than 0.05, with no significant difference. The levels of PDZK1 protein in the three groups by Western blot analysis were as follows: (group A: 0.200±0.082, 0.412±0.032, 0.723±0.063, 1.202±0.021, 1.222±0.023;group B: 0.124±0.064, 0.303±0.081, 0.325±0.062, 0.223±0.071, 0.343±0.052; group C: 0.017±0.022, 0.204± 0.015, 0.187±0.053, 0.302±0.051, 0.404±0.051), The levels of PDZK1 protein in group A treated with different concentrations of IL-37 followed by the concentration of IL-37. The level of PDZK1 protein in group B increased with the increase of IL-37 concentration, but the level of PDZK1 protein in group B was lower than that in the group treated with IL-37 only, and decreased when the concentration of IL-37 was 40 ng/ml.The level of PDZK1 protein in group C increased with the increase of IL-37 concentration, but the level of PDZK1 protein in group C was lower than that in the group treated with IL-37 only, and the same concentration of IL-37 in group A and B. The values of T were 1.83, 1.37, 1.64, 1.57 ,1.49, with P values greater than 0.05. There was no significant difference between the two groups. The values of T were 1.28, 1.37, 1.26, 1.42, 1.39 in the same concentration of IL-37 in group A and C, with P values greater than 0.05, with no significant difference.The results of immunofluorescence analysis showed that the trend of the three groups was basically consistent with that of Western-Blot. Conclusion The pathogenesis of gout induced by IL-37 through PDZK1 protein may not occur at the transcriptional level, but may occur at the level of protein translation.
ABSTRACT
Objective To detect the expression levels of metallothionein1 H(MT1 H)in children and adoles-cents osteosarcoma serums,and to analyze its relationship with clinicopathological features,and to explore the effect of MT1 H on cell proliferation of osteosarcoma cells and its mechanism.Methods Enzyme -linked immuno sorbent assay (ELISA)was performed to detect the expression of MT1 H in children and adolescents osteosarcoma serums and non-neoplastic disease serums.MT1 H vector was transfected into the osteosarcoma U2OS cells.Reverse transcription -poly-merase chain reaction(RT -PCR)and Western blot were used to detect the expression of the mRNA and protein of MT1 H,respectively.Methylthiazolyldiphenyl -tetrazolium bromide(MTT)was used to detect the cell growth.Western blot was performed to detect the expression of nuclear factor(NF)-κB,and inhibitor of κB (IκB)-αprotein. Results The expressions of MT1 H in osteosarcoma serums and nonneoplastic disease serums was (0.51 ± 0.52)μg/L and (2.17 ±0.78)μg/L,respectively,with a significant difference between the 2 groups(t =-8.966, P <0.05).The expression of MT1 H in stage Ⅰ -ⅡA andⅡB -Ⅲ was (1 .98 ±0.69)μg/L and (2.45 ±0.82)μg/L,respectively,showing a gradual increase depending on clinical staging(t =-2.343,P <0.05).The expressions of MT1 H mRNA and protein were elevated in osteosarcoma U2OS cells after MT1 H vector transfection(all P <0.05). MTT assay showed that,the A value in blank control group,blank vector group,MT1 H vector group were 0.38 ±0.03, 0.36 ±0.03,0.42 ±0.03,respectively,the cell proliferation in the MT1 H vector group was significantly promoted when compared with these in the blank vector group and blank control group(F =4.213,P <0.05)from the third day.West-ern blot showed that,the relative expression of NF -κB in blank control group,blank vector group,MT1 H vector group were 0.56 ±0.05,0.53 ±0.05,0.92 ±0.07,respectively,the relative expression of IκB -αprotein were 0.64 ± 0.06,0.62 ±0.09,0.34 ±0.08,respectively,the expression of NF -κB protein was up -regulated and the expression of IκB -αprotein was down -regulated in the MT1 H vector group when compared with those in the blank vector group and blank control group(F =44.581 ,14.927,all P <0.05).Conclusions The expression of MT1 H is increased in children and adolescents osteosarcoma serums compared with that in nonneoplastic disease serums.The clinical stage is later,the expression of MT1 H is higher.MT1 H promotes cell proliferation through regulating the NF -κB pathway.