| Background:Catalpol-puerarin lyophilized powder for injection (C-P, No. of applying for publicas 201010205703.X) is an innovative compound of Chinese herbal monomer recipe developed by our team and amed to use in treatment of ischemic stroke. Our previous research has proved that C-P had protective effect on ischemic stroke, but the molecular mechanism is still unclear. This paper was to analysis the molecular mechanism of the protection of C-P on the hypoxia/reoxygenation injury of astrocytes. This work was supported by the National Natural Science Foundation of China (31402237,81473549), the Ministry of Education Doctoral Program Special Fund (20110182110012), the Key Projects of Chinese Medicine Research of Chongqing Municipal Health Bureau (2010[60] 2010-1-4),the Fundamental and Front Research Funds of Chongqing (CSTC2014jcyjA80023), and the Fundamental Research Funds for Central Universities (XDJK2014C058).Objectives:To expound the molecular mechanism of the protection of the independent innovation of traditional Chinese medicine monomer compound C-P on hypoxia/reoxygenation injury of astrocytes.Methods and Results:1. Establishment and appraisal of the hypoxia/reoxygenation injury model of cortex astrocytes of SD ratsThe cortical astrocytes of SD rats bornwitnin 3 d were originally cultured in DMEM-F12 (1:1) medium contained 10%FBS, then the cells were purified through rotating turbulence for 220rpm×18h×2 times, and identified by immunofluorescence with GFAP combining DAPI, the purity of the cells was analysised by Image-Pro Plus 6.0. The Earle’s solution was used to culture the astrocytes in three gas incubator whith the compound gas ofO2(0.2%) and CO2 (5%) at 37℃ for 6 hours. After that, the Earle’s solution was changed into the complete medium and the modle was cultured in CO2 incubator whith the compound gas of air (95%) and CO2 (5%) at 37℃, and the growing status of the cells was observed and recorded by inverted phase contrast microscope at Oh, 1h,6h,12h,24h, and 48h. The activity of the injuried astrocytes was detected by CCK-8, the apoptosis rate of tested cells was detected by Annexin V-FITC/PI fluorescence, and the expression ofcaspase-3 weredetected by Western Blot (WB).Result:After the purification, the purity of the astrocytes was 97.67±0.10 identified by immunofluorescence with GFAP combining DAPI and analysised by Image-Pro Plus 6.0, which conformed to the requirements of the test. The test of the cells activity indicated that the cells were injured after hypoxia for 1-12 h, and when hypoxia for 6 h, the activity of cells reached about 50% of the normal cells, which was the best station for the reoxygenation process. Compared to the normal cultured cells, hypoxia for 6 h and reoxygenation for 1 h to 48 h lead to significately lessen tested by CCK-8 (P<0.01), the apoptosis rate was significately increased tested by Annexin V-FITC/PI analysised by flow cytometry (P<0.01), and the expression of caspase-3 was significately increased detected by WB, so the injured cells which were treated hypoxia for 6 h and reoxygenation for 1 h to 48 h were all succeed models, whereas the injury peaked at hypoxia for 6 h and reoxygenation for 12 h, which was celected to be the injured station of this research.2. Analysis ofprotection ofC-P on hypoxia/reoxygenation injury of cortex astrocytesThe hypoxia/reoxygenation injury model of astrocytes were produced by hypoxia for 6h and reoxygenation for 1h to 48h, and 31.25μg·mL-1,125μg·mL-1 and 500μg·mL-1 of C-P were used to intervene the injury. The protection of C-P on hypoxia/reoxygenation injury of astrocytes were analysised by the morphology change of the cells, the change of the cells activity was tested by CCK-8, the apoptosis rate change of the cells was tested by Annexin V-FITC/PI, and the expressionchange of caspase-3 was tested by WB.Result:Compared with the hypoxia for 6 h and reoxygenation for 12 hmodle group, after the intervene of 31.25μg·mL-1,125μg·mL-1, and 500μg·mL-1C-P, the morphology of injured cells was improved obviously, the activity of cells was significately increased (P<0.01), the apoptosis rate was significately decreased (P<0.01), and the expressionof caspase-3 was significately decreased(P<0.01), which indicated that the protection of C-P on hypoxia/reoxygenation injury might be associated with anti-apoptosis function.3.Comparison of protection of C-P and the same doce of puerarin and catalpol on hypoxia/reoxygenation injury of astrocyteThe same doce of puerarin and catalpol in the C-P were used as control, andthe protection of C-P as well as puerarin and catalpol on hypoxia/reoxygenation injury of astrocytes were analysised by the morphology change of the cells, the change of the cells activity was tested by trypan blue count and CCK-8, and statistical analysis the protection of C-P, puerarin, and catalpol on hypoxia/reoxygenation injury of astrocytes.Result:The protection of C-P on astrocytes after hypoxia for 6 h and reoxygenation for 1 h to 48 h were significantly higher than that of puerarin and catalpol at the same dose in the C-P (P<0.01, P<0.05), which indicated that the protection of C-P on hypoxia/reoxygenation injury of astrocytes was higher than that of puerarin and catalpol at the same dose.4. Transcriptome analysis on the protection of C-P on hypoxia/reoxygenation injury of astrocytesThe purified astrocytes were injured by hypoxia for 6 h and reoxygenation for 0 h and 12 h, then 500μg·mL-1 of C-P was added into the medium to protect the cells, and the normal cultured cells were used as control. The total RNA of different group of cells was extracted by RNA extraction kit, then cDNA was obtained through reverse transcription. After the cDNA was marked by biotin, the changes of transcriptome were detected by Affymetrix RAT 230 2.0 expression profile chip, and Affymetri 30007G gene chip scanner was used to record the results. The results were analyzed by information biological method. Genes with a change of 2 times on the quantity of mRNA expression were selected to assess the influence of C-P on the transcriptome of the test cells.Results:Compared with the normal control group, the mRNA expression of 404 kinds of genes were up regulated and 454 of them were down regulated in the hypoxia for 6 h model group, and the HIF-1, MAPK signal pathwayswere upregulated and the PI3k/Aktpathway was downregulated. Otherwise, the pathways of steroid biosynthesis, focal adhesion, and other pathways were upregulated, andmineral absorption pathway, tumor associated protein polysaccharide, and other pathways were downregulated.Compared with the hypoxia for 6 h modle group, the mRNA expression of 31 kinds of genes were up regulated and 40 of them were down regulated in the C-P group. However, the change of mRNA expression on proliferation and apoptosis associated factors were all under 2 times. The changed mRNA included upregulated olfactory transduction pathway, and downregulated terpenoid backbone biosynthesis and bile secretionpathways.Compared with the normal control group, the mRNA expression of 515 kinds of genes were up regulated and 634 of them were down regulated in the hypoxia for 6 h and reoxygenation for 12 h model group, and the JAK/STATsignal pathway was upregulated and the PI3K/Akt pathway was downregulated which regulate proliferation or apoptosis of cells. Otherwise, the mRNA of cell cycle,DNAreplication, mismach repair, and other pathways were uprugulated and olfactory transduction,gap junction,nucleotide excision repair and other pathways were downregulated.Compared with the hypoxia for 6 h and reoxygenation for 12 h modle group, the mRNA expression of 36 kinds of genes were up regulated and 88 of them were down regulated in the C-P group, and among them, the JAK/STAT signal pathway was downregulated and the PI3K/Akt pathway was upregulated which regulate proliferation or apoptosis of cells. Otherwise, the mRNAs of olfactory transduction were changed too.The upregulation of PI3k/Akt signal pathways and the downregulation of JAK/STAT signal pathways may be the protection mechanism of C-Pto hypoxia/reoxygenation injury of rat’s cerebral cultured astrocytes.5. qPCR validation of some of the differentially expressed genesThe astrocytes were injured by hypoxia for 6 h and reoxygenation for 0 h and 12 h respectively and protected by 500μg·mL-1 C-P.The total RNA of the tested cells were extracted and purified by respective kits and cDNA was obtained through reverse transcription, then mRNA expression of JAK2, STAT3, PI3K, Akt, and caspase 3 were detected by qPCR to verify the transcriptome chip results.Results:Compared with the normal control group, significantly decrease of mRNA of PI3K and Akt (P<0.01) and significantly increase of mRNA of caspase 3 (P< 0.01) were detected after hypoxia for 6 h, but there was no significant difference on mRNA of JAK2 and STAT3. Compared with the hypoxia for 6 h group, after the addition of C-P, the mRNA expression of PI3K and Akt were significantly increased (P< 0.01) and that of caspase 3 was significantly decreased (P< 0.01), but no significant difference was detected on mRNA of JAK2 and STAT3. Compared with the normal control group, significantly increase of mRNA of JAK2, STAT3, and caspase 3 (P< 0.01) and significantly decrease of mRNA of PI3K and Akt (P< 0.01) were detected after hypoxia for 6 h and reoxygenation for 12 h. Compared with the hypoxia for 6 h and reoxygenation for 12 h group, after the addition of C-P, the mRNA expression of JAK2, STAT3, and caspase 3 were significantly decreased (P< 0.01) and that of PI3K and Akt were significantly increased (P<0.01). These results were consistent with the results of transcriptome chip.Conclusion1. After injured with hypoxia for 1-12h, the injury level of astrocytes were sustained serious, and after hypoxia for 6 h, the activity of cells reached about 50% of the normal cells, which was the best station for the reoxygenation process. After hypoxia for 6 h and reoxygenation for 1 h to 48 h, the cell survival rates were significantly lower, apoptosis rate and expression of caspase 3 were significantly increased, and the injury was upto the peak after reoxygenation for 12 h, and the best condition for hypoxia/reoxygenation injury was hypoxia for 6 h and reoxygenation for 12 h.2. The super-apoptosis of astrocytes andupregulated expressionof caspase-3 induced by hypoxia for 6 h and reoxygenation for 12 hwere inhibited by C-P, and the activity of the injured cells were significantly improved by C-P. Theprotection effect of C-P on hypoxia/reoxygenation injury of astrocytes was significantly higher than that of puerarin andcatalpol in the compound with same doses.3. The protection of C-P on astrocytes after hypoxia for 6 h and reoxygenation for 1 h to 48 h were significantly higher than that of puerarin and catalpol at the same dose in the C-P, which indicated the rationality of the prescription composition。4. The apoptosis of astrocytes induced by hypoxia/reoxygenation was associated with the upregulation of HIF-1, MAPK, and JAK/STATpathways and downregulation of PI3k/Aktpathway.5. The protection mechanism ofC-P on the hypoxia/reoxygenation injury of astrocytes was associated with downregulation of JAK/STAT pathway and upregulation of PI3k/Aktpathway. |
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