| Objective To investigate the effect of Vitamin A in neural function and tissue recovery of hypoxic-ischemic brain damage rats.Methods Eighty-four 7d age wistar rats were randomly divided into VA normal (VAN), VA deficiency (VAD) and sham operation group (control). Different VA intake made VA levels to reach normal or deficiency, and rat were HIBD damage in VAD and VAN groups. Serum VA levels examined with HPLC to evaluate rats VA nutrition, the mNSS were tested in each group in the middle and late recovery stage, the neurocognitive function were assessed with morris water maze and shuttle box. Calcium image was analyzed the NMDA-induced neuronal calcium influx phenomenon in hippocampal CA1 area. The EdU labeling assessed the new cell generation of VAD and VAN groups in the recovery stadium acmes, the Nissl staining investigated the hippocampus atrophy of VAD and VAN groups. Tunel staining was used to assess neural cell apoptosis of VAD and VAN groups. Flow cytometric analyzed the apoptosis and mitochondrial membrane potential of oxygen glucose deprivation (OGD) in primary neurons with various concentrations of all-trans retinoic acid (ATRA) and expression of RARαtreatment. Furthermore, we analyzed the gene and protein level of RA signaling, TGF-βsignaling molecules and mitochondrial apoptosis pathway in hippocampus and primary hippocampal neuronsResults (1) The serum VA levels of VAD, VAN and Control groups rats were respectively controlled at 0.4~0.7μmol/L, 0.7~1.1μmol/L and 0.65~1.05μmol/L during the experiment.(2) Functional results: The neurological injury score of VAD group were significantly higher than that of VAN group in 7, 14, 21 and 28d after damage, control group scores were significantly lower than the other two groups. In the morris water maze test, the average latency of VAD group were significantly longer than VAN group in 2~5d test, and the number of cross platform was significantly lower than VAN group. However the average latency of control group were significantly lower than the other two groups during the test period, and the number of cross platform were significantly higher than the other two groups. In the shuttle box test, the active avoidance response rate (AARR) of VAD group were significantly lower than the VAN group in 2 ~ 5d days test, none avoidance response rate (NARR) were significantly higher than the VAN group, nevertheless, AARR of control group were significantly higher than the other two groups, and NARR were significantly lower than the other two groups. The calcium shock excited 50μmol/LNMDA of pyramidal cells in hippocampal CA1 area of VAD was stronger than VAN group in the acme phase (3~7d), while in the later (after 14~30d) was significantly weaker than VAN group. In summary, the VA normal in vivo could significantly enhance the neural function repair after HIBD.(3) Histological results: The gap between hippocampus CA3 and cortex of VAD group was significantly wider than the VAN group, and the hippocampus is smaller than the VAN group. The newborn cells (EdU positive cells, red fluorescence) of VAD's hippocampal CA1, CA3, DG, and parietal cortex, temporal cortex was significantly less than VAN group after 2,7d, and the newborn cells of hippocampus was seen in pyramidal cells Layer, the cortex region in layer2~5 main areas, the EdU positive cells in 2d was significantly more than 7d. Postoperative 3d, VAD's hippocampal CA1, CA3, DG, and parietal cortex, temporal cortex was more severe apoptosis compared with VAN group (yellow arrow shows green fluorescence) and apoptosis in hippocampus of both groups mainly located in the pyramidal layer, cortical apoptosis was mainly seen in layer4~5, in postoperative 7d, VAD apoptosis also was more serious than VAN group, the district apoptosis in postoperative 7d is spreader. In summary, the VA normal in vivo could significantly promote the neural tissue repair after HIBD.(4) Gene and protein results: We found that the mRNA a levels of RARαin the acme and mediate period (postoperative 3~14d) VAD group were significantly higher than VAN group, in the late stage (postoperative 14~28d) the RARαmRNA and protein levels in VAD group was significantly lower than VAN, RARαis the dominant receptor of RA receptor. Furthermore, we found that the neuronal markers NSE's mRNA levels in VAD group were significantly lower than VAN group in 3, 14d, glial cell marker GFAP's mRNA levels in VAD group was significantly lower than VAN group in 1,7,14d, Neural stem cell markers Nestin's mRNA n levels in VAD was significantly lower than VAN group in 3, 7, 14 d. In 7d, VAD group's GDNF was significantly lower than VAN group, and TGF-β1,2 in VAD was significantly higher than VANM in 1~3d, in 7d VAD group was significantly lower than VAN group. So we examined the apoptosis signaling pathway in VAD and VAN between the two groups in the acme and mediate period. In postoperative 1, 7d, the apoptotic protein caspase-3 mRNA levels in VAD group were significantly higher than VAN group, caspase-8 mRNA and protein levels in postoperative 1, 3, 7d were significantly higher than VAN group, and bax of mitochondrial apoptosis pathway in 1, 3, 7 d were significantly higher than VAN group, bak was significantly higher than the VAN group in 1d, bid were significantly higher than VAN group in 7, 14d, however the anti-apoptotic molecule bcl-2 of the mitochondrial apoptosis pathway mRNA and protein levels in postoperative 7, 14d was significantly lower than VANM group. In summary, the body with VA normal could enhance the treatment of HIBD nerve tissue function recovery, the possible mechanisms is RA signal affect the TGF-βsignaling pathway and further regulate the mitochondrial apoptosis cascade effect to inhibit neuronal apoptosis.(5) RA signal affect the apoptosis of hippocampal neurons with OGD injury: the apoptosis rate in 1~5μmol/LATRA handling groups were significantly lower than other concentrate ATRA groups, We further over-expressed and silenced RARαto investigate whether RARαmediates RA signaling inhibits apoptosis, the apoptosis rate in 1μmol / LATRA treated group was significantly lower than other treatments groups, however overRARα+1μmol/L ATRA and siRARα+1μmol/L ATRA groups were the highest. The abnormal mitochondrial membrane potential in 1μmol/L ATRA group was minimum than other groups, however the overRARα+1μmol/L ATRA groups were the highest. These results suggest that only moderate RA signaling could significantly anti-apoptosis.(6) Possible mechanisms: We found that there is a dose-related increase between the mRNA and protein levels of RARαwith the ATRA concentration.We further examined the expression of key molecules in the mitochondrial apoptosis signal pathway, overRARα+1μmol/L ATRA and siRARα+1μmol/L ATRA groups significantly increased the apoptosis signaling molecules caspase-3, caspase-8, bid, bax, bak expression, and the bax of 1μmol/LATRA group expressed lower than OGD group, while the anti-apoptotic molecules bcl-2 expression in 1μmol/LATRA groups were significantly higher than OGD group, however, the bcl-2 in overRARα+1μmol/L ATRA significantly increased compared with OGD and siRARα+1μmol/L ATRA groups. The TGF-βexpressions in overRARα+1μmol/L ATRA and siRARα+1μmol/L ATRA groups were lower than the 1μmol/L ATRA group, but the OGD group is the highest, however 1μmol/LATRA treatment stayed intermediate level. The GDNF expression in 1μmol/LATRA groups were significantly higher than OGD and siRARα+1μmol/L ATRA group, and overRARα+1μmol/L ATRA group was the highest than the other groups, while over-expression group also had the highest expression of apoptosis signaling molecules. These results suggest RA signaling can anti-apoptotic through an appropriate abundance affected GDNF, TGF-βsignaling, the suitable TGF-βsignaling level affect the mitochondrial apoptosis signal pathway to promote anti-apoptosis.Conclusions The body with suitable VA nutrient levels can significantly promote the neural function and tissue recovery after HIBD, the possible pathologic mechanism is appropriate VA promote nerve cell regeneration and inhibit the brains shrink and apoptosis, its molecular mechanism is suitable RA signaling promotes the appropriate TGF-βsignaling and GDNF and thus inhibit the expression of the mitochondrial apoptosis pathway cascade signaling and increased expression of bcl-2, finally inhibit the damage caused by apoptosis. These suggest the body to maintain the normal VA level in vivo and vitro environment is benefit for the treatment of HIBD. Objective To investigate the effect of Vitamin A in neural function and tissue recovery of hypoxic-ischemic brain damage(HIBD) rats with rMSCs treatment.Methods 144 7d age wistar rats were randomly divided into VA normal with rMSCs translation (VANM), VA deficiency with rMSCs translation (VADM) and HIBD group. Different VA intake made VA levels to reach normal or deficiency, and rat were HIBD damage in VADM and VANM groups, 24h after HIBD every rat was abdominally transplanted 1 * 106rMSCs in VADM and VANM groups. Serum VA levels examined with HPLC to evaluate rats VA nutrition, the mNSS were tested in each group in the middle and late recovery stage, the cognitive function were assessed with morris water maze and shuttle box. Calcium image was analyzed the NMDA-induced neuronal calcium influx phenomenon in hippocampal CA1 area. TTC staining was assessed the cerebral tissue infarction in VADM and VANM groups. The EdU labeling assessed the new cell generation of VADM and VANM groups in the recovery stadium acmes, Tunel staining assessed neural cell apoptosis of VADM and VANM groups. Flow cytometric analyzed the apoptosis and mitochondrial membrane potential of oxygen glucose deprivation (OGD) in primary neurons with various concentrations of all-trans retinoic acid (ATRA) and rMSCs treated. Furthermore, we analyzed the gene and protein level of RA signaling, TGF-βsignaling molecules and mitochondrial apoptosis pathway in hippocampus and primary hippocampal neuronsResults (1) The serum VA levels of VADM, VANM and HIBD groups rats were respectively HIBD led at 0.4 ~ 0.8μmol/L, 0.7 ~ 1.3μmol/L and 0.6~1.1μmol/L during the experiment.(2) Functional results: The neurological injury score of VADM group were significantly higher than that of VANM group in 7, 14, 21 and 28d after damage, HIBD group scores were significantly higher than the other two groups. In the morris water maze test, the average latency and path length of VADM group were significantly longer than VANM group in 2 ~ 5d test, and the number of cross platform was significantly lower than VANM group. However the average latency and path length of HIBD group were significantly longer than the other two groups during the test period, and the number of cross platform were significantly higher than the other two groups In the shuttle box test, the active avoidance response rate (AARR) of VADM group were significantly lower than the VANM group in 2~5d days test, none avoidance response rate (NARR) were significantly higher than the VANM group in 4~5d days test. Nevertheless, AARR of HIBD group were significantly lower than the other two groups, and NARR were significantly higher than the other two groups. The calcium shock excited 50μmol/LNMDA of pyramidal cells in hippocampal CA1 area of VADM was stronger than VANM group in the acme phase (3~7d), while in the later (after 14~30d) was significantly weaker than VANM group. In summary, the VA normal in vivo could significantly enhance the neural function repair after HIBD with rMSCs transplantation treatment. (3) Histological results: postoperative 7d the infarct size of VADM group injury side (left side of the brain) was significantly greater than VANM group, but VADM and VANM's infarct size were significantly smaller than the HIBD group. 2, 7 d after rMSCs transplantation, we assessed the colonization of rMSCs in the brain, colonization rMSCs(green fluorescence)were only found in the two groups cortex, while the hippocampus is not found, and in the temporal lobe and parietal cortex of VANM group had significantly more colonization rMSCs than that of VADM group. The newborn cells (EdU positive cells, red fluorescence) of VADM's hippocampal CA1, CA3, DG, and parietal cortex, temporal cortex was significantly less than VANM group after translation 2,7d, and the newborn cells of hippocampus was seen in pyramidal cells Layer, the cortex region in layer2~5 main areas, the EdU positive cells 2d after transplantation was significantly more than 7d. After rMSCs transplantation 2d, VADM's hippocampal CA1, CA3, DG, and parietal cortex, temporal cortex was more severe apoptosis compared with VANM group (yellow arrow shows green fluorescence) and apoptosis in hippocampus of both groups mainly located in the pyramidal layer and molecular layer of area, cortical apoptosis was mainly seen in layer3~5, in postoperative 7d, VADM apoptosis also was more serious than VANM group, the district apoptosis in postoperative 7d is more narrow. In summary, the VA normal in vivo could significantly promote the neural tissue repair after HIBD with rMSCs transplantation treatment. (4) Gene and protein results: We found that the mRNA and protein levels of RARαin the acme period (postoperative 3~7d, P3~7d) VADM group were significantly higher than VANM group, in the late stage (P14~28d) the RARαmRNA and protein levels in VADM group was significantly lower than VANM, RARαand RXRγreceptor is the dominant receptor of RA receptor. Furthermore, we found that the neuronal markers NSE's mRNA and protein levels in VADM group were significantly lower than VANM group in P14d, glial cell marker GFAP's mRNA and protein levels in VADM group was significantly lower than VANM group in P21~30d, Neural stem cell markers Nestin's mRNA and protein levels in VADM was significantly lower than VANM group in P3, 14 d. In P3, 7, 14 d, VADM group's GDNF was significantly lower than VANM group, and TGF-β1 in VADM was significantly higher than VANM in P3~7d, in P7~14d VADM group was significantly lower than VANM group. So we examined the apoptosis signaling pathway in VADM and VANM between the two groups in the acme and mediate period.In P7d, the apoptotic protein caspase-3, bid, bax and bak, mRNA or protein levels in VADM group were significantly higher than VANM group, caspase-8 mRNA and protein levels in P3,7,14d were significantly higher than VANM group, And the anti-apoptotic molecule bcl-2 of the mitochondrial apoptosis pathway mRNA and protein levels in P7, 14d was significantly lower than VANM group. In summary, the body with VA normal could enhance the treatment of rMSCs transplantation in HIBD nerve tissue function recovery, the possible mechanisms is RA signal affect the TGF-βsignaling pathway and further regulate the mitochondrial apoptosis cascade effect to inhibit neuronal apoptosis.(5) ATRA combined rMSCs affected primary neuronal apoptosis with OGD injury: the apoptosis rate in rMSCs handling groups were significantly lower than OGD group, the apoptosis rate in rMSCs +1μmol / LATRA treated group was significantly lower than other treatments groups. The abnormal mitochondrial membrane potential in rMSCs +1μmol / LATRA group was minimum than other treatments, however the high concentration of ATRA treatment groups were not different with OGD group. These results suggest that moderate RA could significantly promote the rMSCs stimulate anti-apoptotic effect, while high concentrations may counteract this effect.(6) Possible mechanisms: We found that there is a dose-related increase between the mRNA and protein levels of RARαwith the ATRA concentration, the expression of RARαin rMSCs combined 1μmol/LATRA treatment group is the middle levels and RARαexpression in the control group is the minimum. NSE is the highest expression in the control group, rMSCs treatment groups were significantly higher than OGD group, in which the rMSCs+1μmol/LATRA treated had the highest expression compared with other injured groups, and OGD group was the lowest. The GDNF expressions in rMSCs treatment groups were significantly higher than OGD group, in which the GDNF of rMSCs+1μmol/LATRA treated group was the highest than the other injured groups. The TGF-β1, 2 expressions in rMSCs treatment groups were higher than the control group, but lower than OGD group, however rMSCs+1μmol/LATRA treatment stayed intermediate level.We further examined the expression of key molecules in the mitochondrial apoptosis signal pathway, rMSCs treatment groups could significantly decreased the apoptosis signaling molecules caspase-3, caspase-8, bid, bax, bak expression, and the rMSCs+1μmol/LATRA treatment group expressed lower, while high concentrations and no ATRA treatment group s'expression were relatively higher than that of the rMSCs+1μmol/LATRA treatment group; and the anti-apoptotic molecules bcl-2 expression in rMSCs groups were significantly higher than OGD group, which rMSCs+1μmol/LATRA treatment group was the highest expression during them, while the high concentration and no ATRA treatment group was relatively lower than the rMSCs+1μmol/LATRA treatment group. These results suggest that rMSCs treatment significantly anti-apoptosis in OGD injury neurons, combined with different concentrations of ATRA stimulation, found that low concentrations could promote rMSCs anti-apoptotic, high and no concentrations inhibit the anti-apoptosis. In summary, the RA signal has the most suitable abundance to play the role of anti-apoptosis, RA signaling may promote rMSCs anti-apoptotic through an appropriate abundance affected GDNF, TGF-βsignaling, the suitable TGF-βsignaling level affect the mitochondrial apoptosis signal pathway to promote rMSCs anti-apoptosis.Conclusions The body with suitable VA nutrient levels can significantly promote the rMSCs transplantation treatment HIBD, the possible pathologic mechanism is appropriate VA promote nerve cell regeneration and inhibit the brains infarct and superimposed on the enhancing of rMSCs colonization and activity to enhance cell regeneration and inhibit infarct and superimposed, its molecular mechanism is suitable RA signaling unite paracrine effect of rMSCs interact with the appropriate TGF-βsignaling and GDNF and thus inhibit the expression of the mitochondrial apoptosis pathway cascade signaling and increased expression of bcl-2, and ultimately inhibit the damage caused by apoptosis. These suggest the body to maintain the normal VA level in vivo and vitro environment is benefit for the rMSCs transplantation in the treatment of HIBD.
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