| Objective:Female SD rats and SH SY5Y cells as the research object,using water maze,immunohistochemical staining,RT-PCR,Western blot techniques,such as observing animals castration and estrogen replacement therapy and cellular supplementary effect of estrogen on the expression of CD147,explore the estrogen in Aβremoval and the role of learning and memory,to find the treatment of Alzheimer’s disease related strategy and direction to provide theoretical basis.Methods:1 The effect of castration and estrogen supplementation on the learning and memory of SD female rats.A total of 72 female SD rats with healthy females were randomly divided into Sham operation group（Sham）,OVX group（OVX）,OVX group（OVX+E2）,and 24 patients in each group.OVX group and the OVX+E2 to ovarian surgery,Sham group operation steps are the same but retain the ovaries,3 d after intraperitoneal injection of OVX+E2 group rats given E2 10μg（100μg/ml）,the OVX group and the Sham group was given equal by intraperitoneal injection of corn oil.Continuous administration of 30 d.The Morris water maze experiment was conducted at 24 d.First,the navigation experiment was carried out,and the rats were recorded to escape the incubation period,which was carried out once a day for five consecutive days.The space exploration experiment was conducted on the 6th day to record the number of rats crossing the platform in 120 s.2 The effect of both castration and estrogen supplementation on CD147expression in the hippocampal tissue of SD female rats.2.1 Immunohistochemical method to detect the expression of CD147 in hippocampus:each group of 12 rats only,after anesthesia individuation to cerebral perfusion paraformaldehyde after,the supremacy of optic chiasma high interception of brain tissue in paraffin section,immunohistochemistry staining.The positive expression area was observed under the positive microscope,and the average optical density（AOD）was measured.2.2 RT-PCR detection mRNA expression of CD147 in hippocampus:each group of 12 rats only,beheaded stripping hippocampus after anesthesia,take a side in the EP tube in the liquid nitrogen,for RNA extraction,reverse transcription polymerase chain reaction,agarose gel electrophoresis and ultraviolet imaging system testing,eyes only forβ-actin mRNA expression level relatively determination of CD147 in each group.2.3 The protein expression of CD147 in the hippocampal tissue was detected by Western blot:the other half of the hippocampal tissue was crushed in the ultrasonic crusher,and after 5 minutes of ice,the low temperature centrifuge was removed and the liquid was removed.Western blot detection,incubation of CD147,and GAPDH were used to determine the relative expression level of CD147 protein in each group.3 Effect of estrogen on CD147 expression in SH-SY5Y cells.3.1 Immune cell fluorescence staining to detect the expression of CD147 in SH-SY5Y cells:DMEM-F12 medium,under the condition of 37℃and 5%CO2 culture SH-SY5Y cells within 24 orifice.Randomly divided into control group（Con group）,estrogen receptor inhibitor+estrogen group（ICI+E2group）,estrogen group（E2 group）.E2 group was given the function of 100nM E2 and 24 h,and the ICI group was given a pre-treatment of 1μM ICI 182780for 30 min before adding E2 to 24 h,and the control group received the same amount of DMSO.The expression and localization of CD147 in SH-SY5Y cells were observed by immunofluorescence staining.3.2 RT-PCR to detect the mRNA expression of CD147 SH-SY5Y cells:DMEM-F12 medium,under the condition of 37℃and 5%CO2 culture SH-SY5Y cells within 6 orifice.Grouping and processing with 3.1.Test method and 2.2.3.3 Western blot detection of CD147 protein expression in SH-SY5Y cells:DMEM-F12 medium,under the condition of 37℃and 5%CO2 culture SH-SY5Y cells within 6 orifice.Grouping and processing with 3.1.The detection method is the same as 2.3.Results:1.The influence of castration and estrogen supplementation on learning and memory of SD female rats.The results of positioning navigation experiment:during the experiment of 5 d,the avoidance latency of 3 groups of experimental animals could be reduced to different degrees.In the experiment,the differences between the rats in the first and third groups were not significant,and the differences were not statistically significant（P>0.05）.In the fourth day,OVX group（21.83±7.40）and OVX+E2（19.93±7.01）group were higher than the Sham group（12.36±4.24）,and the difference was statistically significant（P<0.05）.In the fifith day,OVX group（22.95±3.91）avoided the incubation period higher than the Sham group（10.34±2.54）and OVX+E2 group（14.77±3.62）,and the difference was statistically significant（P<0.05）.Space exploration experiment results:OVX group rats swimming trajectory around the pool around each quadrant,Sham group,the OVX+E2group preference,show the directivity of the target area and scope of Sham group of swimming is more concentrated near the platform.Compared with the Sham group（11.54±3.72）,the number of the OVX group（6.50±3.02）was significantly reduced across the platform,and the difference was statistically significant（P<0.05）.Compared with the OVX group（6.50±3.02）,the number of the OVX+E2 group（9.67±2.44）increased across the platform,and the difference was statistically significant（P<0.05）.There was no significant difference between the Sham group and OVX+E2 group.2.The influence of castration and estrogen supplementation on the expression of CD147 in the hippocampal tissue of SD female rats.2.1 Immunohistochemical staining results:the expression of CD147 in CA1,CA3,CA4 and dentate gyrus was observed under the microscope,and the positive reaction sites were mainly located in cytoplasm and cytoplasm.The average optical density value analysis showed that:compared with the Sham group regions（CA1 area 0.097±0.013,CA3 area 0.109±0.016,CA4 area0.016±0.015）,district CD147 expression corresponding OVX group was obviously lower（CA1 area 0.052±0.013,CA3 area 0.048±0.011,CA4 area0.011±0.010）,statistically significant difference（P<0.05）.Compared with OVX group（each area CA1 area 0.052±0.013,CA3 area 0.048±0.011,CA4area 0.011±0.010）,the OVX+E2 group corresponding districts CD147expression increased（CA1 area 0.089±0.015,CA3 area 0.078±0.012,CA4area 0.012±0.012）,statistically significant difference（P<0.05）.The expression of CD147 mRNA was detected by RT-PCR:compared with Sham group（0.590±0.133）,the expression of CD147 mRNA was significantly decreased in OVX group（0.310±0.109）,and the difference was statistically significant（P<0.05）.Compared with OVX group（0.310±0.109）,the expression of OVX+E2 group（0.390±0.108）was increased,and the difference was statistically significant（P<0.05）.2.3 The expression of CD147 protein was detected by Western blot:compared with Sham group（0.519±0.1081）,the expression of CD147 protein was significantly decreased in OVX group（0.280±0.087）,and the difference was statistically significant（P<0.05）.The expression of OVX+E2（0.372±0.102）was increased compared with OVX group（0.280±0.087）,and the difference was statistically significant（P<0.05）.3.Effect of estrogen on CD147 expression in SH-SY5Y cells.3.1 The results of immunofluorescence staining:CD147 was expressed in cytoplasm,cell membrane and axon.There was no significant change in the expression of CD147 protein between the group（0.123±0.020）and ICI+E2（0.135±0.019）.The difference was not statistically significant（P>0.05）.The group of E2 group（0.240±0.027）was significantly higher than that of the Con group（0.123 or 0.020）and ICI+E2（0.135±0.019）,and the difference was statistically significant（P<0.05）.The expression of CD147 mRNA was detected by RT-PCR:the expression of CD147 protein in the group of Con group（0.101±0.032）and ICI+E2 group（0.132±0.031）showed no significant change,and the difference was not statistically significant（P<0.05）.The group of E2 group（0.273±0.076）was significantly higher than that in the Con group（0.101±0.032）and ICI+E2（0.132±0.031）,and the difference was statistically significant（P<0.05）.The expression of CD147 protein was detected by Western blot,and there was no significant change in the expression of CD147 protein between the group（0.673±0.040）and ICI+E2（0.683±0.035）.The difference was not statistically significant（P<0.05）.The group of E2 group（1.157±0.156）was significantly higher than that in the Con group（0.673±0.040）and ICI+E2（0.683±0.035）,and the difference was statistically significant（P<0.05）.Conclusions:1.The castration can lead to the decline in the learning and memory ability of female SD rats,and the decrease of the learning and memory ability of rats can be relieved by the addition of estrogen supplementation.2.The castration could lead to the decrease of CD147 expression level in the hippocampal tissues of female SD rats,and the expression level of CD147increased after estrogen supplementation.3.Estrogen can increase the expression of CD147 in SH-SY5Y cells,which can be inhibited by the estrogen receptor inhibitor ICI 182780. |
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