| Objective In vivo and in vitro experiments were used to explore the protective effect of HSYA on mPTP against cerebral mitochondrial functional damage in rats with cerebral ischemia-reperfusion Oxygen deprivation/reoxygenation(OGD/R)injury rat BMECs protective effect and mechanism research for clinical treatment of ischemic Stroke provides pharmacological basis.Method 1.HSYA Alleviates MCAO-Induced I/R in rats Establishment of a rat model of cerebral ischemia and reperfusion with modified suture method;Brain water content by drying method;Brain tissue from ischemic penumbra zone to prepare mitochondria according to the operation of tissue mitochondrial extraction kit;Elisa detects the release of ROS and Cyto C in rats;Determination of the openness of mitochondrial mPTP in damaged brain tissue by chemical colorimetry;ELISA was determined to detect the activity of ATP in rat ischemic penumbra zone;The expression level of MEK、p ERK、Cyp D in rat cortex detected by WB.2.HSYA protects BMECs damaged by OGD/R by inhibiting the opening of mptp The complete medium was replaced with sugar-free medium,combined with hypoxic chamber incubator,to establish an OGD/R model;observe the swelling degree of mitochondrial morphology;Colorimetric method for detecting calcium ion concentration;Detection of mitochondrial ATP activity of BMECs by ELISA;BMECs mitochondrial membrane potential was measured by JC-1 kit;Fluorescence microscope and colorimetric method to detect the openness of BMECs mitochondrial mPTP;Apoptosis was detected by AV-PI double staining respectively;Elisa detects the release of Cyto C in BMECs;Detect the expression of Cyp D in the mitochondria of BMECs through WB;The expression level of Cyto C、Caspase3、Caspase9、Bax in BMECs detected by WB;The expression level of Bcl2、Bax、Cyto C in the mitochondria of BMECs detected by WB;3.HSYA reduced the damage of BMECs caused by OGD/R through MEK/ERK/Cyp D signal pathway.Si RNA transfection of Cyp D;Cells proliferation dected by CCK-8 assay;Fluorescence microscope to detect the openness of BMECs mitochondrial mPTP;The LDH release detects by Microplate reader;The expression level of p ERK、Cyp D in BMECs detected by WB;Results 1.HSYA Alleviates MCAO-Induced I/R in rats(1)HSYA effectively reduces neurobehavioral score,cerebral infarction volume and cerebral water content in I/R rats;(2)Compared with model group,HSYA can effectively reduce the expression of ROS;(3)HSYA can effectively reduce the release of Cyto C compared with model group;(4)HSYA can effectively inhibit the openness of mPTP;(5)HSYA can significantly increase the ATP depletion caused by cerebral ischemia and reperfusion;(6)The expression of MEK and p ERK was significantly up-regulated under the action of HSYA,and the expression of Cyp D protein was significantly down-regulated.2.HSYA protects BMECs damaged by OGD/R by inhibiting the opening of mptp(1)The cells were cultured normally,and they appeared to be "paving stones";The factor VIII fluorescence was positive under a fluorescence microscope.(2)CCK-8 results showed that the cell viability of the model group decreased to about 50%,while HSYA significantly improved cell damage and increased cell viability.(3)From the results of transmission electron microscopy,mitochondrial swelling in the model group was significantly increased,and HSYA can effectively reduce mitochondrial swelling,restore damaged mitochondrial morphology,and reduce mitochondrial vacuolation.(4)The calcium ion results showed that compared with the model group,the intracellular calcium ion concentration in the HSYA group was lower.(5)Compared with model group,HSYA can significantly increase the ATP depletion caused by OGD/R.(6)JC-1 results showed that the HSYA group can significantly reduce the increase of green fluorescence caused by OGD/R,and significantly increase the red fluorescence.(7)The mPTP openness of the HSYA group had a significant downward trend.(8)The expression of Cyp D protein was significantly down-regulated under the action of HSYA.(9)Elisa results showed that HSYA significantly inhibited the mass release of Cyt C into the cytoplasm in the model group.(10)HSYA effectively reduced the apoptotic cells to 3.79%.(11)The expression of caspase-3,caspase-9,Cyto C and Bax proteins was significantly down-regulated under the action of HSYA;the results of protein expression in mitochondria were just the opposite.3.HSYA reduced the damage of BMECs caused by OGD/R through MEK/ERK/Cyp D signal pathway.(1)Fluorescence results show that after siRNA transfection to Cyp D,the transfection conditions of 30 nm and 24 h are the most suitable.(2)CCK-8 results show that HSYA can significantly increase the decline of cell viability caused by U0126;and it also has a certain recovery effect on the decline of cell viability caused by Cyp D transfection.(3)Fluorescence results show that HSYA can significantly reduce the pathological phenomenon of abnormal mPTP opening caused by U0126;and it has a certain recovery effect on mPTP opening caused by Cyp D transfection.(4)The HSYA group can significantly reduce the pathological phenomenon of LDH mass release caused by U0126;and has a certain recovery effect on the mass release of LDH caused by Cyp D transfection.(5)WB results showed that HSYA had a recovery effect on the abnormal expressions of MEK,p ERK,and Cyp D after U0126;but the recovery of the abnormal expressions of MEK,p ERK,and Cyp D after Si Cyp D was not obvious.Conclusion Based on the above results,HSYA has a protective effect on cerebral ischemia-reperfusion injury rats by inhibiting the mitochondrial apoptotic pathway,and this protective effect is related to the regulation of the MEK/ERK/Cyp D signaling pathway by HSYA to inhibit the opening of mPTP. |
