Effects Of Radix Astragali Extractives On Mitochondria Function In Cerebral Ischemia Reperfusion Rats

Ischemic cerebrovascular disease is a common disease, with frequentlyoccurring, high mortality and morbidity. Reperfusion injury with blood reflowis an important pathophysiological process, so the prevention andmedication of reperfusion injury after cerebral ischemia is a necessary part ofthe treatment of ischemic cerebrovascular disease. Clinically, Radix Astragaliare used commonly, especially in treating ischemic cerebrovascular disease, byits effect on reducing neuronal death and promoting nerve renovations. RadixAstragali contain saponins, polysaccharides, flavonoids and other biologicallyactive substances, but which effective parts or components play a major role inreducing neuronal death still remains unclear. Therefore, Radix Astragali hasthe value of full exploitation and utilization research.Our previous study found that Radix Astragali injection providesprotection of hippocampal CA1against neuronal apoptosis. In the experiment,transient middle cerebral artery occlusion model was made adopting modifiedLonga suture method in rats.With Xuesetong as a positive control, detectionsof cerebral infarct area, neurologic score, brain weight and moisture werefulfilled, to evaluate and elect the better effective group between the aqueousextractives of Radix Astragali and ethanol extractives; Cyt C, Cyp D, p53, baxand bcl-2expressions at optic chiasma cortex penumbra were measured byimmunohistochemical staining, and quantification of cytoplasmic andmitochondrial Cyt C, mitochondrial Cyp D was with protein blotting, isolatedmitochondria swelling was inspected by UV spectrophotometer, whichprovides the basis for Radix Astragali preventing cerebral ischemiareperfusion injury, and the foundation for further screening of effective partsor composition.The First Part The research of Radix Astragali extractives reducing cerebral ischemia reperfusion injury in ratsObjective:Compare Radix Astragali water extractives’ effects with alcoholextractives’ on cerebral ischemia reperfusion injury in rats.Method:1Preparation and groupMale, SPF grade, SD rats were randomly divided into sham group (S),the left middle cerebral artery ischemia reperfusion model group (MIR)(M),Radix Astragali water extractives low does (WL), medium does (WM) andhigh does (WH) group, Radix Astragali alcohol extractives low does (AL),medium does (AM) and high does (AH) group, Xuesetong group (B). Ratsfrom WL, WM, WH, AL, AM, AH were of ig administration, qd,6d, andonce more at20h reperfusion. S and M rats were given the same amount ofwater. At6th day, rats underwent transient middle cerebral artery occlusionsurgery. Modified Longa suture method was applied in making transientmiddle cerebral artery occlusion model, and successful model was selected fortest indexes.2Detection of cerebral infarct areaAt24h reperfusion, rats were decapitated, and the cerebrum was cut into5sections along the coronal plane. Sections were stained in TTC solution.Take pictures and calculate infarct area.3Detection of neurologic scoreRats were made scores according to their neurologic deficit signs,including experiments of spontaneous ipsilateral rotation, contralateralhindlimb retraction, going through bars and the double front paws gripping.4Brain weight and moisture contentWeigh wet cerebrum and wet ischemic brain hemisphere. Dry ischemichemisphere, calculate the ischemic hemisphere moisture content, brainmoisture content (%)=(wet weight-dry weight)/wet weight×100%.Results:Radix Astragali water extractives and alcohol extractives protect brain agaist cerebral ischemia reperfusion injury significantly, by reducingneurologic dysfunction, decreasing infarct area and losing brain weight andmoisture content, caused by cerebral ischemia reperfusion in rats.1Effect of Radix Astragali extractives on neurologic scoreThe score in M is higher than S (P<0.05). WM, WH, AM, AH, B is lessthan M (P<0.05); B, WH, AH have no difference (P>0.05).2Effect of Radix Astragali extractives on cerebral infarct areaThat in M is apparent while there is no infarct area in S (P<0.05). WM,WH, AM, AH, B have significant decreases compared to M (P<0.05);wherein, AH is the minimum (P<0.05).3Effect of Radix Astragali extractives on brain weight and moisturecontentThe brain weight and moisture content of M has significant increase,compared with S (P<0.05). WM, WH, AM, AH and B is less than M(P<0.05); there is no difference in B, WH, AH (P>0.05).Summary:1Radix Astragali water extractives and alcohol extractives significantlyreduce neurologic dysfunction, decrease infarct area and lose brain weight andmoisture content caused by cerebral ischemia reperfusion in rats，protectingbrain agaist cerebral ischemia reperfusion injury.2Radix Astragali alcohol extractives make better effect on preventing ratbrain from cerebral ischemia reperfusion injury.The Second Part Effects of Radix Astragali extractives on mitochondriafunction after cerebral ischemia reperfusion in ratObjective:Investigate effects of Radix Astragali extractives on mitochondriafunction in cerebral ischemia reperfusion injury rats.Method:Rats were randomly divided into sham operation group (S), the left middle cerebral artery ischemia reperfusion model group(MIR)(M), RadixAstragali extractives medication group (E), CsA control group (C), RadixAstragali extractives and CsA group (EC). Cyt C, Cyp D, p53, bax and bcl-2expression at optic chiasma cortex penumbra were measured byimmunohistochemical staining, content of cytoplasmic Cyt C, mitochondrialCyt C and mitochondrial Cyp D was detected by Western Blotting.Spectrophotometer inspected the swelling of isolated mitochondria.Result:Each layer cell in cerebral cortex from sham operation group is ofneat arrangement. Nuclear membrane and nucleolus of nerve cells areclear. Pyramidal cells and granular cells contain a small amount of buffyparticles. In model group, the lateral cortex has a buffer zone (corticalpenumbra) between normal area and infarction area. Cortical penumbrathickens, cells of a bit disorder arrangement, inflammatory cellsinfiltration and interstitial edema are apparent. However, nuclearmembrane and nucleolus are clear, corrugate membrane and irregularform are in a few neuron cells. A large amount of buffy particles aresituated in pyramidal cells and granular cells. RA medication group, RAplus CsA group and the CsA control group have similar cerebral cortex inmodel group, but the degree lightens. RA can obviously downregulateexpression of Cyt C, Cyp D, p53and bax, upregulate bcl-2expression,lessen the swelling of isolated mitochondria, and reduce the release ofmitochondrial Cyt C into cytoplasm, led from cerebral ischemiareperfusion.1Effect of Radix Astragali extractives on Cyt C expressionCyt C is expressed in the cytoplasm of cerebral cortex neurons.Cerebral ischemia reperfusion induces the increasing expression of CytC, and RA restrains the induced reaction. Compared with S, Cyt Cexpression of M increased significantly (P<0.05). E, C and EC are lower thanM (P<0.05). There is no significant difference among E, C and EC (P>0.05).2Effect of Radix Astragali extractives on Cyp D expression Cyp D is mainly expressed in cytoplasm of cortical neurons, andcerebral ischemia reperfusion induces its expression raising. RA reducesthe raising Cyp D expression. Compared with S, Cyp D expression in Mincreases significantly (P<0.01). In comparison to M, E, C and EC decreasesignificantly (P<0.05). C have a significant decrease than E (P<0.05).3Effect of Radix Astragali extractives on p53expressionP53expression is in cytoplasm of cerebral cortex neurons, enhanced bycerebral ischemia reperfusion, and decreased by RA. P53in M increasessignificantly in comparison with S (P<0.01). Compared to M, E, C and ECare decreased significantly (P<0.05). E has a significant decrease with C(P<0.05). EC decreases significantly in comparison to C (P<0.05).4Effect of Radix Astragali extractives on bax expressionBax is expressed in cytoplasm of cerebral cortex neurons, enhanced bycerebral ischemia reperfusion, but decreased by RA. Bax expression in M isincreased significantly (P<0.01), in comparison to S. E has a significantdecrease than M (P<0.05).5Effect of Radix Astragali extractives on bcl-2expressionBcl-2is mainly expressed in cytoplasm of cortical neurons, and itsraising expression is induced by cerebral ischemia reperfusion. RAreduces the raised expression. There is significant difference for bcl-2expression in all groups. Compared to S, bcl-2in M is decreased significantly(P<0.01). Bcl-2content of E is more than M (P<0.05).6Effect of Radix Astragali extractives on Cyt C contentNormal Cyt C exists in mitochondria, cerebral ischemia reperfusioninduces high expression of Cyt C and the release of mitochondria Cyt C intocytoplasm. RA downregulates Cyt C expression and decreases the release ofmitochondria Cyt C into cytoplasm. Compared to S, cytoplasmic Cyt Cprotein of M increases significantly (P<0.001). E, C and EC have significantdecrease (P<0.05) compared to M. E and C decrease significantly comparedwith EC (P<0.05).There is a significant decrease in E rather than C (P<0.05).There is a significant increase in mitochondrial Cyt C content of M (P<0.01), in comparison to S. E and C are decreased significantly (P<0.05) comparedwith M.There is no significant difference between E and C (P>0.05).7Effect of Radix Astragali extractives on Cyp D contentCyp D expressses in mitochondria, and cerebral ischemia reperfusionleads to Cyp D enhanced expression. Cyp D content in M increasessignificantly (P<0.05) compared with S. In comparison to M, E, C and ECdecrease significantly (P<0.01). E has a significant decrease with E (P<0.01)or EC (P<0.05).8Effect of Radix Astragali extractives on mitochondria swelling inducedby Ca2+The more decrease of light transmission value in isolated mitochondria, themore mitochondria swelling. That in S shows a mild decrease compared within M, a violent decrease (P<0.01). E, C and EC are smaller than Msignificantly (P<0.01). E is smallest among the three groups (P<0.01).Summary:Radix Astragali extractives reduce mitochondria swelling, downregulateexpressions of Cyt C, Cyp D, p53and bax, enhanced bcl-2expression,decrease the leakage of mitochondrial Cyt C into cytoplasm significantly,preventing mitochondria from dysfunction resulting from cerebral ischmiareperfusion injury.Conclusion:1Radix Astragali extractives significantly reduce neurologic dysfunction,decrease infarct area, lose brain weight and moisture content, caused bycerebral ischemia reperfusion injury in rats, and Radix Astragali alcoholextractives make better effects than water extractives.2Radix Astragali extractives reduce mitochondria swelling,downregulate expressions of Cyt C, Cyp D, p53and bax, enhanced bcl-2expression, decrease the leakage of mitochondrial Cyt C into cytoplasmsignificantly, protecting mitochondria against dysfunction leading from cerebral ischmia reperfusion injury.