To Explore The Protective Mechanism Of Huoxue Rongluo Fang On Cerebral Ischemia-reperfusion Injury Based On PINK1/Parkin Signaling Pathway

Cerebrovascular disease is the world’s largest cause of death and a common cause of disability,characterized by high mortality,disability and recurrence rates.The goal of acute cerebral infarction is to restore blood supply to ischemic brain tissue.However,rapid reperfusion can have deleterious effects on brain function.Previous studies have shown that cerebral ischemia/reperfusion injury activates mitochondrial autophagy,and moderate mitochondrial autophagy activation can reduce cerebral ischemia/reperfusion injury.Therefore,mitochondrial autophagy is an important potential target for the intervention treatment of cerebral ischemia/reperfusion injury.This study first focused on "Rong Qi Theory",and explored the traditional Chinese medicine understanding of mitochondrial autophagy after cerebral infarction from the pathogenesis of Rong Qi deficiency stagnation.In vivo and in vitro experimental researches on the Huo Xue Rong Luo Decoction based on the theory of "Rong Qi Deficiency and Stagnation",focusing on the PINK1/Parkin signaling pathway to explore the possible mechanism of Huo Xue Rong Luo Decoction against cerebral ischemia reperfusion injury.Part 1 Theoretical discussionThis part aims to explore the correlation between the transformation of Rong Qi and mitochondrial function from the perspective of "Rong Qi deficiency and stagnation" by combining Chinese and Western medicine theories.Rong Qi contains qi,blood,fluid,liquid and other subtle substances,and is the source of subtle substances.Rong Qi is closely related to the generation and physiological function of the brain,which is used for gasification and circulation,internalizing the brain-marrow mechanism and showing various physiological phenomena.The pathogenesis of "Rong Qi deficiency and stagnation" in cerebral infarction is characterized by hepatic and renal Yin deficiency,stirring wind due to yin deficiency,combined with endogenous pathogenic qi,and its clinical mani-festations are blockage of cerebral collaterals,absence of spirit and qi,loss of filling of brain marrow and damage of miraculous mechanism.As the most basic life activity of the body,the dysfunction of Rong Qi gasification runs through the whole onset of cerebral infarction.Based on the understanding of the theory of "Rong Qi deficiency and stagnation" from the function of mitochondria,this paper expounds the consistency of physiology and pathology between mitochondria and Rong Qi,and dialectically treats the regulation of mitochondrial autophagy from the perspective of imbalance of gasification.Combined with typical clinical medical records,it provides a new direction for the treatment of cerebral infarction.Part 2 In vitro experiments Objective:Rat adrenal pheochromocytoma cells(PC12 cell line)were used to establish an oxygen-glucose deprivation/reoxygenation(OGD/R)model.To investigate the protective effect of serum containing Huo Xue Rong Luo Decoction on PC12 cell injury model induced by OGD/R and its possible mechanism.Methods:Hypoxia combined with glucose-free medium was used to induce hypoxia and glucose deficiency in PC12 cells,and then reoxygenation and glucose in normal culture medium was used to establish OGD/R damage model.Firstly,the optimal concentration of serum in Huo Xue Rong Luo Decoction(H-HYXQ)was screened.Mitochondrial autophagy activator rapamycin(RAP)was used as positive control drug.Mitochondrial membrane potential(JC-1),immune-fluorescence staining and western blot(WB)were used to detect PINK1/Parkin pathway and the expression of mitochondrial autophagy-related proteins.Results:1.CCK8 and LDH results indicated that OGD/R damage could significantly reduce the survival rate of PC12 cells and increase the leakage rate of LDH.When the concentrations were 10% and 15%,the serum containing Huo Xue Rong Luo Decoction could significantly increase the survival rate of PC12 cell model injured by OGD/R(P<0.05 or P<0.01).However,5% and 10% concentrations of drug-containing serum could reduce the LDH leakage rate of PC12 cells(P<0.05 or P<0.01).And compared with the KBXQ group,the LDH leakage rate of 10%drug-containing serum concentration was significantly reduced(P<0.05),while the LDH leakage rate of 5% drug-containing serum group had no significant difference(P>0.05).2.Mitochondrial membrane potential(JC-1)detection: Compared with the normal control group,the red fluorescence in the model group was decreased,and the ratio of green fluorescence to red fluorescence in the model group was significantly increased,with statistical significance(P<0.01),suggesting that the mitochondrial membrane potential in the model group was decreased.Compared with model group,the ratio of green fluorescence to red fluorescence in KBXQ group,H-HYXQ group and RAP group was significantly decreased,with statistical significance(P<0.01).And compared with KBXQ group,mitochondrial membrane potential in H-HYXQ group and RAP group was more significantly up-regulated(P<0.05).At the same time,the up-regulation of mitochondrial membrane potential in the RAP group was more significant than that in the H-HYXQ group(P<0.05).3.Immunofluorescence co-localization analysis of mitochondrial(Mito)-LC3B: The results showed that the Pearson correlation coefficient(Rr)and overlap coefficient(R)in H-HYXQ group and RAP group were significantly higher than those in model group,suggesting that LC3B-Mito co-localization was enhanced.4.Expression of mitochondrial autophagy-related proteins: Compared with the model group,the expression of LC3B and p62 protein in KBXQ group was not significantly different(P>0.05),but the expression of TOMM20 protein was decreased(P<0.05).However,the expression of LC3B protein in H-HYXQ group and RAP group was higher than that in model group,while the expression of p62 and TOMM20 protein was lower(P<0.05 or P<0.01).Compared with KBXQ group,the expression of LC3B protein increased and the expression of p62 and TOMM20 decreased(P<0.05)in H-HYXQ group and RAP group,but the down-regulation of TOMM20 in RAP group was more significant than that in H-HYXQ group(P<0.05).5.Mito-PINK1-Parkin fluorescence co-localization analysis: The results showed that the Rr and R coefficients of the H-HYXQ group and RAP group were significantly increased,suggesting that the co-localization of Mito-PINK1-Parkin was enhanced.6.Expression of PINK1/Parkin pathway proteins: Compared with the normal control group,the expression of PINK1 and Parkin proteins in the model group were up-regulated(P<0.05 or P<0.01),while there was no significant difference in the expression of PINK1 and Parkin proteins in the KBXQ group compared with the model group(P>0.05).Compared with model group and KBXQ group,the expression of PINK1 and Parkin protein in H-HYXQ group and RAP group were up-regulated(P<0.05).And there was no significant difference between H-HYXQ group and RAP group(P>0.05).Conclusions:The serum containing of Huo Xue Rong Luo Decoction can significantly reduce the PC12 cell damage induced by OGD/R.The mechanism may be related to the regulation of the PINK1/Parkin pathway,which increases the mitochondrial translocation of related proteins,up-regulates LC3B,down-regulates the expression of p62 and TOMM20,to activate mitochondrial autophagy.Part 3 In vivo experiment Objective:The Middle Cerebral Artery Occlusion Reperfusion(MCAO/R)model of rats was established by line plugging method to investigate the protective effect of Huoxue Rong Luo Decoction on MCAO/R model rats and its possible mechanism.Methods:SPF grade SD rats were randomly divided into sham operation group,model group,Huo Xue Rong Luo Decoction(HXRLF)group and autophagy activator(RAP)group.MCAO/R model was established by suture method in model group,HXRLF group and RAP group,while no suture was inserted in sham operation group.The rats in the HXRLF group were given intragastric administration(11.7g/kg/d)for 3 times 36 hours before the establishment of the model,24h/times,the sham operation group,the model group and the RAP group were given the same amount of distilled water,the RAP group was injected intraperitoneally at the same time of reperfusion(1.5mg/kg),and the other three groups were given the same amount of normal saline intraperitoneal injection.After 2 hours of ischemia and 24 hours of reperfusion,the neurological deficit score was performed.And the volume of cerebral infarction,the number of intact neurons in the ischemic cortex were detected by TTC staining,Nissl’s staining.And the number of autophagosomes was observed by transmission electron microscope.Immunocytochemistry(IHC)and WB assay were used to detect the expression levels of autophagy related proteins p62,LC3B and TOMM20 in mitochondria of rats in each group.Also,the expression of PINK1/Parkin pathway protein was detected by WB.Results:1.The neuroprotective effect of Huo Xue Rong Luo Decoction: Compared with the sham operation group,the neurological function score of the model group was significantly higher and the neurological deficit was more serious(P<0.01).The neurological deficit was significantly alleviated by HXRLF(P<0.01).The results of TTC staining showed that Huo Xue Rong Luo Decoction and RAP could significantly reduce the cerebral infarction volume of MCAO/R model rats(P<0.01),and Nissl staining showed that Huo Xue Rong Luo Decoction and RAP significantly reduced the ischemic injury of neurons(P<0.01).2.Regulation of Huoxue Rongluo Decoction on mitochondrial autophagy and related proteins in MCAO/R model rats: Under electron microscope,the number of autophagosomes in bilayer membrane structure increased in HXRLF group and RAP group.The results of IHC and WB showed that Huoxue Rongluo Decoction and RAP could up-regulate the expression of LC3B protein and down-regulate the expression of p62 and TOMM20 protein,and the difference was statistically significant(P<0.05 or P<0.01).However,there was no significant difference in observed indexes between HXRLF group and RAP group(P>0.05).3.Regulation of Huoxue Rongluo Decoction on PINK1/Parkin pathway protein in MCAO/R model rats: The results of WB showed that the expression of PINK1 and Parkin proteins in the ischemic cortex of the model group was higher than that of the sham operation group(P<0.05 or P<0.01).Compared with the model group,the expression of PINK1 and Parkin proteins in the ischemic cortex of HXRLF group and RAP group increased(P<0.05 or P<0.01),but there was no significant difference between HXRLF group and RAP group(P>0.05).Conclusion:Huo Xue Rong Luo Decoction may activate PINK1/Parkin signal pathway,up-regulate mitochondrial autophagy and reduce neurological damage in rats with cerebral ischemia-reperfusion injury,so as to play a neuroprotective effect.