Study On Mechanism Of Pyroptosis In Experimental Intracerebral Hemorrhage Mice By The Method Of Activating Blood And Removing Stasis And Supplementing Essence And Marrow Based On NLRP3/Caspase-1/GSDMD Signal Pathway

Objective:1.By sorting out the representative works and literatures of ancient physicians on the understanding of hemorrhagic stroke,this paper clarifies its etiology,pathogenesis,therapeutic principles and methods,analyzes the influence of toxin on hemorrhagic stroke,research progress on the treatment of hemorrhagic stroke with Didang decoction which is a classic prescription of traditional Chinese medicine,and lays a theoretical foundation for further study of the pathogenesis of hemorrhagic stroke’marrow deficiency and toxin damage’.2.Under the guidance of the pathogenesis of’activating blood and removing stasis and supplementing essence and marrow’,the treatment method is’activating blood and removing stasis and supplementing essence and marrow’as the treatment method and legal prescription,the intracerebral hemorrhage mouse model was intervened by the legal prescription to explore that the method of activating blood and removing stasis and supplementing essence and marrow may regulate NLRP3 inflammasome corpuscles through Nrf2,inhibit the occurrence of pyroptosis of downstream cells,and reduce the secondary injury of intracerebral hemorrhage.It provides a reliable basis for clinical application of this method in the treatment of intracerebral hemorrhage.Methods:1.Literature researchBy consulting the works and kinds of literature of representative doctors in the past dynasties,sorting out the etiology,pathogenesis,and treatment principles of hemorrhagic stroke,finding out the influence of toxin on hemorrhagic stroke and the development and change process of toxins,as well as the related research of Didang Decoction in the treatment of hemorrhagic stroke.To explain the theoretical basis of the treatment of hemorrhagic stroke by the method of activating blood and removing stasis and supplementing essence and marrow under the guidance of the pathogenesis of’marrow deficiency and toxic damage’.2.Experimental researchExperiment 1:Establishment of a mouse model of intracerebral hemorrhage.The mice were randomly divided into sham operation group,model group,low dose group,middle dose group and high dose group.Each group of traditional Chinese medicine was given a certain proportion of traditional Chinese medicine decoction for activating blood and removing stasis and supplementing essence and marrow,and the sham operation group was given an equal volume of normal saline.The neurological deficit scores of mice were scored every day,and the mice were sacrificed by anesthesia after three days of gavage.After sampling,the brain water content of mice was detected.The brain tissue sections of mice were stained with hematoxylin-eosin to observe the pathological changes of brain tissue in mice.The content of ROS in the brain tissue of mice was detected by immunofluorescence.The contents of Nrf2,HO-1,NLRP3,ASC,and Caspase-1 related proteins in the brain tissue of mice were detected by Western blotting.Experiment 2:The mouse model of intracerebral hemorrhage was established by using Nrf2 gene knockout mice.C57 mice were randomly divided into sham operation group,model group and Chinese medicine group.Nrf2^-/-mice were also randomly divided into sham operation group,model group and Chinese medicine group.The two Chinese medicine groups were given intragastric administration of Chinese medicine decoction for activating blood and removing stasis and supplementing essence and marrow.The sham operation group was given equal volume of normal saline.The neurological deficit scores of mice were scored every day,and the mice were sacrificed by anesthesia after three days of gavage.The brain water content of mice was detected.The pathological changes of brain tissue in mice were observed by hematoxylin-eosin staining.The protein contents of NLRP3,ASC,Caspase-1,and GSDMD in the brain tissue of mice in each group were detected by immunohistochemistry.The m RNA expression levels of NLRP3,ASC,Caspase-1 and GSDMD in the brain tissue of mice in each group were detected by real-time fluorescence quantitative PCR.The contents of IL-1βand IL-18 in the serum of mice were detected by enzyme-linked immunosorbent assay.Results:1.Literature resultsThe discussion of the etiology and pathogenesis of hemorrhagic stroke by ancient physicians is nothing more than starting from external wind and internal wind.This disease is caused by wind induced internal circulation,qi and blood circulation disorder,blocking the qi flow,crossing the meridians,ascending and attacking the brain,the pulse not binding blood,blood not returning to the meridians,overflowing outside the pulse,and blocking the brain meridians.’Poison’is crucial in the occurrence and development of hemorrhagic stroke.Poison is a pathogenic factor of hemorrhagic stroke and also all harmful substances produced after hemorrhagic stroke,such as edema and hematoma.This disease can be treated with the addition and subtraction of Dudang Tang,which can break blood and remove stasis.2.Experimental resultsExperiment 1:（1）Neurological function defect score:Before modeling,the neurological function defect score of each group of mice was 0.Three hours after modeling,compared with the sham operation group,both the model group and the traditional Chinese medicine group showed significant neurological dysfunction（P<0.01）.On the first day,compared with the model group,the improvement of neural function in each Chinese medicine group was not significant（P>0.05）.On the second day,compared with the model group,the neural function of mice in the middle dose group and the high dose group of traditional Chinese medicine began to gradually recover（P<0.01）.On the third day,compared with the model group,the neurological function of mice in each group of traditional Chinese medicine recovered,while the recovery rate in the low-dose group of traditional Chinese medicine was slower（P<0.05）.（2）Mouse brain water content:After three days,compared with the sham operation group,the brain water content in each group increased（P<0.01）.Compared with the model group,there was no significant decrease in brain water content in the low-dose group of traditional Chinese medicine（P>0.05）.The brain water content in the middle dose group and the high dose group of traditional Chinese medicine decreased（P<0.05）.（3）Brain tissue sections of mice with intracerebral hemorrhage:Brain tissue sections can visually observe the hematoma size of brain tissue after intracerebral hemorrhage in mice.The hematoma in the sham operation group is not obvious,and a large dark red hematoma can be seen in the model group.After treatment,the hematoma in each group was absorbed,and the hematoma absorption in the middle and high dose groups of Chinese medicine was more obvious.（4）Hematoxylin-eosin staining:In the sham operation group,the number of neurons was abundant and closely arranged,the morphological structure of neurons was normal,the boundary between nucleus and cytoplasm was clear,and the nucleolus was obvious.The model group showed bleeding,more red blood cells,neutrophil infiltration,loose surrounding tissue,and a reduced number of neurons.The bleeding in the low-dose group was reduced but not obvious.The bleeding in the middle dose group and the high dose group was significantly reduced,with a small number of red blood cells,accompanied by a small amount of neutrophil infiltration,and the surrounding tissue was slightly loose and lightly stained.After the treatment of traditional Chinese medicine,the bleeding area and the morphology of each nerve cell were improved.（5）Immunofluorescence detection of ROS content in brain tissue of mice with intracerebral hemorrhage:Compared with the sham operation group,the ROS content in both the model group and the traditional Chinese medicine group increased（P<0.05）.Compared with the model group,there was no significant change in the ROS content in the low dose group of traditional Chinese medicine（P>0.05）,while the ROS content in the middle and high dose groups of traditional Chinese medicine decreased（P<0.05）.The ROS content in the high dose group of traditional Chinese medicine was significantly lower than that in the middle dose group of traditional Chinese medicine（P<0.01）.（6）The protein expression levels of Nrf2,HO-1,NLRP3,ASC,and Caspase-1 in mice with intracerebral hemorrhage:Compared with the sham operation group,the protein levels of Nrf2 and HO-1 in the model group increased（P<0.05）,while the protein levels of NLRP3,ASC,and Caspase-1 increased（P<0.05）.The levels of Nrf2 and HO-1 protein in each Chinese medicine group increased（P<0.05）,while the levels of NLRP3,ASC,and Caspase-1 protein decreased（P<0.05）.Compared with the model group,the protein levels of Nrf2,NLRP3,and Caspase-1 in the low-dose group of traditional Chinese medicine did not significantly change（P>0.05）,while the protein level of HO-1 increased（P<0.05）,while the protein level of ASC decreased（P<0.05）;The protein levels of Nrf2 and HO-1 in the middle and high dose groups of traditional Chinese medicine increased（P<0.05）,while the protein levels of NLRP3,ASC,and Caspase-1 decreased（P<0.05）.Experiment 2:（1）Neurological deficit score:Before the operation,the neurological deficit score of mice with intracerebral hemorrhage in each group was 0.At 3h after modeling,the mice in the two sham operation groups had normal activities and no neurological deficits.The mice in the two model groups and the two Chinese medicine groups showed different degrees of neurological deficits,such as the incomplete extension of the front claw,flexion posture,rotation,and paralysis.On the first day,the difference between the Chinese medicine group and the Nrf2^-/-Chinese medicine group was statistically significant（P<0.05）.On the second day,compared with the model group,the neurological function of the Chinese medicine group began to recover（P<0.01）,and the neurological function of the Nrf2^-/-Chinese medicine group recovered slowly（P>0.05）.There was no difference in the scores between the Chinese medicine group and the Nrf2^-/-Chinese medicine group（P>0.05）.On the third day,compared with the model group,the neurological function of the Chinese medicine group and the Nrf2^-/-Chinese medicine group recovered（P<0.01）.（2）Brain water content of mice:After three days,compared with the sham operation group,the brain water content of the two model groups and the Chinese medicine group increased（P<0.01）.Compared with the model group,the brain water content of the two Chinese medicine groups was significantly reduced（P<0.01）.The brain water content of the Nrf2^-/-Chinese medicine group was significantly higher than that of the Chinese medicine group（P<0.05）.（3）Hematoxylin-eosin staining:The number of neurons in the sham operation group and Nrf2^-/-sham operation group was abundant and closely arranged.The morphological structure of neurons was normal,the boundary between the nucleus and cytoplasm was clear,and the nucleolus was obvious.Hemorrhage was observed in the model group,and the Nrf2^-/-model group had obvious bleeding.Both groups had more red blood cells,accompanied by neutrophil punctate infiltration,loose surrounding tissues,and a reduced number of neurons.The bleeding was reduced in the Chinese medicine group,and the bleeding was reduced but not obvious in the Nrf2^-/-Chinese medicine group.A small number of red blood cells and a small amount of neutrophil infiltration were observed in both groups,and the surrounding tissues were slightly loose and lightly stained.（4）Expression of NLRP3,ASC,Caspase-1,and GSDMD proteins in the brain tissue of mice with intracerebral hemorrhage in each group:The expression of NLRP3,ASC,Caspase-1,and GSDMD in the brain tissue of mice was most obvious in the Nrf2^-/-model group,with a large number of positive cells and deep staining.The expression of positive cells in the sham operation group was the weakest,with a small number of cells.Compared with the sham operation group,there were differences in NLRP3,ASC,Caspase-1,and GSDMD proteins between the traditional Chinese medicine group and the model group（P<0.05）.The expression level of positive cells in Nrf2^-/-mice in each group was higher than that in Nrf2 gene non knockout mice.The number of Caspase-1 positive cells in Nrf2^-/-model group was significantly different from that in the model group（P<0.05）.There was no difference in the number of NLRP3 positive cells between the Nrf2^-/-traditional Chinese medicine group and the traditional Chinese medicine group（P>0.05）.The number of caspase-1,ASC,and GSDMD positive cells in the Nrf2^-/-traditional Chinese medicine group was significantly different from that in the traditional Chinese medicine group（P<0.05）.（5）Expression of NLRP3,ASC,Caspase-1,and GSDMD m RNA in mouse brain tissue in each group:Compared with the sham operation group,the m RNA levels of NLRP3,Caspase-1,ASC,and GSDMD in mouse brain tissue were significantly increased（P<0.05）.Compared with the model group,the m RNA levels of NLRP3,Caspase-1,ASC,and GSDMD in mouse brain tissue were significantly decreased（P<0.05）.After Nrf2 gene knockout,the m RNA levels of NLRP3,Caspase-1,ASC,and GSDMD in mouse brain tissue were increased.（6）The expression of IL-1βand IL-18 in the serum of mice in each group:Compared with the sham operation group,the serum levels of IL-1βand IL-18 in each group were significantly increased（P<0.05）.Compared with the model group,the serum levels of IL-1βand IL-18 in the Chinese medicine group were significantly decreased（P<0.05）.After Nrf2 gene knockout,the levels of IL-1βand IL-18 in the serum of mice increased.Chinese medicine can reduce the levels of IL-1βand IL-18 in the serum of mice with intracerebral hemorrhage.Conclusion:1.Literature research:The deficiency of hemorrhagic stroke is the emptiness of the sea of marrow,and the excess of the sea of marrow is the accumulation of wind,fire,phlegm,blood stasis,and deficiency into poison,which poisons the brain and marrow.Poison is a product of all damage to brain tissue.The method of activating blood and removing stasis and supplementing essence and marrow is an effective method for the treatment of hemorrhagic stroke.It can treat’marrow deficiency’caused by’toxic damage’.2.Experimental study:The model of intracerebral hemorrhage was established by autologous blood injection in mice.The model of intracerebral hemorrhage was stable,and the hematoma was obvious.The core factor of secondary injury after intracerebral hemorrhage was’toxic damage’.The decoction of traditional Chinese medicine can improve the neurological function injury of mice with intracerebral hemorrhage,reduce hematoma and edema,and play an’attenuated’effect.The decoction of traditional Chinese medicine can inhibit the formation of NLRP3 inflammasome by regulating Nrf2 and affecting the occurrence of pyroptosis in downstream cells.The target of traditional Chinese medicine decoction for the treatment of intracerebral hemorrhage may be Nrf2.