| [Objective]1.To further investigate the active ingredients and targets in the treatment of intracerebral hemorrhage(ICH)via ferroptosis,network pharmacology was performed.To find out the potential molecular mechanisms and pathways of Zhi Long Huo Xue Tong Yu capsule,the enrichment of GO and KEGG pathways were carried out.And then finally screen out the key targets for validation in animal experiments,so as to provide a new research direction and data support for the clinical application of Zhi Long Huo Xue Tong Yu capsule.2.To verify the ameliorative effect of Zhi Long Huo Xue Tong Yu capsule on behavioral outcomes in rats with ICH,and the regulation of Zhi Long Huo Xue Tong Yu capsule on m RNA and protein expression of related targets of iron metabolism and lipid peroxidation accumulation in brain tissue of rats after ICH was studied,so as to further verify the regulatory effect of Zhi Long Huo Xue Tong Yu capsule on TP53,a key target after network pharmacology screening,and ferroptosis related targets.Verify the mechanism of Zhi Long Huo Xue Tong Yu capsule in treating ICH by inhibiting ferroptosis,summarize the specific neuroprotective effect of Zhi Long Huo Xue Tong Yu capsule on ICH rats,and provide in vivo experimental data support for the clinical application of Zhi Long Huo Xue Tong Yu capsule.[Methods]1.Network pharmacologyTCMSP,TCMID,Swiss ADME and Swiss Target Prediction databases were used to obtain the active ingredients and targets of Zhi Long Huo Xue Tong Yu capsule,and the target names were standardized and unified in the Uniprot database.The targets of ICH disease and ferroptosis mechanism were obtained from Gene Cards database and the Online Mendelian Inheritance in Man(OMIM)database.The three targets were intersected to obtain the intersecting targets,and the intersecting targets were imported into STRING to create a PPI network map,and then was imported into Cytoscape for data analysis and visualization.The intersecting targets were imported into Metascape for GO enrichment and KEGG pathway enrichment analysis,and the results were visualized and analyzed.Finally,the active ingredients,key targets and potential pathways of Zhi Long Huo Xue Tong Yu capsule for the treatment of ICH were screened.2.Experimental research120 male SD rats(250±20g)around the age of 3 months were used to establish a rat model of ICH by autologous blood injection method.There were 20 rats in each group,and the rats were divided into each group by random number table method.The rats were divided into sham group(Sham group),model group(Ich group),normal saline group(Ns group),low dose group(Zll group),medium dose group(Zlm group)and high dose group(Zlh group)of Zhi Long Huo Xue Tong Yu capsule.The gavage doses were calculated according to the body surface area exchange method as low dose(0.5g/kg/day),medium dose(1 g/kg/day)and high dose(2 g/kg/day)for 21 days,and the normal saline group was given equal dose of saline by gavage.2.1Behavioural effects of Zhi Long Huo Xue Tong Yu capsule on rats after ICH In this study,behavioral scores(NSS scores,Zea-Longa scores)were performed on rats at 1,3,5,7,10,14 and 21 days after the modeling operation,and various behavioral tests were performed on rats,including the open field test,Y-maze test,morris watermaze test,and rotards test.2.2Effects of Zhi Long Huo Xue Tong Yu capsule on brain tissue damage and ferroptosis in rats after ICHAfter 21 days,morphological and molecular detections were performed.HE staining and Nissle staining were used to detect changes in brain tissue structure and neuronal cell death in rats.Ferroptosis detection included iron deposition and lipid peroxidation detection,perls stain was used to detect changes in iron deposition around the hematoma,oxidative stress kits including SOD,MDA and GSH were used to detect lipid peroxidation changes in rats after ICH.2.3Mechanisms of Zhi Long Huo Xue Tong Yu capsule inhibition on ferroptosis in rats after ICHReal-time quantitative PCR was performed to detect the changes of TFR1,SLC40A1,TP53,NOX4,SESN2,SCL7A11 and GPX4 m RNA expression in rats;immunofluorescence was performed to detect the changes of NOX4 expression in cortex;western bloting was performed to detect the changes of TP53,NOX4,SESN2 and GPX4 protein expression in rats.[Results]1.Network pharmacology resultsZhi Long Huo Xue Tong Yu capsule could alleviate ICH through ferroptosis via mutiingredients,muti-targets and muti-pathway,the important active ingredients of Zhi Long Huo Xue Tong Yu capsule for the treatment of ICH through ferroptosis mainly include quercetin,crocetin,kaempferol,beta-sitosterol,formononetin,isorhamnetin.PTGS2,RELA,JUN,TP53,TNF,CASP8 and GSK3 B were the important targets of Zhi Long Huo Xue Tong Yu capsule for the treatment of ICH through ferroptosis,the main pathways included ferroptosis pathway,IL-17 pathway and VEGF pathway.After reviewing the literature and combining the results of network pharmacology,TP53 was finally selected as the key target for in vivo animal experiments to validate.2.Results of in vivo experiments on animals2.1Behavioral effects of Zhi Long Huo Xue Tong Yu capsule on rats after ICH2.1.1Behavioral scoresCompared with the Sham group,the NSS scores and Zea-Longa scores of rats in the Ich group and Ns group were significantly higher and statistically different(P<0.05);compared with the Ns group,the NSS scores and Zea-Longa scores of rats had a significant decreasing trend after treatment with Zhi Long Huo Xue Tong Yu capsule and there was a statistical difference(P<0.05).2.1.2Open field testCompared with the Sham group,the number of grooming and standing,duration of grooming and standing,total distance travelled and number of central area entries were significantly reduced in the Ich group and Ns group with statistical differences(P<0.05);the number of grooming and standing,duration of grooming and standing,total distance travelled and central area entries were significantly increased in rats after treatment with Zhi Long Huo Xue Tong Yu capsule,and was statistically different in the Zlh group(P<0.05).2.1.3Y-maze testCompared with the Sham group,the rats in the Ich group and Ns group showed an increase in the duration and number of entries of the initial arm and wrong arm,and a decrease in the duration and number of entries of the food arm,and there was a statistical difference(P<0.05);after treatment with Zhi Long Huo Xue Tong Yu capsule,compared with the Ns group,the rats showed a decrease in the duration and number of entries of the initial arm and wrong arm,and an increase in the duration and number of entries of the food arm,and there was a statistical difference(P<0.05).2.1.4Morris water maze testCompared with the Sham group,the rats in the Ich group and the Ns group showed a significant decrease in the number of target crossing and the ratio of the third quadrant to the total distance travelled,and a significant increase in escape latency with statistical differences(P < 0.05);after treatment with Zhi Long Huo Xue Tong Yu capsule,compared with the Ns group,the number of target crossing and the ratio of the third quadrant to the total distance travelled increased significantly,and the Zlh group showed statistical differences(P<0.05),and the rats escape latency was significantly reduced,and there was a statistical difference between the Zlh group on fifth day(P<0.05).2.1.5Rotards testCompared with Sham group,the duration of rotards test in Ich group and Ns group was significantly reduced(P<0.05);after treatment with Zhilong Huoxue Tongyu capsule,compared with the Ns group,the duration of rotards test increased and there was significant difference in the Zlh group(P<0.05).2.2Effects of Zhi Long Huo Xue Tong Yu capsule on brain tissue damage and ferroptosis in rats after ICHCompared with the Sham group,the brain tissues of the Ich group and Ns group were loose and lattice-like in HE staining and Nissle staining,and the structural damage of the brain tissues of the rats was significantly improved and the structure was more intact after the treatment with Zhilong Huoxue Tongyu capsule.Nissle staining showed a statistically significant increase in the number of neuronal cell death in the Ich group and Ns group compared with the Sham group(P <0.05);the number of neuronal cell death in the rats decreased significantly after treatment with Zhilong Huoxue Tongyu capsule compared with the Ns group,and there was a statistically significant difference in the Zlh group(P <0.05).The area of iron deposition around the hematoma in the Ich group and Ns group increased significantly compared with the Sham group by Persl stain,and there was a statistical difference(P <0.05);the area of iron deposition around the hematoma in rats decreased significantly after treatment with Zhilong Huoxue Tongyu capsule compared with the Ns group and there was a statistical difference(P<0.05)in the Zlh group.For the detection of lipid peroxidation level,compared with the Sham group,the cortical MDA content in rats decreased significantly and the SOD and GSH content increased significantly after treatment with Zhilong Huoxue Tongyu capsule compared with the Ns group,with statistical differences(P <0.05).2.3Mechanisms of Zhi Long Huo Xue Tong Yu capsule inhibition on ferroptosis in rats after ICH2.3.1 Zhi Long Huo Xue Tong Yu capsule improves iron deposition in rats after ICH by regulating TFR1 and SLC40A1Compared with the Sham group,the expression levels of TFR1 m RNA brain tissues of the Ich group and Ns group increased significantly and the expression levels of SLC40A1 m RNA decreased significantly in rats after ICH with statistical differences(P <0.05).After treatment with Zhilong Huoxue Tongyu capsule,the expression levels of TFR1 m RNA decreased significantly and the expression levels of SLC40A1 m RNA increased significantly in rats compared with the Ns group with statistical differences(P <0.05).2.3.2 Zhilong Huoxue Tongyu capsule regulates the expression of TP53 and lipid peroxidation related targets of ferroptosis in rats after ICHCompared with the Sham group,the expressions of SLC7A11,GPX4 and SESN2 m RNA decreased significantly,the protein expression levels of GPX4 and SESN2 decreased significantly,the expressions of NOX4,TP53 m RNA and protein increased significantly in Ich group and Ns group after ICH with statistical difference significantly(P < 0.05).Immunofluorescence results showed that the NOX4 content in rats increased significantly in Ich group and Ns group compared with the Sham group,(P <0.05).After treatment with Zhilong Huoxue Tongyu capsule,the expression levels of SLC7A11,GPX4 and SESN2 m RNA increased significantly,the protein expression levels of GPX4 and SESN2 increased significantly,the expressions of NOX4,TP53 m RNA and protein decreased significantly compared with the Ns group with statistical differences(P <0.05).Immunofluorescence results showed that the NOX4 content in rats decreased significantly compared with the Ns group with statistical differences(P<0.05).It proved that Zhilong Huoxue Tongyu capsule can improve the ability of rats to resist oxidative stress injury by regulating the expression of SESN2 and NOX4,increase the ability of rats to eliminate lipid peroxidation by regulating the expression of GPX4,decrease the expression of TP53 to inhibit ferroptosis,and finally protect rats from brain injury after ICH.[Conclusion]1.The results of network pharmacology showed that the molecular mechanism of the therapeutic effect of Zhilong Huoxue Tongyu capsule on ICH was mainly related to oxidative stress,inflammatory response apoptosis and ion metabolism,and pathways were mainly related to Ferroptosis and IL-17 signaling pathway.Zhilong Huoxue Tongyu capsule can alleviate ICH through muti-ingredients,muti-targets and mutipathways.The key target TP53 is closely related to ICH and ferroptosis,and further studies are needed to figure out the role of TP53.2.Zhilong Huoxue Tongyu capsule can improve the behavioral outcomes of rats after ICH,reduce the behavioral impairment,stabilize the anxiety of rats,and increase the spatial memory and learning function of rats.3.Zhilong Huoxue Tongyu capsule can alleviate cell damage and reduce the area of iron deposition around the hematoma in rats after intracerebral hemorrhage.The mechanism is related to the regulation of TFR1 and SLC40A1 expression by Zhilong Huoxue Tongyu capsule.4.Zhilong Huoxue Tongyu capsule could improve oxidative stress,inhibit lipid peroxidation and reduce the occurrence of ferroptosis in the rats after ICH,and the mechanism was related to the regulation of SESN2/NOX4 pathway and TP53/GPX4 pathway by Zhilong Huoxue Tongyu capsule. |
