| Purpose:The network pharmacology method was used to construct the network of "Chinese medicine composition-target-disease" with Searching wind and expelling phlegm Chinese herbal as the research carrier,and the target was enriched and analyzed to predict the mechanism of Searching wind and expelling phlegm Chinese herbal in treating coronary heart disease.Under the condition of cell culture in vitro,Rat Cardiac microvascular endothelial cells(RCMECs)is taken as the research object.Observing the protective effect of serum containing Searching wind and expelling phlegm Chinese herbal on the injury of RCMECs induced by homocysteine(Hcy)in rats,and to explore the effect of serum containing Searching wind and expelling phlegm Chinese herbal on the inflammatory injury and mitochondrial apoptosis of Cardiac microvascular endothelial cells induced by homocysteine from the perspective of molecular biology.The mechanism of Coronary microvascular dysfunction can provide experimental basis for the application on searching wind and eliminating phlegm methods,and enrich the scientific connotation of wind phlegm theory.Material and method:(1)Study on the mechanism of Searching wind and expelling phlegm Chinese herbal in treating coronary heart disease based on network pharmacology : Through literature search and multiple databases,we searched every component target and disease related target of Searching wind and expelling phlegm Chinese herbal.In order to explain the molecular mechanism of Searching wind and expelling phlegm Chinese herbal,we used the software of Cytoscape3.6.0 to analyze the topology of the network of effective components intersection target points,and further analyzed the go function and the enrichment of KEGG pathway.(2)Study on the dose effect relationship of serum containing Searching wind and expelling phlegm Chinese herbal on Hcy induced injury of myocardial microvascular endothelial cells: Forty SPF Sprague-Dawley rats were divided into groups,and different doses of normal saline and Searching wind and expelling phlegm Chinese herbal were administered to the stomach to prepare drug-containing serum.The purchased RCMECs were subcultured,frozen,and resuscitated.The morphology of RCMECs was observed under an inverted microscope.Cells were identified by CD31 immunofluorescence method,and the positive rate was more than 95%.They were divided into blank control group,model group and low,medium and high concentration group of Searching wind and expelling phlegm Chinese herbal.Except blank control group,other groups intervened with homocysteine on RCMECs for24 hours.The relevant indexes were detected after the cells were collected.The morphology of RCMECs was detected by MTT method.The levels of ET-1,IL-6,TNF-α,IL-8 were detected by ELISA.Western blot was used to detect the expression of Bax,Bcl-2,Cyto-c,caspase 9,caspase 3,e NOS proteins in each group.(3)The effect of serum containing Searching wind and expelling phlegm Chinese herbal on the PI3K/Akt signaling pathway in Hcy injured cardiac microvascular endothelial cells was studied : RCMECs were divided into blank control group,model control group,LY294002(PI3K specific inhibitor)group and Searching wind and expelling phlegm Chinese herbal + LY294002 group.Among them,model control group,LY294002 group and Searching wind and expelling phlegm Chinese herbal +LY294002 group were induced in RCMECs injury with Hcy,then LY294002 group and Searching wind and expelling phlegm Chinese herbal+ LY294002 group were used with 20μmol/L LY294002,20μmol/L LY294002 + middle dose of Searching wind and expelling phlegm Chinese herbal;They were cultured with cells for 24 hours.The relevant indexes were detected after the cells were collected.Apoptosis was detected by TUNEL method.Number of ROS positive cells detected by active oxygen kit.The expression of PI3 K P85,Akt m RNA was detected by RT-PCR.Western blot was used to detect the expression of PI3 K P85,P-PI3 K P85,Akt,P-Akt in each group.(4)The regulatory effect of serum containing Searching wind and expelling phlegm Chinese herbal on IKK/NF-k B signaling pathway in Hcy injured cardiac microvascular endothelial cells was studied:RCMECs were divided into blank control group,model control group,IMD0354(IKK specific inhibitor)group and Searching wind and expelling phlegm Chinese herbal + IMD0354 group.Among them,model control group,IMD0354 group and Searching wind and expelling phlegm Chinese herbal + IMD0354 group were induced in RCMECs injury with Hcy,then IMD0354 group and Searching wind and expelling phlegm Chinese herbal+ IMD0354 group were used with 0.5μmol/L IMD0354,0.5μmol/L IMD0354 + middle dose of Searching wind and expelling phlegm Chinese herbal;They were cultured with cells for24 hours.The relevant indexes were detected after the cells were collected.Western blot was used to detect the expression of NF-k B p65,P-NF-k B p65,Ik B-α,P-Ik B-α,IL-6 and IL-8 in each group.The expression of P-NF-k B p65 protein was detected by immunofluorescence.Results:1 Internet pharmacology In this study 583 compounds,777 component targets,1229 disease targets and 313 intersection targets of Searching wind and expelling phlegm Chinese herbal were collected,and a component target network was constructed.Through the network analysis by the software of Cytoscape,it was found that these compounds have anti-inflammatory.Further pathway enrichment analysis showed that the biological processes of these target genes were mainly related to apoptosis and inflammation.PI3K/Akt signal pathway,NF-k B signal pathway are often involved in the pathway.PI3 K,Akt,Bcl-2,Ik B-α,NF-k B and e NOS are included.2 Morphology and identification of RCMECs in rats When the purchased RCMECs were cultured to the 2nd-3rd day,the single microvascular endothelial cells were fusiform,star shaped or polygonal under the inverted microscope in the unfused state.When the tissue block was removed and cultured to the 5th day,the aggregation of cells into paving stones could be seen.The results of CD31 immunofluorescence showed that the purity of RCMECs was more than 95%.3 The effect of serum containing Searching wind and expelling phlegm Chinese herbal on Cell activity in injuried RCMECs induced by Hcy Compared with the blank control group,the OD value of model group weredecreased significantly(P<0.01);Compared with the model group,the OD value of each concentration groups were increased significantly(P<0.01).4 The effect of serum containing Searching wind and expelling phlegm Chinese herbal on the content of ET-1,IL-6,TNF-α,IL-8 in injuried of RCMECs induced by Hcy Compared with the blank control group,the content of ET-1,TNF-α,IL-6 and IL-8 were increased in model group(P<0.01);Compared with the model group,the content of ET-1,TNF-α,IL-6 and IL-8 of each concentration groups were decreased significantly(P<0.01).5 The effect of Searching wind and expelling phlegm Chinese herbal medicine on the expression of Bax,Bcl-2,Cyto-c,Caspase9,Caspase3,e NOS proteins in Hcy induced RCMECs injury5.1 Proteins expression of e NOS and Cyto-c in each groups Compared with the blank control group,the expression of e NOS were decreased significantly and the expression of Cyto-c were increased significantly(P<0.01)in model group;Compared with the model group,the expression of e NOS were increased significantly(P<0.01)in each concentration groups and the expression of Cyto-c were decreased significantly(P<0.01)in the middle and high concentration groups.5.2 Proteins expression of Bax,Bcl-2,Caspase9 and Caspase3 in each groups Compared with the blank control group,the protein expression of Bax,Bcl-2,Caspase9 and Caspase3 were increased significantly in the model group(P<0.01);Compared with the model group,the expression of Caspase9 and Caspase3 proteins were decreased significantly and the expression of Bcl-2 protein were increased significantly(P<0.01)in each concentration groups.The expression of Bax protein was decreased significantly in the middle and high concentration groups(P<0.01).6 Effect of serum containing Searching wind and expelling phlegm Chinese herbal medicine on apoptosis of RCMECs induced by Hcy Only a few TUNEL positive cells were found in the blank control group;Compared with the blank control group,the number of apoptotic cells in model group were increased(P<0.01);Compared with model group,the number of apoptotic cells in LY294002 group and LY294002 + Chinese herbal medicine group were decreased(P<0.01);Compared with LY294002 group,the number of apoptotic cells in LY294002 + Chinese herbal medicine group were decreased(P<0.01).7 Effect of serum containing Searching wind and expelling phlegm Chinese herbal medicine on ROS of RCMECs injury induced by Hcy Only a few ROS positive cells were found in the blank control group;Compared with the blank control group,the number of ROS positive cells in model group were increased significantly(P<0.01);Compared with model group,the number of ROS positive cells in LY294002 group and LY294002 +Chinese herbal medicine group were decreased(P<0.01);Compared with LY294002 group,the number of ROS positive cells in LY294002 +Chinese herbal medicine group were decreased(P<0.01).8 The effect of serum containing Chinese herbal medicine on the expression of PI3 K P85 and Akt m RNA in RCMECs injured by Hcy after adding LY294002 Compared with the blank control group,PI3 K P85 and Akt m RNA expression in model group were decreased(P<0.01);Compared with the model group,PI3 K P85 and Akt m RNA expression in LY294002 group,Chinese herbal medicine+LY294002 group were decreased(P<0.01);Compared with LY294002 group,PI3 K P85 and Akt m RNA expression in Chinese herbal medicine+LY294002 group were increased(P<0.01).9 The effect of serum containing Chinese herbs on the expression of PI3 K P85,P-PI3 K P85,Akt,P-Akt proteins in RCMECs injured by Hcy after adding LY294002 Compared with the blank control group,P-PI3 K P85,P-Akt proteins expression and the ratio of P-PI3K/PI3 K,P-Akt/Akt were decreased in the model group(P<0.01);Compared with the model group,P-PI3 K P85,P-Akt proteins expression and the ratio of P-PI3K/PI3 K,P-Akt/Akt were decreased in the LY294002 and LY294002 +Chinese herbal medicine group(P<0.01);Compared with the LY294002 group,the expression of P-PI3 K P85 and P-Akt proteins were increased,and the ratio of P-PI3K/PI3 K and P-Akt/Akt was increased in LY294002 +Chinese herbal medicine group(P<0.01).10 The effect of serum containing Chinese herbs on the expression of NF-k B p65,P-NF-k B P65,Ik B-α,P-Ik B-α,IL-6 and IL-8 proteins in Hcy injured RCMECs after adding IMD035410.1 Protein expression of P-NF-KB P65、NF-k B P65、P-Ik B-α、Ik B-α in each groups of cells Compared with the blank control group,the proteins expression of P-NF-k B P65、NF-k B P65、P-Ik B-α、Ik B-α in model group were increased(P<0.01);Compared with model group,the protein expression of P-NF-k B P65、P-Ik B-α in IMD0354 group,Chinese herbal medicine+IMD0354 group were decreased(P<0.01);Compared with IMD0354 group,the protein expression of P-NF-k B P65、P-Ik B-α in Chinese herbal medicine + IMD0354 group were decreased(P<0.01).10.2 Protein expression of IL-6 and IL-8 in each groups of cells Compared with the blank control group,the protein expression of IL-6 and IL-8 in model group were increased(P<0.01);Compared with model group,the proteins expression of IL-6 and IL-8 in IMD0354 group,Chinese herbal medicine +IMD0354 group were decreased(P<0.01);Compared with IMD0354 group,the proteins expression of IL-6 and IL-8 in Chinese herbal medicine + IMD0354 group were decreased(P<0.01).11 The effect of serum containing Chinese medicine on nuclear transfer of P-NF-k B P65 protein in RCMECs injured by Hcy after adding IMD0354 The expression of P-NF-k B P65 protein in the blank control group mostly occurred in the cytoplasm,only a few in the nucleus is exited;Compared with the blank control group,the expression of P-NF-k B P65 protein in the model group were increased significantly in the nucleus(P<0.01);IMD0354 group,Chinese herbal medicine + IMD0354 group significantly were inhibited the transfer of P-NF-k B P65 protein to the nucleus,and the expression in the nucleus were significantly less than that in the model group(P<0.01);The expression of P-NF-k B P65 in the nucleus of Chinese herbal medicine + IMD0354 group were less than that of IMD0354 group(P<0.01).Conclusion:1 Based on the network pharmacology,the main active components and potential molecular mechanism of Searching wind and expelling phlegm Chinese herbal medicine in the treatment of coronary heart disease were predicted from the molecular level,mainly involving inflammatory response and cell apoptosis,most of which involve PI3K/Akt signal pathway and NF-k B signal pathway.2 The serum of Searching wind and expelling phlegm Chinese herbal medicine can inhibit the inflammatory injury and apoptosis of RCMECs induced by Hcy,improve the dysfunction of coronary microvascular,and exits dose-response relationships.3 The serum of Searching wind and expelling phlegm Chinese herbal medicine can inhibit the apoptosis of mitochondria,improve the dysfunction of coronary microvascular.The mechanism may be related to the regulation of PI3K/Akt signaling pathway.4 The serum containing traditional Chinese medicine of Searching wind and expelling phlegm Chinese herbal medicine can inhibit the inflammatory reaction,improve the dysfunction of coronary microvascular.The mechanism may be related to the regulation of IKK/ NF-k B signal pathway. |
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