Study On The Mechanism Of DAPK1 Involved In Qingda Granule Regulating Hypertension

Objective:The current study was to investigate the effect and underlying mechanisms of Qingda Granules（QDG）on hypertension,vascular function and structure;To explore the role of DAPK1 in hypertension,vascular function and structure,as well as the activation of downstream myosin light chain（MLC）pathway,and clarify its regulation mechanism on hypertension;To explore the role of rhynchophylline in hypertension,vascular function and structure,as well as the regulation of DAPK1/p-MLC pathway;To further enrich the underling mechanism and material basis of QDG on anti-hypertension,which might provide experimental basis for clinical application of QDG and further research.Methods:1.The effect of QDG on hypertension and its underlying mechanisms in spontaneously hypertensive rats（SHRs）:（1）SHRs were intragastrically administrated with QDG for 10 weeks;（2）A noninvasive blood pressure system,ultrasound,hematoxylin and eosin staining were performed to determine the blood pressure,vascular function,and pathological changes in abdominal aortas of SHRs,respectively;（3）RNA sequencing was used to identify the differentially expressed transcripts and enrichment pathway in the abdominal aorta of SHRs after QDG treatment;（4）Immunohistochemistry was used to verify the differentially expressed transcripts and activation of associated signaling pathways in abdominal aortas of SHRs.2.The effect of DAPK1 knockdown on hypertension and its underlying mechanisms in SHRs:（1）DAPK1-knockdown adeno-associated virus was injected into the tail vein of SHRs to establish the DAPK1-knockdown SHRs model;（2）Small animal imaging system was used to detect the expression of virus;（3）A noninvasive blood pressure system,ultrasound,hematoxylin and eosin staining were performed to determine the blood pressure,vascular function,and pathological changes in abdominal aortas of SHRs,respectively;（4）Immunohistochemistry was used to verify the effect of DAPK1 knockdown on the expression of DAPK1 and the activation of MLC pathway.3.The effects of DAPK1 knockout on hypertension and its underlying mechanisms in Ang Ⅱ infused mice:（1）Confocal laser scanning was used to detect the Ca²⁺release in Ang Ⅱ stimulated VSMCs;（2）QPCR and Western blotting were used to analyze the effect of Ang Ⅱ on the m RNA and protein expression of DAPK1,the phosphorylation of p-DAPK1（S308）and the activation of MLC pathway,respectively;（3）Immunohistochemistry was used to verify the expression of DAPK1,p-DAPK1（S308）in abdominal aorta of Ang Ⅱ infused mice;（4）DAPK1^-/-mice were generated,the DAPK1^+/+and DAPK1^-/-mice were infused with Ang Ⅱ;（5）A noninvasive blood pressure system,ultrasound,hematoxylin and eosin staining were performed to determine the blood pressure,vascular function,and pathological changes in Ang Ⅱ infused mice,respectively;（6）RNA sequencing was used to detect the differential gene expression and enriched pathways in the abdominal aorta of Ang Ⅱ infused mice after DAPK1knockout.（7）Vascular tensiometer was used to detect the effect of DAPK1 knockout on the contraction of abdominal aortic ring of mice-stimulated with Ang Ⅱ;（8）Immunohistochemistry was used to verify the effect of DAPK1 knockout on MLC pathway activation in abdominal aorta of Ang Ⅱ infused mice.4.The effects of DAPK1 inhibitor（TC-DAPK6）on hypertension and its underlying mechanisms in Ang Ⅱ infused mice:（1）C57BL/6 mice were infused with Ang Ⅱ,and intragastrically administrated with TC-DAPK6 daily（1μg/kg/day,and 10μg/kg/day,respectively）;（2）A noninvasive blood pressure system,ultrasound,hematoxylin and eosin staining were performed to determine the blood pressure,vascular function,and pathological changes in Ang Ⅱ infused mice,respectively;（3）Immunohistochemistry was used to verify the effect of TC-DAPK6 on the expression of DAPK1,p-DAPK1（S308）and the activation of MLC pathway in abdominal aorta of Ang Ⅱ infused mice;（4）Confocal laser scanning microscopy was used to detect the effect of TC-DAPK6 on Ang Ⅱ-induced cytoskeleton（5）Western blotting was used to analyze the effect of TC-DAPK6 on the expression of DAPK1,p-DAPK1（S308）,and the activation of MLC pathway in Ang Ⅱ stimulated VSMCs.5.The effects of rhynchophylline on Ang Ⅱ induced hypertension and its underlying mechanisms:（1）Molecular docking analysis of the binding between the main components of QDG and DAPK1;（2）C57BL/6 mice were infused with Ang Ⅱ,and intragastrically administrated with rhynchophylline daily;（3）Immunohistochemistry was used to verify the effect of rhunchophylline on the expression of DAPK1,p-DAPK1（S308）and the activation of MLC pathway in abdominal aorta of Ang Ⅱ infused mice;（4）A noninvasive blood pressure system,ultrasound,hematoxylin and eosin staining were performed to determine the blood pressure,vascular function,and pathological changes in Ang Ⅱ infused mice,respectively.Results:1.The effect of QDG on hypertension and its underlying mechanisms in SHRs:（1）QDG treatment significantly reduced systolic blood pressure,diastolic blood pressure and mean arterial pressure of SHRs,but had no significant effect on body weight.（2）QDG treatment significantly reduced the pulse wave propagation velocity and the thickness of abdominal aorta of SHRs;（3）RNA sequencing showed that QDG could reverse the expression of 738differentially expressed transcripts including DAPK1 in the abdominal aorta,which were significantly enriched in the vascular smooth muscle contraction pathway and calcium ion signaling pathway.（4）QDG treatment significantly reduced the expression of DAPK1 protein,promote the phosphorylation of p-DAPK1（S308）and inhibit the activation of MLC pathway in abdominal aorta of SHRs.2.The effect of DAPK1 knockdown on hypertension and its underlying mechanisms in SHRs:（1）DAPK1 knockdown SHRs model were constructed successfully;（2）DAPK1 knockdown adeno-associated virus still expressed after 10 weeks;（3）DAPK1 knockdown significantly reduced systolic blood pressure,diastolic blood pressure and mean arterial pressure in SHRs,but had no significant effect on body weight.（4）DAPK1 knockdown significantly reduced the pulse wave propagation velocity and the thickness of abdominal aorta of SHRs.（5）DAPK1knockdown significantly inhibited the expression of DAPK1 protein and the activation of MLC pathway in abdominal aorta of SHRs.3.The effects of DAPK1 knockout on hypertension and its underlying mechanisms in Ang Ⅱ infused mice:（1）Ang Ⅱ stimulation increased the concentration of calcium ion in vascular smooth muscle cells;（2）Ang Ⅱ stimulation had no effect on the m RNA expression of DAPK1in vascular smooth muscle cells;（3）Ang Ⅱ stimulation had no effect on DAPK1 protein in vascular smooth muscle cells,but inhibited p-DAPK1（S308）phosphorylation,and promotes the activation of MLC pathway in VSMCs;（4）Ang Ⅱ induction had no effect on the expression of DAPK1 protein in the abdominal aorta of mice,but significantly inhibited the phosphorylation of p-DAPK1（S308）;（5）DAPK1 knockout transgenic hypertensive mice model were constructed successfully;（6）DAPK1 knockout significantly reduced systolic blood pressure,diastolic blood pressure and mean arterial pressure,but had no significant effect on body weight in Ang Ⅱ infused mice;（7）DAPK1 knockout significantly reduced the pulse wave propagation velocity and the thickness in abdominal aorta of Ang Ⅱ infused mice;（8）RNA sequencing revealed that DAPK1 knockout reversed the expression of 539 genes in abdominal aorta of Ang Ⅱ infused mice,which were significantly enriched in vascular smooth muscle contraction,calcium signaling and cytoskeleton regulatory signaling pathways;（9）DAPK1knockout inhibited Ang Ⅱ-stimulated contraction of abdominal aortic ring in mice;（10）DAPK1knockout significantly reduced the activation of MLC pathway in abdominal aorta of Ang Ⅱ infused mice.4.The effects of DAPK1 inhibitor（TC-DAPK6）on hypertension and its underlying mechanisms in Ang Ⅱ infused mice:（1）The hypertensive mouse models with DAPK1inhibition were constructed successfully;（2）TC-DAPK6 significantly decreased systolic blood pressure,diastolic blood pressure and mean arterial pressure in Ang Ⅱ infused mice,but had no significant effect on body weight;（3）TC-DAPK6 significantly reduced the pulse wave propagation velocity and the thickness in abdominal aorta of Ang Ⅱ infused mice.（4）TC-DAPK6 had no effect on the expression of DAPK1 protein in abdominal aorta of Ang Ⅱ infused mice,but significantly promoted the phosphorylation of p-DAPK1（S308）;（5）TC-DAPK6significantly inhibited the activation of MLC pathway in abdominal aorta of Ang Ⅱ infused mice;（6）TC-DAPK6 significantly reduced the number of myofilaments of VSMCs cytoskeleton induced by Ang Ⅱ;（7）TC-DAPK6 had no effect on the expression of DAPK1 in VSMCs induced by Ang Ⅱ,but promoted the phosphorylation of p-DAPK1（S308）;（8）TC-DAPK6 significantly inhibited Ang Ⅱ-induced activation of MLC pathway in VSMCs.5.The effects of rhynchophylline on Ang Ⅱ induced hypertension and its underlying mechanisms:（1）DAPK1 combine with various main components of QDG,and has a high affinity with rhynchophylline;（2）Rhynchophylline had no effect on the expression of DAPK1protein in abdominal aorta of Ang Ⅱ infused mice,but significantly promoted the phosphorylation of p-DAPK1（S308）;（3）Rhynchophylline significantly inhibited the activation of MLC pathway in abdominal aorta of Ang Ⅱ infused mice;（4）Rhynchophylline significantly decreased systolic blood pressure,diastolic blood pressure and mean arterial pressure of Ang Ⅱ-induced hypertensive mice;（5）Rhynchophylline significantly reduced the pulse wave propagation velocity and the thickness in abdominal aorta of Ang Ⅱ infused mice.Conclusion:The DAPK1/p-MLC（S19）pathway plays an important role in the regulation of hypertension,and one of the important mechanisms of QDG and rhynchophylline in prevention and treatment of hypertension is through the regulation of DAPK1/p-MLC（S19）pathway.This study not only enrich the molecular mechanism of hypertension pathogenesis,but also provide potential new targets for its clinical treatment.Meanwhile,it also elucidate the mechanism of action of QDG and rhynchophylline in preventing and treating hypertension,and provide further experimental basis for its clinical application.