| Objective: To explore the potential key components,targets and pathways of Qingda granule in alleviating hypertension-induced renal damage by RNA sequencing and network pharmacological analysis,as well as experimental verification of in vitro and in vivo.To further clarify the molecular mechanism and material basis of Qingda granule in regulating hypertension and protecting kidney damage,and provide further theoretical and experimental basis for the clinical application of Qingda granule.Method:1.Research on the effect of Qingda granules in preventing hypertension and attenuating kidney damage: 8-week-old C57BL/6 mice were selected and divided into Control group,Ang II group,Ang II + QDG group(n=12).The control group were implanted with saline osmotic pump subcutaneously,and given saline gavage.Ang II group and Ang II + QDG group were implanted with Ang II osmotic pump(500ng/kg/min)for 4 weeks,and normal saline and Qingda granules were given gavage,respectively.A non-invasive blood pressure detector for mice was used to observe the effect of Qingda granules on blood pressure.The pathological changes of kidney tissues were observed by HE staining.TUNEL staining was applied to detect cell apoptosis in kidney tissues of mice.Masson staining were used to detect the kidney fibrosis of mice.2.Research on potential key targets and pathways of Qingda granules in alleviating hypertension-induced renal damage based on network pharmacology and renal gene sequencing:the network pharmacology was applied to construct a multi-level network of drug components-targets-genes-signaling pathways of Qingda granules for the treatment of hypertension.RNA sequencing was used to detect the differential gene expression of Qingda granules in hypertensive kidney tissues.The sequencing results were intersected with the results of network pharmacology of Qingda granules.The common genes of Qingda granules were analyzed by GO and KEGG enrichment analysis to find potential key targets and pathways of Qingda granules in preventing hypertensive kidney damage.3.Research on verifying the key targets and pathways of Qingda granules in the prevention of hypertensive kidney damage:1)In vivo experiment: The contents of SOD and MDA in kidney tissues of mice were detected by colorimetry;The expression of NOX1 in kidney tissues of mice was detected by Western Blot;The m RNA levels of TNF-α,IL-6 and IL-1βin the kidney tissues of mice were detected by q-PCR;immunohistochemistry was used to detect the expression of NF-k B signaling pathway-related proteins in kidney tissues of mice;The expressions of apoptosisrelated proteins Cleaved caspase-3,Cleaved caspase-9,Bax,Bcl-2 and p53 were detected by Western-blot analysis.2)In vivo experiment:A cell model of hypertensive renal injury was established by stimulating NRK-52 E renal tubular epithelial cells with Ang II;CCK8 was used to detect the cell viability of NRK-52 E cells;ROS probe was used to detect ROS levels in NRK-52 E cells;Colorimetry was used to detect the contents of MDA and SOD in NRK-52 E cells;The m RNA levels of IL-6,TNF-α and IL-1β in NRK-52 E cells were determined by q-PCR;The secretion levels of IL-6,TNF-α and IL-1β in the supernatant of NRK-52 E cells were determined by ELISA;Apoptosis of NRK-52 E cells was observed by TUNEL and Annexin V/PI staining.;The expressions of NOX1 protein,apoptosis-related protein,NF-k B signaling pathwayrelated protein and p53 protein in NRK-52 E cells were detected by Western-blot.Results:1.Qingda granules have the effect of lowering blood pressure and reducing kidney injury.1)Compared with Control group,systolic blood pressure,diastolic blood pressure and mean arterial pressure in AngⅡ group were significantly increased(P < 0.05).Compared with AngⅡ group,systolic blood pressure,diastolic blood pressure and mean arterial pressure in AngⅡ+QDG group were significantly decreased(P < 0.05).There was no significant difference in body weight among all groups(P > 0.05).2)Compared with Control group,glomerular atrophy,basement membrane thickening,renal tubule arrangement disordered,interstitial inflammatory cell infiltration increased,and renal index increased significantly in AngⅡ group(P < 0.05);Compared with AngⅡ group,the pathological morphology of kidney in Ang Ⅱ +QDG group was significantly improved,and the renal index was significantly decreased(P < 0.05).3)Compared with Control group,renal fibrosis and collagen volume fraction in AngⅡgroup were significantly increased(P < 0.05);Compared with AngⅡ group,the renal fibrosis and collagen volume fraction in Ang Ⅱ +QDG group were significantly decreased(P < 0.05).4)Compared with Control group,the apoptosis of kidney cells in AngⅡ group was significantly increased(P < 0.05).Compared with AngⅡ group,the apoptosis of kidney cells in AngⅡ+QDG group was significantly decreased(P <0.05)..2.To explore the potential key components,targets and signaling pathways of Qingda granules in alleviating hypertensive renal damage by RNA sequencing and network pharmacology.1)The results of Network pharmacological analysis showed that quercetin,baicalein,luteolin,etc.were the key active ingredients of Qingda granules in the treatment of hypertension.2)The results of renal RNA sequencing showed that 1,427 genes were up-regulated and 1,173 were down-regulated in the kidney tissues of Ang II group.After the intervention of Qingda granules,261 down-regulated genes and 401up-regulated genes could be reversed;3)GO and KEGG enrichment analysis showed that multiple biological processes,including oxidative stress,inflammatory response and apoptosis,and multiple signaling pathways,including NF-k B and p53 signaling pathways,were enriched.3.Regulatory effect of Qingda granules on oxidative stress,inflammatory response and apoptosis of kidney(1)Qingda granules alleviated oxidative stress in hypertensive renal injury: the results of in vivo experiment showed as follows: compared with Control group,NOX1 protein expression,the level of MDA and ROS in kidney tissue of AngⅡ group were significantly up-regulated(P < 0.05),while SOD level was significantly downregulated(P < 0.05).Compared with AngⅡ group,NOX1 protein expression,the level of MDA and ROS in kidney tissue of AngⅡ+QDG group were significantly downregulated(P < 0.05),while SOD level was significantly up-regulated(P < 0.05);The results of in vitro experiment showed as follows: Compared with Control group,NOX1 protein expression,the level of MDA and ROS in NRK-52 E cells of AngⅡ group were significantly up-regulated(P < 0.05),while SOD level was significantly downregulated(P < 0.05).Compared with AngⅡ group,NOX1 protein expression,the level of MDA and ROS in NRK-52 E cells of AngⅡ+QDG group were significantly downregulated(P < 0.05),while SOD level was significantly up-regulated(P < 0.05).(2)Qingda granules inhibited the inflammatory response in hypertensive kidney:the results of in vivo experiment showed as follows: Compared with Control group,the m RNA and protein levels of IL-6,TNF-αand IL-1βin kidney tissue of AngⅡ group were significantly increased(P < 0.05).Compared with AngⅡ group,the m RNA and protein levels of IL-6,TNF-α and IL-1β in kidney tissue of AngⅡ+QDG group were significantly decreased(P < 0.05).The results of in vitro experiment showed as follows: Compared with Control group,the m RNA levels of IL-6,TNF-αand IL-1βin NRK-52 E cells of AngⅡ group were significantly increased(P < 0.05),and secretion levels of IL-6,TNF-αand IL-1βin supernatant were also significantly increased(P < 0.05).Compared with AngⅡ group,the m RNA levels of IL-6,TNF-αand IL-1β in NRK-52 E cells of AngⅡ+QDG group were significantly decreased(P< 0.05),and secretion levels of IL-6,TNF-αand IL-1βin supernatant were also significantly decreased(P < 0.05).(3)Qingda Granules inhibited the activation of NF-k B signaling pathway in hypertensive kidney: the results of in vivo experiment showed as follows: Compared with Control group,the expressions of p65,p-P65,p-IKB in kidney tissue of AngⅡgroup were significantly increased(P < 0.05),while the Ik B expression was significantly decreased(P < 0.05).Compared with AngⅡ group,the expression of p65,p-P65,p-IKB in kidney tissue of AngⅡ+QDG group was significantly decreased(P <0.05),while the Ik B expression was significantly increased(P < 0.05).The results of in vitro experiment showed as follows,Compared with Control group,the expression of p65,p-P65,p-IKB in NRK-52 E cells of AngⅡ group was significantly increased(P < 0.05),while the Ik B expression was significantly decreased(P < 0.05).Compared with AngⅡ group,the expression of p65,p-P65,p-IKB in NRK-52 E cells in AngⅡ+QDG group was significantly decreased(P < 0.05),while the Ik B expression was significantly increased(P < 0.05).(4)Qingda Granules inhibited cell apoptosis in hypertensive kidney: the results of in vivo experiment showed as follows: Compared with Control group,the number of cell apoptosis in kidney tissue of AngⅡ group was significantly increased(P < 0.05),the protein expressions of P53,Bax,Cleaved caspase-3 and Cleaved caspase-9 were significantly up-regulated(P < 0.05),and the Bcl-2 expression was significantly downregulated(P < 0.05).Compared with AngⅡ group,the number of cell apoptosis in kidney tissue of AngⅡ+QDG group was significantly decreased(P < 0.05),the protein expressions of P53,Bax,Cleaved caspase-3 and Cleaved caspase-9 were significantly decreased(P < 0.05)and the Bcl-2 expression was significantly up-regulated(P < 0.05).The results of in vitro experiment showed as follows: Compared with Control group,the number of apoptosis of NRK-52 E cells in AngⅡ group was significantly increased(P < 0.05),the protein expressions of P53,Bax,Cleaved caspase-3 and Cleaved caspase-9 were significantly up-regulated(P < 0.05),and the Bcl-2 expression was significantly down-regulated(P < 0.05).Compared with AngⅡ group,the number of apoptosis of NRK-52 E cells in AngⅡ+QDG group was significantly increased(P <0.05),the protein expressions of P53,Bax,Cleaved caspase-3 and Cleaved caspase-9in NRK-52 E cells of AngⅡ+QDG group were significantly down-regulated(P < 0.05),and the protein expression of Bcl-2 was significantly up-regulated(P < 0.05).Conclusion: Qingda granules not only inhibit the increase of blood pressure in AngⅡinduced hypertensive mice,but also alleviate the renal pathological morphology and inhibit renal fibrosis in AngⅡ-induced hypertensive mice.Qingda granules may inhibit oxidative stress,inflammatory response and apoptosis in renal tissues,by which through regulating NF-k B and P53 signaling pathways,and thus exert its antihypertensive and renal protective effects. |
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