ABSTRACT
Previously, Carthami Flos and Lepidii Semen(CF-LS) drug pair has been proved effective in inhibiting myocardial fibrosis(MF) by blunting the activity of cardiac fibroblasts. The present study explored the underlying mechanism of CF-LS in inhibiting MF by improving the cardiac microenvironment based on network pharmacology combined with experimental verification. Active compounds and potential targets of CF-LS were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the potential targets of MF were obtained from GeneCards, Online Mendelian Inheritance in Man(OMIM), and Pharmacogenetics and Pharmacogenomics Knowledge Base(PharmGKB). The "active component-target-MF" network was constructed and analyzed by Cytoscape 3.8.1. The protein-protein interaction(PPI) network was constructed by STRING. The Gene Ontology(GO) biological process enrichment analysis was performed by CluoGO plug-in. Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analysis was performed by R 4.0.2 and Funrich. Subsequently, the inhibitory effect of CF-LS on MF was investigated based on angiotensin Ⅱ(Ang Ⅱ)-induced MF rats. RT-PCR and ELISA were used to verify the effect of CF-LS on the targets of signaling pathways related to vascular endothelial cells predicted by the network pharmacology. Thirty-one active components and 204 potential targets of CF-LS, 4 671 MF-related targets, and 174 common targets were obtained. The network analysis showed that the key targets of CF-LS against MF included RAC-alpha serine/threonine-protein kinase(AKT1), transcription factor AP-1(JUN), mitogen-activated protein kinase 1(MAPK1), cellular tumor antigen p53(TP53), transcription factor p65(RELA), and mitogen-activated protein kinase 8(MAPK8). Biological processes mainly involved regulation of blood vessel diameter, regulation of blood vessel endothelial cell migration, cell death in response to oxidative stress, etc. Advanced glycation end products(AGE)-receptor for advanced glycation end products(RAGE) signaling pathway, phosphoinositide 3-kinase(PI3 K)-serine/threonine protein kinase(AKT) signaling pathway, hypoxia-inducible factor-1(HIF-1) signaling pathway, integrin signaling pathway, transforming growth factor-β(TGF-β) signaling pathway, etc. were involved in signaling pathway enrichment. Literature retrieval confirmed that some of these signaling pathways were closely related to vascular endothelial cells, including AGE-RAGE, PI3 K-AKT, HIF-1α, p53, the transcription factor activator protein-1(AP-1), integrin, p38 MAPK, and TGF-β. Animal experiments showed that CF-LS inhibited MF induced by Ang Ⅱ in rats by suppressing the expression of RAGE, HIF-1α, integrin β6, and TGF-β1. The inhibitory effect of CF-LS on MF has the characteristics of multiple components, multiple targets, and multiple pathways. CF-LS can inhibit MF by regulating the activity of vascular endothelial cells in the cardiac microenvironment.
Subject(s)
Animals , Rats , Animal Experimentation , Drugs, Chinese Herbal/pharmacology , Endothelial Cells , Fibrosis , Medicine, Chinese Traditional , Molecular Docking Simulation , Network Pharmacology , Phosphatidylinositol 3-Kinases , SemenABSTRACT
Objective:To explore the effect and mechanism of Guizhitang with different proportions of Cinnamomi Ramulus and Paeoniae Alba Radix on overactive cardiac sympathetic nerves in salt-sensitive hypertensive rats. Method:Randomly divide the forty male 6-week-old salt-sensitive hypertensive rats into five groups： the normal control group， the model group， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶2 group，and the Cinnamomi Ramulus and Paeoniae Alba Radix 2∶1 group， each group has 8 animals， the normal control group was fed with low-salt feed， and the remaining four groups were fed with 8% high-salt feed. After 4 weeks of feeding， gastric feeding was started. Give both the normal control group and model group saline and the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group， the 1∶2 group，and the 2∶1 group， were given Guizhitang aqueous solution at 4.0， 5.5 and 5.5 g·kg-1， respectively. Continuous gavage intervention was held for 4 weeks. IITC multi-channel non-invasive sphygmomanometer was used to detect changes of systolic blood pressure before and after treatment in rats. Left ventricular anterior wall end-diastolic thickness （LVAWd） and interventricular septal diastolic thickness （IVSd） were detected by echocardiography. Hematoxylin-eosin（HE）staining and Masson staining were used to observe the myocardial morphological changes of rats in each group， Western blot was used to detect the expression of nerve growth factor （NGF）， growth-associated protein 43 （GAP43） and tyrosine hydroxylase （TH） protein. Result:After 4 weeks of intervention with Guizhitang， compared with the normal control group， the blood pressure， LVAWd and IVSd of the model group were significantly increased， and the expressions of NGF， TH and GAP43 protein were significantly increased （P<0.01）. HE and Masson staining results showed that the model group had myocardial cell edema， a large number of inflammatory cell infiltration， myocardial fiber hyperplasia and disordered arrangement， and a large amount of collagen deposition could be seen in the intercellular substance. Compared with model group， the systolic blood pressure of rats in each Guizhitang group increased slowly， and the expression of NGF， TH and GAP43 protein decreased （P<0.05，P<0.01），the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group has the best effect. The results of echocardiography shows that the 1∶1 Guishao group could reduce LVAWd and IVSd levels （P<0.05，P<0.01）， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶2 group could reduce IVSd level （P<0.05）， there was no statistical difference in LVAWd， there was no statistical difference in LVAWd and IVSd in 2∶1 group. In terms of myocardial morphology， each group of Guizhitang can reduce cell edema and inflammatory cell infiltration， reduce myocardial fiber hyperplasia and collagen deposition， and improve the disorder of myocardial fiber arrangement. Among them， the 1∶1 group has the best effect. Conclusion:Guizhitang can inhibit the overactive activation of cardiac sympathetic nervous system，reduce the extent of myocardial fibrosis， inflammatory infiltration and myocardial hypertrophy， and protect salt-sensitive hypertension rats， whose mechanism may be related to regulating the expression of heart NGF.Among them， the Cinnamomi Ramulus and Paeoniae Alba Radix 1∶1 group is better than the 1∶2 and 2∶1 group in reducing myocardial fibrosis， inflammatory infiltration and myocardial hypertrophy.
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<b>Objective::To observe the effect of Guizhitang with different proportions of Cinnamomi Ramulus and Paeoniae Alba Radix on the expressions of transforming growth factor-<italic>β</italic><sub>1</sub>(TGF-<italic>β</italic><sub>1</sub>)/Smads signaling pathway and interleukin-10(IL-10), IL-6 and tumour necrosis factor-<italic>α</italic>(TNF-<italic>α</italic>)related inflammatory cytokines in salt-sensitive hypertensive rats, in order to explore the mechanism of Guizhitang in improving myocardial fibrosis in salt-sensitive hypertensive rats. <b>Method::Totally 40 male 6-week-old salt-sensitive rats were randomly divided into 5 groups: the normal control group, the model group, the 1∶1(RC/peony)Guishao group, the 1∶2 Guishao group, and the 2∶1 Guishao group, with 8 in each group. The normal control group was fed with normal salt diet, while the other four groups were fed with high-salt diet. After 4 weeks of feeding, the rats were given intragastric administration, the normal control group and the model group were given the same amount of normal saline, and the 1∶1 Guishao group, the 1∶2 Guishao group and the 2∶1 Guishao group were given 4.0, 5.5, 5.5 g·kg<sup>-1</sup> of Guizhitang by gavage for 4 weeks. Blood pressure was measured once a week, left ventricular end systolic diameter(LVESD), left ventricular end diastolic diameter (LVEDD), left ventricular ejection fraction (LVEF) and left ventricular short axis shortening fraction (LVFS) were detected by using echocardiogram. The pathological changes of myocardial morphology were observed by htoxylin eosin(HE)and Masson staining. The expressions of type Ⅰ and Ⅲ collagen in myocardial tissue of each group was detected by immunohistochemistry. The mRNA expression levels of IL-10, IL-6 and TNF-<italic>α</italic> in myocardial tissue of each group were detected by quantitative real-time fluorescence polymerase chain reaction(Real-time PCR). The protein expression levels of TGF-<italic>β</italic><sub>1</sub>, <italic>α</italic>-smooth muscle actin(<italic>α</italic>-SMA), Smad2, Smad3 and Smad7 in myocardial tissue of each group were detected by Western blot. <b>Result::Compared with the normal control group, the blood pressure was increased in the model group at 8-15 weeks, LVESD, LVEDD were increased in the model group, while LVFS, LVEF were decreased in the model group. The collagen volume fraction was increased, immunohistochemistry showed the expression levels of type Ⅰ and Ⅲ collagen were increased, mRNA expression levels of IL-10, IL-6 and TNF-<italic>α</italic> were increased, the protein expression levels of TGF-<italic>β</italic><sub>1</sub>, Smad2, Smad3 and <italic>α</italic>-SMA were increased, whereas the protein expression of Smad7 was decreased (<italic>P</italic><0.01). Compared with the model group, the blood pressure rise of each group of Guizhitang was delayed in 12-15 weeks, LVESD and LVEDD were decreased in Guizhitang group (<italic>P</italic><0.01), LVFS, LVEF were increased in Guizhitang group (<italic>P</italic><0.01), the collagen volume fraction was decreased in Guizhitang group (<italic>P</italic><0.01), and the expressions of type Ⅰ and Ⅲ collagen were decreased in Guizhitang group (<italic>P</italic><0.01). At the same time, the mRNA expression of IL-10 was increased in Guizhitang group (<italic>P</italic><0.05, <italic>P</italic><0.01), the mRNA expressions of IL-6 and TNF-<italic>α</italic> were decreased in Guizhitang group (<italic>P</italic><0.01), and the protein expressions of TGF-<italic>β</italic><sub>1</sub>, Smad2, Smad3 and <italic>α</italic>-SMA were decreased in Guizhitang group (<italic>P</italic><0.05, <italic>P</italic><0.01), and the protein expression of Smad7 was increased in Guizhitang group (<italic>P</italic><0.01). Compared with the 2∶1 Guishao group, the effect of the 1∶1 Guishao group in improving the above indicators was more obvious (<italic>P</italic><0.05, <italic>P</italic><0.01). <b>Conclusion::Guizhitang with different proportions of Ramulus Cinnamomi and Poeny can alleviate the degree of myocardial fibrosis in salt-sensitive hypertensive rats. The mechanism may be related to the regulation of TGF-<italic>β</italic><sub>1</sub>/Smads signaling pathway and the reduction of inflammatory response. Besides, the 1∶1 Guishao group showed the optimal effect in reducing inflammation and improving myocardial fibrosis.
ABSTRACT
<p><b>OBJECTIVE</b>To observe the preventive effect of different compatibilities of Ramulus Cinnamomi (RC) and Radix Paeomiae alba (RPA) in Guizhi Decoction (GZD) on neurotransmitters and their rate-limiting enzymes, and neurotrophic factors of cardiac sympathetic denervation model rats induced by 6-hydroxydopamine (6-OHDA).</p><p><b>METHODS</b>Totally 54 male Wistar rats were randomly divided into 6 groups, i.e., the blank control group, the model group, the methycobal group, the 2:1 (RC/RPA) Guishao group, the 1:2 Guishao group, and the 1:1 Guishao group, 9 in each group. Sympathetic denervation was induced by intraperitoneal injection of 6-OHDA for three successive days. Rats in the methycobal group and GZD groups were administered with corresponding decoction by gastrogavage 1 week before modeling (methycobal at the daily dose 0.15 mg/kg; GZD at the daily dose of 4.0, 5.5, 5.5 g crude drugs/kg for GZD 1:1, 1:2, and 2:1 groups). All medication lasted for 10 successive days. Levels of norepinephrine (NE), tyrosine hydroxylase (TH), choline acetyl-transferase (ChAT), nerve growth factor (NGF), growth associated protein43 (GAP-43) and ciliary neurotrophic factor (CNTF) in myocar- dial homogenates of right atrium and ventricular septum were detected by ELISA.</p><p><b>RESULTS</b>Compared with the blank control group, levels of NE, TH, TH/ChAT ratio, and GAP-43 in myocardial homogenates of right atrium and ventricular septum decreased in the model group, and level of NGF increased (P < 0.01, P < 0.05). Compared with the model group, levels of NE and GAP-43 increased in the right atrium and interventricular septum; NGF level of the ventricular septum decreased in the methycobal group and each GZD groups. TH and TH/ChAT ratio in the right atrium increased in the 2:1 Guishao group and the 1:2 Guishao group (P < 0.01, P < 0.05); NGF levels in the right atrium and interventricular septum decreased only in the 1:1 Guishao group (P < 0.01, P< 0.05). Compared with the methycobal group, levels of NE, TH, and GAP-43 in the right atrium and interventricular septum increased, and NGF levels in the right atrium and interventricular septum decreased in the 1:1 Guishao group (P < 0.05). Compared with the methycobal group, levels of NE and GAP-43 in interventricular septum increased in the 2:1 Guishao group (P < 0.05).</p><p><b>CONCLUSION</b>GZD (with the proportion between RC and RPA 2:1 and 1:1) could improve contents of neurotransmitters and their rate-limiting enzymes, as well as neurotrophic factors in cardiac sympathetic denervation model rats induced by 6-OHDA, alleviate cardiac sympathetic denervation induced by 6-OHDA, and maintain the balance of sympathetic-vagal nerve system.</p>
Subject(s)
Animals , Male , Rats , Choline O-Acetyltransferase , Metabolism , Ciliary Neurotrophic Factor , Metabolism , Drugs, Chinese Herbal , Pharmacology , GAP-43 Protein , Metabolism , Heart , Myocardium , Metabolism , Nerve Growth Factor , Metabolism , Norepinephrine , Metabolism , Oxidopamine , Random Allocation , Rats, Wistar , Sympathectomy , Tyrosine 3-Monooxygenase , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To observe the preventive effect different compatibilities of Ramulus Cinnamomi (RC) and peony in Guizhi Decoction (GD) on diabetic cardiac autonomic neuropathy (DCAN).</p><p><b>METHODS</b>Totally 60 male rats were randomly divided into 5 groups, i.e., the blank control DM group, the model group, the methycobal group, the 1:1 (RC/peony) Guishao group, the 2:1 Guishao group, and the 1:2 Guishao group, 10 in each group. Rats were pretreated with corresponding drugs for 1 week, and then induced diabetes by intraperitoneal injection of STZ. Drugs were administrated by gastrogavage for 4 more weeks after STZ-injection. Enzyme-linked immunosorbent assay (ELISA) was employed to detect levels of tyrosine hydroxylase (TH), choline acetyltransferase (CHAT), nerve growth factor. (NGF), and ciliary neurotrophic factor (CNTF) in myocardial homogenates.</p><p><b>RESULTS</b>After 4-week modeling, body weight (BW) was obviously lower, but blood glucose (BG) was higher in STZ rats than in rats of the blank control DM group. There was no statistical difference in BW or BG among the 5 groups (P >0.05). Compared with the blank control group, TH, TH/CHAT, and NGF in left ventricle and ventricular septum increased, CHAT and CNTF increased in the model group (P < 0.05, P < 0.01). Compared with the model group, TH and TH/CHAT in left ventricle decreased (P < 0.05, P < 0.01), CNTF in left ventricle increased (P < 0.05), CHAT in left ventricle and ventricular septum increased (P < 0.05, P < 0.01) in the methycobal group. TH and TH/CHAT in left ventricle and ventricular septum decreased, CNTF in left ventricle and ventricular septum increased (P < 0.05, P < 0.01), CHAT in left ventricle and ventricular septum increased (P < 0.01), NGF in ventricular septum decreased (P < 0.01) in the 1:1 Guishao group. TH/CHAT in left ventricle decreased (P < 0.01), CHAT and CNTF in left ventricle and ventricular septum increased (P < 0.05, P < 0.01) in the 1:2 Guishao group. Compared with the methycobal group, CHAT in left ventricle decreased, TH and TH/CHAT in left ventricle increased in the 2:1 Guishao group (P < 0.05, P < 0.01). TH and TH/CHAT in ventricular septum decreased (P < 0.05), CHAT and CNTF in left ventricle and ventricular septum increased (P < 0.05, P < 0.01) in the 1:1 Guishao group. Compared with the 1:2 Guishao group and the 2:1 Guishao group, CHAT in left ventricle increased, TH/CHAT in left ventricle decreased, TH and TH/CHAT in ventricular septum decreased, CHAT in ventricular septum increased, CNTF in left ventricle and ventricular septum also increased in the 1:1 Guishao group (all P < 0.01).</p><p><b>CONCLUSIONS</b>STZ model rats had autonomic neural injury, manifested as lowered vagal nerve activity and hyperactive sympathetic nerves. GD could effectively suppress hyperactive cardiac sympathetic nerves and protect the vagus. Besides, GD (1:1) showed the optimal effect in regulating the balance of cardiac autonomic nerves and could be used in early prevention of DCAN.</p>
Subject(s)
Animals , Male , Rats , Blood Glucose , Choline O-Acetyltransferase , Diabetic Neuropathies , Drug Therapy , Drugs, Chinese Herbal , Therapeutic Uses , Heart , Heart Ventricles , Myocardium , Nerve Growth Factor , Paeonia , Tyrosine 3-MonooxygenaseABSTRACT
<p><b>OBJECTIVE</b>To observe abnormalities in heart rate variability (HRV) in diabetic rats and to explore the effects of treatment with Guizhi Decoction ([symbols; see text]) on cardiac autonomic nervous (CAN) imbalance.</p><p><b>METHODS</b>A radio-telemetry system for monitoring physiological parameters was implanted into rats to record electrocardiac signals and all indictors of HRV [time domain measures: standard deviation of all RR intervals in 24 h (SDNN), root mean square of successive differences (RMSSD), percentage of differences between adjacent RR intervals greater than 50 ms (PNN50), and standard deviation of the averages of RR intervals (SDANN); frequency domain measures: low frequency (LF), high frequency (HF), total power (TP), and LF/HF ratio]. The normal group was randomly selected, and the remaining rats were used to establish streptozocin (STZ)-induced diabetic model. After 4 weeks, the model rats were divided into the model group, the methycobal group, and the Guizhi Decoction group, 9 rats in each group. Four weeks after intragastric administration of the corresponding drugs, the right atria of the rats were collected for immunohistochemical staining of tyrosine hydroxylase (TH) and choline acetyltransferase (CHAT) to observe the distribution of the sympathetic and vagus nerves in the right atrium. The myocardial homogenate from the interventricular septum and the left ventricle was used for determination of TH, CHAT, growth-associated protein 43 (GAP-43), nerve growth factor (NGF), and ciliary neurotrophic factor (CNTF) levels using an enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>(1) STZ rats had elevated blood glucose levels, reduced body weight, and decreased heart rate; there was no difference between the model group and the drug treated groups. (2) Compared with the model group, only RMSSD and TP increased in the methycobal group significantly (P<0.05); SDNN, RMSSD, PNN50, LF, HF, and TP increased, LF/HF decreased (P<0.05), and SDANN just showed a decreasing trend in the Guizhi Decoction group (P>0.05). TH increased, CHAT decreased, and TH/CHAT increased in the myocardial homogenate of the model group (P<0.05). Compared with the model group, left ventricular TH reduced in the methycobal group; and in the Guizhi Decoction group CHAT increased, while TH and TH/CHAT decreased (P<0.05). Compared with the model group, CNTF in the interventricular septum increased in the methycobal group (P<0.05); GAP-43 increased, NGF decreased, and CNTF increased (P<0.05) in the Guizhi Decoction group. There were significant differences in the reduction of NGF and elevation of CNTF between the Guizhi Decoction group and the methycobal group (P<0.05). (3) Immunohistochemical results showed that TH expression significantly increased and CHAT expression significantly decreased in the myocardia of the model group, whereas TH expression decreased and CHAT expression increased in the Guizhi Decoction group (P<0.05).</p><p><b>CONCLUSION</b>Guizhi Decoction was effective in improving the function of the vagus nerve, and it could alleviate autonomic nerve damage.</p>