Mechanism of Zhenwu Decoction modulating TLR4/NF-κB/HIF-1α loop through miR-451 to delay renal fibrosis in type 2 CRS.

BACKGROUND: Type 2 cardiorenal syndrome (CRS) is a progressive renal insufficiency in patients with chronic heart failure, but its pathophysiology is still unclear. The Chinese medicine Zhenwu Decoction plays an important role in the prevention and treatment of 2-CRS, however, its mechanism of action remains unknown. PURPOSE: The aim of this study was to investigate whether the ameliorative effect of ZWD on 2-CRS renal fibrosis is related to the modulation of miR-451 expression and thus mediating the TLR4/NF-κB/HIF-1α loop. STUDY DESIGN AND METHODS: A type 2 CRS rat model was constructed using ligation of the left anterior descending branch of the coronary artery + 3/4 nephrectomy, and randomly divided into Control, Sham, Model, Captopril, ZWD-L, ZWD-M and ZWD-H groups.After 4 weeks of ZWD intervention, its effects on cardiac and renal functions of type 2 CRS rats were observed by hematuria and cardiac ultrasonography. Changes in kidney tissue morphology were observed by HE, Masson and PASM staining. The protein and mRNA expression of TLR4, NF-κB, HIF-1α and IκBα in kidney tissues were detected by immunohistochemistry and qPCR. Immunofluorescence was used to detect the protein expression of NF-κB and HIF-1α in renal tissues. Western blot and qPCR were used to detect the protein expression of MCP-1, ICAM-1, IL-1ß, IL-6, TGF-ß, α-SMA, FN, Smad2, Smad3, and E-cadherin in renal tissues. PCR was used to detect the protein expression of miR-451mRNA expression level in kidney tissues. RESULTS: In this study, we found that ZWD was able to reduce the expression of Scr, BUN, NT-proBNP, and 24-hour quantitative urine protein, elevate LVEF, FS, CO, and reduce the level of LVIDS in type 2 CRS rats, as well as attenuate renal interstitial fibrosis and improve tubular swelling. In addition, Zhenwu Decoction up-regulated the expression of miR-451 in renal tissues and inhibited the expression of TLR4, NF-κB, and HIF-1α proteins and genes, which in turn inhibited the expression of inflammatory factors and fibrosis-related factors. CONCLUSION: ZWD was able to up-regulate the expression of miR-451 in renal tissues, inhibit the TLR4/NF-κB/HIF-1α response loop, and then inhibit the expression of inflammatory factors and fibrosis-related factors, improve renal fibrosis, and delay the pathological process of type 2 CRS.

Modified Zhenwu Decoction suppresses chronic colitis via targeting macrophage CCR2/Fyn/p38 MAPK signaling axis.

BACKGROUND: Ulcerative colitis (UC) is associated with intestinal macrophage infiltration due to disruption of the mucosal barrier and bacterial invasion. Therefore, it is crucial to identify therapeutic agents capable of attenuating the macrophage-induced inflammatory response to preserve mucosal homeostasis and immune tolerance. The modified Zhenwu decoction (CDD-2103) is a novel herbal formulation developed based on the principles of Traditional Chinese medicine. To date, there are no clinically approved herbal formulations for UC with a well-known mechanism of action on macrophages. PURPOSE: The objective of this study was to systematically investigate the inhibitory effect of the active fraction of CDD-2103 in a mouse model of chronic colitis and delineate the mechanisms underlying its inhibitory action. METHODS: CDD-2103 was extracted into four fractions using organic solvents with increasing polarity. A chronic 49-day dextran sulfate sodium (DSS)-induced colitis mice model, closely resembling human clinical conditions, was used to examine the effect of CDD-2103 on chronic colitis. To confirm the effect of CDD-2103 on macrophages in this chronic colitis model, adoptive macrophage transfer and CCL2 supplementation were conducted. The mechanisms of action of CDD-2103 were further elucidated utilizing bone marrow-derived macrophages (BMDMs). Transcriptome analysis was conducted to gain insights into the underlying mechanism of action of CDD-2103 in BMDMs. RESULTS: Our in vitro and in vivo findings demonstrated that the ethanol-enriched fraction of CDD-2103 exhibited significant anti-inflammatory effects, leading to the suppression of colitis severity. This effect was associated with diminished accumulation of colonic macrophages in the lamina propria of CDD-2103-intervened colitis mice. Specifically, CDD-2103 inhibited CCR2/L2-mediated proinflammatory macrophage infiltration into the colon without affecting macrophage proliferation. Mechanistically, CDD-2103 inhibited Fyn expression-mediated p38 MAPK activation and subsequently suppressed CCR2 expression in BMDMs. CONCLUSIONS: Collectively, our study supports the potential use of CDD-2103 to limit macrophage infiltration, thereby reducing inflammation during UC treatment. CDD-2103 and the components in the ethanolic fraction are promising candidates for the development of novel drugs for UC management. Additionally, our study underscores Fyn-mediated CCR2 expression as a potential therapeutic target for the management of UC.

UPLC-ESI-Q-TOF-MS/MS based metabolomics investigation on chemical constituent consistency of Zhenwu Decoction before and after compatibility.

Zhenwu Decoction (ZWD), a classic formula from Zhang Zhongjing's "Treatise on Typhoid Fever" in the Han Dynasty, consists of five traditional Chinese medicines: Aconiti Lateralis Radix Praeparata (ALRP), Paeoniae Radix Alba, Poria Cocos, Ginger, and Rhizoma Atractylodis Macrocephalae. To evaluate the chemical constituent consistency of ZWD before and after compatibility, an ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry was established to comprehensively study the constituents of ZWD. By normalizing the peak area, the pairwise compatibility of ALRP and the other four medicinal herbs, as well as the compatibility of the entire formula were studied, respectively. Multivariate statistical analysis was used to identify the differences. The processed data were analyzed by principal component analysis and supervised orthogonal partial least squared discriminant analysis, and an S-plot was generated to compare the differences in the chemical composition of the two types of decoction samples. The results showed that during the decoction process of ZWD, a total of seven components were recognized as differential compounds before and after compatibility of ZWD, namely 6-gingerol, zingerone, benzoylhypaconine, hypaconitine, benzoylaconine, paeoniflorin and fuziline. The results of this study provide basic data reference for understanding the law of ZWD compatibility and are valuable for the compatibility study of other herbal medicines.

System pharmacology-based determination of the functional components and mechanisms in chronic heart failure treatment: an example of Zhenwu decoction.

Chronic heart failure (CHF) is the primary cause of death among patients with cardiovascular diseases, representing the advanced stage in the development of several cardiovascular conditions. Zhenwu decoction (ZWD) has gained widespread recognition as an efficacious remedy for CHF due to its potent therapeutic properties and absence of adverse effects. Nevertheless, the precise molecular mechanisms underlying its actions remain elusive. This study endeavors to unravel the intricate pharmacological underpinnings of five herbs within ZWD concerning CHF through an integrated approach. Initially, pertinent data regarding ZWD and CHF were compiled from established databases, forming the foundation for constructing an intricate network of active component-target interactions. Subsequently, a pioneering method for evaluating node significance was formulated, culminating in the creation of core functional association space (CFAS). To discern vital components, a novel dynamic programming algorithm was devised and used to determine the core component group (CCG) within the CFAS. Enrichment analysis of the CCG targets unveiled the potential coordinated molecular mechanisms of ZWD, illuminating its capacity to ameliorate CHF by modulating genes and related signaling pathways involved in pathological remodeling. Notable pathways encompass PI3K-Akt, diabetic cardiomyopathy, cAMP and MAPK signaling. Concluding the computational analyses, in vitro experiments were executed to assess the effects of vanillic acid, paradol, 10-gingerol and methyl cinnamate. Remarkably, these compounds demonstrated efficacy in reducing the production of ANP and BNP within isoprenaline-induced AC 16 cells, further validating their potential therapeutic utility. This investigation underscores the efficacy of the proposed model in enhancing the precision and reliability of CCG selection within ZWD, thereby presenting a novel avenue for mechanistic inquiries, compound refinement and the secondary development of TCM herbs.

The cardioprotective effect of Shengmai Zhenwu decoction on patients with chronic heart failure based on the levels of soluble interleukin 1 receptor-like 1.

Objective: The purpose of this study was to evaluate the protective effect of Shengmai Zhenwu decoction on patients with chronic heart failure (CHF) based on the levels of soluble interleukin 1 receptor-like 1 (ST2). Methods: We included a total of 80 outpatients and inpatients with CHF who were undergoing treatment at the Shanghai Municipal Hospital of Traditional Chinese Medicine between March 2020 and March 2022. We randomly divided them into the observation group (n = 40) and the control group (n = 40). Patients in the control group received treatments as per conventional Western medicine, while those in the observation group were treated with the Shengmai Zhenwu decoction in conjunction with Western medicine for eight consecutive weeks. We then compared the pre- and post-treatment levels of ST2 and N-terminal pro-brain natriuretic peptide (NT-proBNP) of the patients in the two groups. Results: There were no significant differences in the pre-treatment levels of ST2 and NT-proBNP indexes between the two groups (P > 0.05), while the post-treatment comparison between the two groups in terms of ST2 and NT-proBNP levels suggested that the effect in the observation group was better, with statistical significance (P < 0.05). Conclusion: Shengmai Zhenwu decoction was beneficial in patients with CHF, suggesting that it could be a promising and effective method for the treatment of CHF.

[Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway].

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1ß, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

[Effect of Zhenwu Decoction on electrical remodeling of cardiomyocytes in heart failure via I_(to)/Kv channels].

This study aimed to investigate the underlying mechanism of Zhenwu Decoction in the treatment of heart failure by regulating electrical remodeling through the transient outward potassium current(I_(to))/voltage-gated potassium(Kv) channels. Five normal SD rats were intragastrically administered with Zhenwu Decoction granules to prepare drug-containing serum, and another seven normal SD rats received an equal amount of distilled water to prepare blank serum. H9c2 cardiomyocytes underwent conventional passage and were treated with angiotensin Ⅱ(AngⅡ) for 24 h. Subsequently, 2%, 4%, and 8% drug-containing serum, simvastatin(SIM), and BaCl_2 were used to interfere in H9c2 cardiomyocytes for 24 h. The cells were divided into a control group [N, 10% blank serum + 90% high-glucose DMEM(DMEM-H)], a model group(M, AngⅡ + 10% blank serum + 90% DMEM-H), a low-dose Zhenwu Decoction-containing serum group(Z1, AngⅡ + 2% drug-containing serum of Zhenwu Decoction + 8% blank serum + 90% DMEM-H), a medium-dose Zhenwu Decoction-containing serum group(Z2, AngⅡ + 4% drug-containing serum of Zhenwu Decoc-tion + 6% blank serum + 90% DMEM-H), a high-dose Zhenwu Decoction-containing serum group(Z3, AngⅡ + 8% drug-containing serum of Zhenwu Decoction + 2% blank serum + 90% DMEM-H), an inducer group(YD, AngⅡ + SIM + 10% blank serum + 90% DMEM-H), and an inhibitor group(YZ, AngⅡ + BaCl_2 + 10% blank serum + 90% DMEM-H). The content of ANP in cell extracts of each group was detected by ELISA. The relative mRNA expression levels of ANP, Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 were detected by real-time quantitative PCR. The protein expression of Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 was detected by Western blot. I_(to) was detected by the whole cell patch-clamp technique. The results showed that Zhenwu Decoction at low, medium, and high doses could effectively reduce the surface area of cardiomyocytes. Compared with the M group, the Z1, Z2, Z3, and YD groups showed decreased ANP content and mRNA level, increased protein and mRNA expression of Kv4.2, Kv4.3, DPP6, and KChIP2, and decreased protein and mRNA expression of Kv1.4, and the aforementioned changes were the most notable in the Z3 group. Compared with the N group, the Z1, Z2, and Z3 groups showed significantly increased peak current and current density of I_(to). The results indicate that Zhenwu Decoction can regulate myocardial remodeling and electrical remodeling by improving the expression trend of Kv1.4, Kv4.2, Kv4.3, KChIP2, and DPP6 proteins and inducing I_(to) to regulate Kv channels, which may be one of the mechanisms of Zhenwu Decoction in treating heart failure and related arrhythmias.

Exploring the possible mechanism(s) underlying the nephroprotective effect of Zhenwu Decoction in diabetic kidney disease: An integrated analysis.

BACKGROUND: Diabetic kidney disease (DKD) is one of the major chronic microvascular complications of diabetes and the main cause of end-stage renal failure. Zhenwu Decoction (ZWD), an ancient classic herbal formula in Chinese medicine, has been clinically used for the treatment of kidney disease in China for many years. However, there is currently limited research investigating the application of ZWD in the treatment of DKD and the underlying chemical and biochemical mechanisms involved. Therefore, in the present study, we aimed to identify active components in ZWD and unravel the possible mechanism(s) of action for ZWD in treating DKD. METHODS: The protective effect of ZWD against DKD was evaluated utilizing an in vitro model of diabetic renal proximal tubulopathy. The major chemical components from ZWD were identified by LC-MS/MS. Drug targets were predicted by submitting the SMILES (Simplified Molecular Input Line Entry System) of the compounds to SEA (Similarity Ensemble Approach) search server and SwissTargetPrediction. The differentially expressed genes (DEGs) of the disease were collected and integrated from GeneCards. The constructions of "Compounds-potential targets interaction" (CTI) network and Protein-Protein Interaction (PPI) network, as well as topology analysis were conducted by Cytoscape. Gene Ontology (GO) enrichment and Metacore pathway enrichment analysis were also performed. Lastly, molecular docking and experimental studies were adopted to validate the core target and identify an active component(s) of ZWD. RESULTS: We demonstrated that the ZWD extract could significantly rescue the palmitic acid (PA) and high glucose-induced apoptotic cell death in HK-2 cells, and the cytoprotection was accompanied by decreases in the extent of reactive oxygen species (ROS) production, mitochondrial membrane depolarization and ATP depletion. Fifty-seven compounds in the aqueous extract of ZWD were identified by LC-MS. The results of PPI analysis showed that top hub genes involved epidermal growth factor receptor (EGFR), Signal Transducer and Activator of Transcription 3 (STAT3), Serine/Threonine Kinase 1 (AKT1), Vascular Endothelial Growth Factor A (VEGFA) and Fibroblast Growth Factor 2 (FGF2). Pathway enrichment analysis revealed the involvement of S1P1 receptor signaling and EGFR pathways. The results of molecular docking analysis showed that albiflorin has a high binding affinity to EGFR. Albiflorin could also exert protective effects in an HK-2 cell model of DKD, which may be related to the inhibition of the high glucose/high lipid-induced EGFR and Akt phosphorylation. CONCLUSION: ZWD has been shown to be effective in ameliorating cell death in an experimental model of DKD. The beneficial effect of ZWD against DKD was associated with the interactions between the active ingredients and the hub genes, such as EGFR, STAT3, AKT1, and VEGF-A. Albiflorin may be one of the active components responsible for the nephroprotective effect in ZWD.

[Zhenwu Decoction:connotation interpretation based on severe cases and modern pathophysiological mechanisms and application in treatment of heart failure in critical care unit].

Zhenwu Decoction is recorded in Treatise on Febrile Diseases by an outstanding physician ZHANG Zhong-jing in the Han dynasty. With effect of warming yang, transforming Qi, and promoting urination, Zhenwu Decoction is mainly used to treat edema due to yang deficiency. The studies of the severe and critical cases and the pathophysiological mechanisms have demonstrated that the record of Zhenwu Decoction in Treatise on Febrile Diseases describes the clinical symptoms and therapeutic regimen of acute heart failure. The syndrome treated by this formula may be related to the misdiagnosis and wrong treatment. Due to the difficult distinguishing between cardiogenic dyspnea and pulmonary dyspnea, high doses of Ephedrae Herba may be misused for inducing sweating, which may finally lead to the acute aggravation of heart failure, electrolyte disorder, and pulmonary infection. The syndrome treated by Zhenwu Decoction can illustrate the lack of experience of ancient physicians in treating acute heart failure. The description of "trembling and shivering" may be the clinical manifestation of heart failure, which is an upgraded version of "trembling and shaking" treated by Linggui Zhugan Decoction.(1)In terms of diseases, Zhenwu Decoction is suitable for the treatment of acute or chronic heart failure, cardiorenal syndrome, and diuretic resistance. The decoction is especially suitable for treating whole heart failure, acute heart failure, heart failure with reduced ejection fraction, and heart failure with the syndrome of sold and dampness. In addition, it can be used to treat both type Ⅱ and type Ⅳ cardiorenal syndrome.(2)In terms of symptoms, Zhenwu Decoction can be used for treating chest tightness, palpitations, lower limb edema, difficult urination or increased urine output, fear of cold, pale fat tongue with teeth marks, white and slippery tongue fur, and deep or slow pulse.(3)In terms of the pharmacological mechanism, Zhenwu Decoction treats heart failure following the principle of promoting urination, expanding blood vessels, and invigorating heart in modern medicine. Aconiti Lateralis Radix Praparata is the sovereign herb in the formula, with the recommended dosage of 30-60 g. However, arrhythmia may be caused by high doses of Aconiti Lateralis Radix Praparata, which should be used with concern. In addition to Zhenwu Decoction, Shenqi Pills, Renshen Decoction, Wuling Powder, and Fangji Huangqi Decoction with the effect of invigorating spleen, replenishing Qi, warming Yang, and promoting urination can be used in the recovery stage. The therapy of reinforcing Yang was the last choice for critical cases due to the lack of medical conditions, unclear clinical diagnosis in history, which should be treated objectively now.

Integrating Network Pharmacology and an Experimental Model to Investigate the Effect of Zhenwu Decoction on Doxorubicin-Induced Heart Failure.

BACKGROUND: Doxorubicin-induced heart failure is a clinical problem that needs to be solved urgently. Previous studies have confirmed that Zhenwu Decoction, a traditional Chinese medicine compound, can effectively improve chronic heart failure. However, its interventional effect on Doxorubicin-induced heart failure has not yet been investigated. In this study, we investigated the therapeutic effect and potential mechanism of Zhenwu Decoction on Doxorubicininduced heart failure through animal experiments and network pharmacology. OBJECTIVE: The study aimed to investigate the therapeutic effect and potential mechanism of Zhenwu Decoction (ZWD) on Doxorubicin-induced heart failure. METHODS: A heart-failure mouse model was established in 8-week-old male C57/BL6J mice using Doxorubicin, and the mice were then treated with ZWD for a 4-week period. Firstly, network pharmacology was conducted to explore the potential active components and molecular mechanisms of ZWD on Doxorubicin-induced heart failure. Next, we conducted an in vivo study on the effect of ZWD on Doxorubicin-induced heart failure. After the intervention, the cardiac function and levels of cardiac function injury marker in serum were measured to evaluate the therapeutic effect of ZWD on cardiac function. Then HE staining and Masson staining were used to evaluate the effect of ZWD on myocardial pathology, and biochemical method was used to detect the effect of ZWD on total antioxidant capacity and inflammation, and finally, Western blot was used to detect TGFß, Smad-3, and collagen I protein expression levels to evaluate its effect on myocardial fibrosis. RESULTS: In Doxorubicin-induced heart failure mice, ZWD improved cardiac function and reduced the levels of CK-MB, NT-proBNP, and BNP in the serum, improved myocardial pathology, and reduced TGFß, Smad-3 and collagen I protein expression levels to improve myocardial fibrosis. Network pharmacological analysis showed that ZWD has 146 active ingredients and 248 candidate targets. Moreover, 2,809 genes were found to be related to Doxorubicin-induced heart failure, and after screening, 74 common targets were obtained, mainly including IL-6, AKT1, caspase-3, PPARG, PTGS2, JUN, HSP90AA1, and ESR1. KEGG analysis confirmed that PI3K/AKT and IL- 6/NF-κB signaling pathways were the two main pathways underlying the cardioprotective effects of ZWD. Finally, in vivo experiments showed that ZWD improved the total antioxidant capacity, reduced the SOD level, increased the protein expression of PI3K, Akt, Bcl-2, Bax, and caspase-3, reduced the levels of TNF-α, IL-6, and IL-1ß, and decreased the NF-κB p65, IL-6, and TNF-α protein expression levels. CONCLUSION: In Doxorubicin-induced heart-failure mice, Zhenwu Decoction improved the cardiac function and myocardial pathology, and improved myocardial fibrosis through the TGFß/Smad-3 signaling pathway. According to the prediction of network pharmacology, in vivo experiments demonstrated that Zhenwu Decoction can improve the oxidative stress response, improve myocardial cell apoptosis through the PI3K/AKT signaling pathway, and improve myocardial inflammation by reducing the levels of inflammatory factors and by reducing the protein expression of NF- κB p65, IL-6, and TNF-α.

Zhenwu decoction ameliorates cardiac hypertrophy through activating sGC (soluble guanylate cyclase) - cGMP (cyclic guanosine monophosphate) - PKG (protein kinase G) pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhenwu Decoction (ZWD) is a traditional Chinese medicine (TCM) formula which has wide scope of indications related to Yang deficiency and dampness retention in TCM syndrome. Cardiac hypertrophy can induce similar symptoms and signs to the clinical features of Yang deficiency and dampness retention syndrome. ZWD can increase the left ventricular ejection fraction, reduce cardiac hypertrophy of patients with chronic heart failure. However, its underlying pharmacological mechanism remains unclear. AIM OF THE STUDY: The study aimed to confirm the protective effects of ZWD on cardiac hypertrophy and explore the underlying mechanisms. MATERIALS AND METHODS: The potential targets and pathways of ZWD in cardiac hypertrophy were highlighted by network pharmacology and validated by mechanistic and functional studies. RESULTS: Our network pharmacology analysis suggests that the protective effects of ZWD on cardiac hypertrophy are related to cyclic guanosine monophosphate (cGMP) - protein kinase G (PKG) pathway. Subsequent animal studies showed that ZWD significantly ameliorated cardiac function decline, cardiac hypertrophy, cardiac fibrosis and cardiomyocyte apoptosis. To explore the underlying mechanisms of action, we performed Western blotting, immunohistochemical analysis, and detection of inflammatory response and oxidative stress. Our results showed that ZWD activated the soluble guanylate cyclase (sGC) - cGMP - PKG signaling pathway. The sGC inhibitor ODQ that blocks the sGC-cGMP-PKG signaling pathway in zebrafish abolished the protective effects of ZWD, suggesting sGC-cGMP-PKG is the main signaling pathway mediates the protective effect of ZWD in cardiac hypertrophy. In addition, three major ingredients from ZWD, poricoic acid C, hederagenin and dehydrotumulosic acid, showed a high binding energy with prototype sGC. CONCLUSION: ZWD reduces oxidative stress and inflammation and exerts cardioprotective effects by activating the sGC-cGMP-PKG signaling pathway.

Effect of Zhenwu Decoction on electrical remodeling of cardiomyocytes in heart failure via I_(to)/Kv channels / 中国中药杂志

This study aimed to investigate the underlying mechanism of Zhenwu Decoction in the treatment of heart failure by regulating electrical remodeling through the transient outward potassium current(I_(to))/voltage-gated potassium(Kv) channels. Five normal SD rats were intragastrically administered with Zhenwu Decoction granules to prepare drug-containing serum, and another seven normal SD rats received an equal amount of distilled water to prepare blank serum. H9c2 cardiomyocytes underwent conventional passage and were treated with angiotensin Ⅱ(AngⅡ) for 24 h. Subsequently, 2%, 4%, and 8% drug-containing serum, simvastatin(SIM), and BaCl_2 were used to interfere in H9c2 cardiomyocytes for 24 h. The cells were divided into a control group [N, 10% blank serum + 90% high-glucose DMEM(DMEM-H)], a model group(M, AngⅡ + 10% blank serum + 90% DMEM-H), a low-dose Zhenwu Decoction-containing serum group(Z1, AngⅡ + 2% drug-containing serum of Zhenwu Decoction + 8% blank serum + 90% DMEM-H), a medium-dose Zhenwu Decoction-containing serum group(Z2, AngⅡ + 4% drug-containing serum of Zhenwu Decoc-tion + 6% blank serum + 90% DMEM-H), a high-dose Zhenwu Decoction-containing serum group(Z3, AngⅡ + 8% drug-containing serum of Zhenwu Decoction + 2% blank serum + 90% DMEM-H), an inducer group(YD, AngⅡ + SIM + 10% blank serum + 90% DMEM-H), and an inhibitor group(YZ, AngⅡ + BaCl_2 + 10% blank serum + 90% DMEM-H). The content of ANP in cell extracts of each group was detected by ELISA. The relative mRNA expression levels of ANP, Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 were detected by real-time quantitative PCR. The protein expression of Kv1.4, Kv4.2, Kv4.3, DPP6, and KChIP2 was detected by Western blot. I_(to) was detected by the whole cell patch-clamp technique. The results showed that Zhenwu Decoction at low, medium, and high doses could effectively reduce the surface area of cardiomyocytes. Compared with the M group, the Z1, Z2, Z3, and YD groups showed decreased ANP content and mRNA level, increased protein and mRNA expression of Kv4.2, Kv4.3, DPP6, and KChIP2, and decreased protein and mRNA expression of Kv1.4, and the aforementioned changes were the most notable in the Z3 group. Compared with the N group, the Z1, Z2, and Z3 groups showed significantly increased peak current and current density of I_(to). The results indicate that Zhenwu Decoction can regulate myocardial remodeling and electrical remodeling by improving the expression trend of Kv1.4, Kv4.2, Kv4.3, KChIP2, and DPP6 proteins and inducing I_(to) to regulate Kv channels, which may be one of the mechanisms of Zhenwu Decoction in treating heart failure and related arrhythmias.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway / 中国中药杂志

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

A Data Mining-Based Study on the Medication Rules of Chinese Medicine to Treat Heart Failure with Diuretic Resistance / 世界科学技术-中医药现代化

Objective To explore the medication rules of clinical prescriptions of Chinese medicine in the treatment of heart failure with diuretic resistance based on data mining method.Methods Seven domestic and foreign databases,including CNKI and Wanfang,were searched for literature on Chinese medicine for heart failure with diuretic resistance from the establishment of the database to October 2021.Frequency analysis,association rule analysis and cluster analysis were used for statistical analysis.Results A total of 41 literatures were included,including 39 prescriptions.Yang deficiency and water retention were the main symptoms of the disease.A total of 72 single herbs were used,with a total of 438 times of use.There are 30 Chinese medicines with a frequency of more than 5 times,involving 9 efficacy categories,mainly tonic medicines,diuresis and dampness medicines,blood-activating and stasis-removing medicines,and interior-warming medicines.The association rule analysis yielded drug compatibility with Poria as the core,such as Atractylodis Macrocephalae Rhizoma→Poria and Cinnamomi Ramulus→Poria.Cluster analysis yielded six new formula combinations with efficacy in warming Yang,inducing diuresis,benefiting qi and invigorating Blood.Conclusion Heart failure with diuretic resistance is a syndrome of root vacuity and tip repletion,cold-heat complex,and the core pathogenesis is"deficiency","water"and"stasis".The treatment of this disease occurs by warming yang and excreting water,supplemented by replenishing qi and activating blood circulation.Poria with Atractylodis Macrocephalae Rhizoma and Cinnamomi Ramulus are commonly used combinations,and Zhen Wu Decoction is the basic formula for the treatment of this disease.

Zhenwu Decoction:connotation interpretation based on severe cases and modern pathophysiological mechanisms and application in treatment of heart failure in critical care unit / 中国中药杂志

Zhenwu Decoction is recorded in Treatise on Febrile Diseases by an outstanding physician ZHANG Zhong-jing in the Han dynasty. With effect of warming yang, transforming Qi, and promoting urination, Zhenwu Decoction is mainly used to treat edema due to yang deficiency. The studies of the severe and critical cases and the pathophysiological mechanisms have demonstrated that the record of Zhenwu Decoction in Treatise on Febrile Diseases describes the clinical symptoms and therapeutic regimen of acute heart failure. The syndrome treated by this formula may be related to the misdiagnosis and wrong treatment. Due to the difficult distinguishing between cardiogenic dyspnea and pulmonary dyspnea, high doses of Ephedrae Herba may be misused for inducing sweating, which may finally lead to the acute aggravation of heart failure, electrolyte disorder, and pulmonary infection. The syndrome treated by Zhenwu Decoction can illustrate the lack of experience of ancient physicians in treating acute heart failure. The description of "trembling and shivering" may be the clinical manifestation of heart failure, which is an upgraded version of "trembling and shaking" treated by Linggui Zhugan Decoction.(1)In terms of diseases, Zhenwu Decoction is suitable for the treatment of acute or chronic heart failure, cardiorenal syndrome, and diuretic resistance. The decoction is especially suitable for treating whole heart failure, acute heart failure, heart failure with reduced ejection fraction, and heart failure with the syndrome of sold and dampness. In addition, it can be used to treat both type Ⅱ and type Ⅳ cardiorenal syndrome.(2)In terms of symptoms, Zhenwu Decoction can be used for treating chest tightness, palpitations, lower limb edema, difficult urination or increased urine output, fear of cold, pale fat tongue with teeth marks, white and slippery tongue fur, and deep or slow pulse.(3)In terms of the pharmacological mechanism, Zhenwu Decoction treats heart failure following the principle of promoting urination, expanding blood vessels, and invigorating heart in modern medicine. Aconiti Lateralis Radix Praparata is the sovereign herb in the formula, with the recommended dosage of 30-60 g. However, arrhythmia may be caused by high doses of Aconiti Lateralis Radix Praparata, which should be used with concern. In addition to Zhenwu Decoction, Shenqi Pills, Renshen Decoction, Wuling Powder, and Fangji Huangqi Decoction with the effect of invigorating spleen, replenishing Qi, warming Yang, and promoting urination can be used in the recovery stage. The therapy of reinforcing Yang was the last choice for critical cases due to the lack of medical conditions, unclear clinical diagnosis in history, which should be treated objectively now.

Tentative exploration of pharmacodynamic substances: Pharmacological effects, chemical compositions, and multi-components pharmacokinetic characteristics of ESZWD in CHF-HKYd rats.

The chemical components of Xin'an famous prescription Ershen Zhenwu Decoction (ESZWD) are still unclear. The results showed that ESZWD could significantly reduce left ventricular end diastolic diameter, decrease N-terminal pro-brain natriuretic peptide (NT-proBNP), angiotensinII, aldosterone, reactive oxygen species, and malondialdehyde, increase serum superoxide dismutase, while had no significant effect on inflammatory factors. Ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis detected 30 prototype components in model rats' serum, mainly including alkaloids, saponins, terpenoids, tanshinones, phenols. UPLC-MS/MS successfully detected the pharmacokinetic parameters of four components, and correlation analysis shows that there are negative correlations between four compounds and serum NT-proBNP. Thirty components of ESZWD may play a therapeutic role in chronic heart failure with heart-kidney Yang deficiency (CHF-HKYd) by improving myocardial injury, reducing oxidative stress levels, and inhibiting activation of the RAAS system in rats. Salsolinol, aconitine, paeoniflorin, and miltrione are equipped with potential characteristics as pharmacodynamic substances for ESZWD in treating CHF-HKYd. Additionally, the constituents of ESZWD in CHF-HKYd rats are different from normal rats, which provided a reference for the selection of subjects for further study.

Mechanisms of Zhenwu decoction for the treatment of renal fibrosis at various stages: What is the role of Corynebacterium?

Many studies demonstrated that Zhenwu decoction (ZWD) is effective in the treatment of kidney fibrosis, whereas the mechanism remains unclear. In this work, a microbiomics-based strategy was used to investigate the mechanism of protective effects of ZWD on kidney fibrosis. Unilateral ureteral obstruction was used to replicate a rat model of renal fibrosis, and rats were divided into prophylactic, early, and progression stages according to the timing of administration. Feces was collected to perform microbiota evaluation by high-throughput 16S DNA sequencing. The results indicated that Corynebacterium, Alistipes, Dorea, and Lactonifactor were highlighted as key targeted flora of ZWD in the treatment of renal fibrosis, and their biological functions were related to inflammation, immunity, and renal excretion. Especially, Corynebacterium presented a significant positive correlation with the concentration of Cys-C, Scr, and BUN. The studies on the changes in inflammatory cytokines (INF-γ, IL-1ß, IL-4, and TNF-α) and immunoglobulin (IgA, IgM, and IgG) confirmed the beneficial effects of ZWD on kidney fibrosis. Therefore, this study confirmed the protective effect of ZWD against renal fibrosis at various disease stages, and its mechanism was associated with re-establishing dysbiosis of the intestinal microbiota, reducing inflammation, as well as regulating immune functions. In particular, Corynebacterium may be a key flora in the treatment of renal fibrosis.

Identification of chemical constituents in vitro and in vivo of Er Shen Zhenwu Decoction by utilizing ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

Er Shen Zhenwu Decoction is a prescription for treating chronic heart failure of heart and kidney yang deficiency, while its active ingredients remain unclear and difficult to identify. This paper aims to apply a rapid assay strategy of ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to collect the mass spectrometry data of Er Shen Zhenwu Decoction and its decomposed recipes (monarch, minister, and assist). By comparing with retention time and MSE fragmentation patterns, 67 and 34 components in vitro and in vivo were identified, respectively, the main ingredients include saponins, terpenes, alkaloids, phenolic acids, tanshinone, urea, steroids, aromatics, organic acids, carbohydrates, and so forth, of which the monarch medicine > minister medicine > assist medicine. By comparison with reference standards, paeoniflorin, rosmarinic acid, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and atractylenolide III were identified in vitro and paeoniflorin, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 were identified in vivo. In this study, the chemical ingredients of Er Shen Zhenwu Decoction were analyzed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry technology and each compound was grouped into the decomposed recipes. The identified substances can be used as references for Er Shen Zhenwu Decoction quality control and potential medicinal substances in chronic heart failure of heart and kidney yang deficiency treatment.

Evaluation and Clinical Implication of Zhenwu Decoction on Seven Cytochrome P450 Enzyme in Chronic Heart Failure Rats.

BACKGROUND: Recently, the combination of Traditional Chinese Medicine (TCM) formulae and other drugs has been used frequently in clinical practice, while the possibility of herb-drug interaction (HDI) risk remains a challenge. Since metabolic enzymes mediate the majority of drug interactions, evaluating the effects of formulae on metabolic enzymes is therefore instructive for the rational formulation of drug delivery plans. OBJECTIVE: Herein, we are devoted to estimating the effects of Zhenwu detection (ZWD) on activities and mRNA expression of 7 cytochrome P450 (CYP450) isoenzymes in chronic heart failure (CHF) rats. METHODS: The CHF rats were replicated by coronary artery ligation and were randomly divided into sham operation group, model group, ZWD low- (2.188 g/kg), middle- (4.375 g/kg), and high- (8.750 g/kg) dose groups, n=6. After 8 weeks, rats were administrated with ZWD and normal saline (NS) for four weeks, and the mixed solution of 7 probe drugs (1 mL/kg) was subsequently injected into 30 rats through the caudal vein after the last administration. Pharmacokinetic parameters and mRNA expression of 7 probe drugs were measured by using UPLC-MS/MS and RT-qPCR, respectively. RESULTS: Activities and mRNA expression of CYP1A2, CYP2B1, CYP2C6, CYP2C11, CYP3A1 were inhibited in CHF rats, and ZWD could reverse this effect except for CYP2B1. CONCLUSION: Overall, these findings underscore that for CHF patients, the HDI should be taken into consideration when ZWD is used on its own or combined with drugs meditated by CYP1A2 (CYP1A2 in rats), CYP2C9 (CYP2C6 in rats), CYP2C19 (CYP2C11 in rats) and CYP3A4 (CYP3A1 in rats). Furthermore, since the apparent volume of distribution (Vd) of amodiaquine, dextromethorphan and bupropion has been proved to be far greater than the total volume of body fluids, we speculate that the dose adjustment and potential organotoxicity of these substrates may need further consideration.

[Analysis on quality value transfer of substance benchmarks of Zhenwu Decoction].

By preparing 15 batches of Zhenwu Decoction substance benchmarks,the characteristic map,index component content and paste-forming rate were determined to define the peak attribution,similarity range,paste-forming rate range,paeoniflorin and6-gingerol content range and transfer rate range. The similarity between the substance benchmark characteristic map and the control map R generated from the 15 batches of substance benchmarks was higher than 0. 970. There were 19 characteristic peaks in total. By further summarization of the characteristic peaks,it could be seen that tuckahoe had 3 characteristic peaks,white peony root had 10 characteristic peaks,atractylodes had 3 characteristic peaks,ginger had 1 characteristic peak,and Aconite root had 3 characteristic peaks; among them,white peony root and aconite root had 1 common peak. The contents and transfer rates of the 15 batches were0. 50%-0. 93 and 16. 11%-26. 20%; those for 6-gingerol were 0. 018 2%-0. 033 9% and 13. 16%-24. 10%,respectively. The pasteforming rate ranged from 10. 00% to 14. 85%. In this study,the transfer process of substance benchmark value of classic formula Zhenwu Decoction was analyzed based on the characteristic map,the paste-forming rate and the content of the index components; a scientific and stable substance benchmark quality evaluation method was preliminarily established to provide a basis for subsequent development of classic formula Zhenwu Decoction and quality control of relevant preparations.