Network Pharmacology and Experimental Verification Strategies to Illustrate the Mechanism of Jian-Pi-Yi-Shen Formula in Suppressing Epithelial-Mesenchymal Transition.

Jian-Pi-Yi-Shen formula (JPYSF), a traditional Chinese medicine, has been recommended to treat renal fibrosis for decades. Previous studies had shown that JPYSF could inhibit epithelial-mesenchymal transition (EMT), an important regulatory role in renal fibrosis. However, the mechanism of JPYSF action is largely unknown. In this study, network pharmacology and experimental verification were combined to elucidate and identify the potential mechanism of JPYSF against renal fibrosis by suppressing EMT at molecular and pathway levels. Network pharmacology was first performed to explore the mechanism of JPYSF against renal fibrosis targeting EMT, and then a 5/6 nephrectomy (5/6 Nx)-induced rat model of renal fibrosis was selected to verify the predictive results by Masson's trichrome stains and western blot analysis. Two hundred and thirty-two compounds in JPYSF were selected for the network approach analysis, which identified 137 candidate targets of JPYSF and 4,796 known therapeutic targets of EMT. The results of the Gene Ontology (GO) function enrichment analysis included 2098, 88, and 133 GO terms for biological processes (BPs), molecular functions (MFs), and cell component entries, respectively. The top 10 enrichment items of BP annotations included a response to a steroid hormone, a metal ion, oxygen levels, and so on. Cellular composition (CC) is mainly enriched in membrane raft, membrane microdomain, membrane region, etc. The MF of JPYSF analysis on EMT was predominately involved in proximal promoter sequence-specific DNA binding, protein heterodimerization activity, RNA polymerase II proximal promoter sequence-specific DNA binding, and so on. The involvement signaling pathway of JPYSF in the treatment of renal fibrosis targeting EMT was associated with anti-fibrosis, anti-inflammation, podocyte protection, and metabolism regulation. Furthermore, the in vivo experiments confirmed that JPYSF effectively ameliorated interstitial fibrosis and inhibited the overexpression of α-SMA, Wnt3a, and ß-catenin, and increased the expression of E-cadherin by wnt3a/ß-catenin signaling pathway in 5/6 Nx-induced renal fibrosis rats. Using an integrative network pharmacology-based approach and experimental verification, the study showed that JPYSF had therapeutic effects on EMT by regulating multi-pathway, among which one proven pathway was the Wnt3a/ß-catenin signaling pathway. These findings provide insights into the renoprotective effects of JPYSF against EMT, which could suggest directions for further research of JPYSF in attenuating renal fibrosis by suppressing EMT.

Jian-Pi-Yi-Shen Formula Alleviates Chronic Kidney Disease in Two Rat Models by Modulating QPRT/NAD+/SIRT3/Mitochondrial Dynamics Pathway.

OBJECTIVE: Jian-Pi-Yi-Shen formula (JPYSF) is a traditional Chinese herbal decoction and has been used for treating chronic kidney disease (CKD) in clinics for decades. However, the potential mechanisms have not been fully elucidated. This study was designed to test the efficacy of JPYSF in treating CKD and explore the underlying mechanism. METHODS: Two CKD rat models were established by 5/6 nephrectomy (5/6 Nx) and feeding with adenine-containing feed, respectively. The intervention dose of JPYSF was 10.89 g/kg/d by gastric irrigation. Renal function was assessed by serum creatinine (Scr) and blood urea nitrogen (BUN). Periodic acid-Schiff (PAS) and Masson's trichrome staining were used to evaluate renal histopathological changes. The levels of nicotinamide adenine dinucleotide (NAD+) were measured by using the enzyme-linked immunosorbent assay kit. The proteins expressions of renal fibrosis, quinolinate phosphoribosyltransferase (QPRT), sirtuin 3 (SIRT3), and mitochondrial dynamics were determined and quantified by Western blot analysis. RESULTS: The results show that administration of JPYSF significantly lowered Scr and BUN levels, improved renal tubular atrophy and interstitial fibrosis, and decreased renal extracellular matrix deposition in two CKD rat models. In addition, CKD rats exhibited suppressed QPRT/NAD+/SIRT3 signal, increased mitochondrial fission, and decreased mitochondrial fusion. JPYSF treatment promoted QPRT/NAD+/SIRT3 signal and restored mitochondrial fission/fusion balance. CONCLUSION: In conclusion, administration of JPYSF effectively alleviated CKD progression in two rat models, which may be related with regulation of the QPRT/NAD+/SIRT3/mitochondrial dynamics pathway.

Impaired Nicotinamide Adenine Dinucleotide Biosynthesis in the Kidney of Chronic Kidney Disease.

Chronic kidney disease (CKD) is a global public health problem with high morbidity and mortality. Decreased nicotinamide adenine dinucleotide (NAD+) levels were found to be associated with aging, cancer, and neurodegenerative and metabolic disorders. However, the alteration of renal NAD+ levels and biosynthesis pathways in CKD is less known. In the present study, we aimed to evaluate renal NAD+ levels and tested the expression of key enzymes in three NAD+ biosynthesis pathways in two different types of CKD rat model. CKD rat models were established by 5/6 nephrectomy (5/6 Nx) and feeding with adenine-containing feed, respectively. Renal function was assessed by serum creatinine (Scr) and blood urea nitrogen (BUN). Renal pathology was evaluated by periodic acid-Schiff (PAS) and Masson's trichrome staining. The expression of key enzymes in three NAD+ biosynthesis pathways was determined and quantified by Western blot analysis. The results showed CKD rat models were successfully established as evidenced by increased Scr and BUN levels, upregulation of neutrophil gelatinase-associated lipocalin (NGAL), glomerular hypertrophy, and renal fibrosis. Renal NAD+ and NADH content were both declined in two CKD rat models, and NAD+ levels were negatively correlated with Scr and BUN levels in CKD rats. Three key enzymes involved in NAD+ biosynthesis were significantly downregulated in the kidney of both of the two CKD models. They were quinolinate phosphoribosyltransferase (QPRT) in the de novo pathway, nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), and NMNAT3 in the salvage pathway. Moreover, the expression of NAD+-consuming enzymes sirtuin 3 (SIRT3) and CD38 decreased significantly in CKD rats. In conclusion, NAD+ biosynthesis was significantly impaired in CKD, which may attribute to downregulation of QPRT and NMNAT 1/3.

Jian-Pi-Yi-Shen Regulates EPO and Iron Recycling Protein Expressions in Anemic Rats with Chronic Kidney Disease: Accumulation of Hypoxia Inducible Factor-2α via ERK Signaling.

Jian-Pi-Yi-Shen (JPYS), the traditional Chinese medicine (TCM) decoction, has been commonly used to treat chronic kidney disease (CKD) and its complications such as anemia. JPYS has been previously found to induce erythropoietin (EPO) production in HEK293T cells and CKD rats. However, the mechanism of JPYS in treating anemia of CKD rats has remained largely unknown. Here, we further extend our effort to investigate the translational control of hypoxia inducible factor- (HIF-) α protein via ERK signaling and the effect on iron recycling-related protein expression by JPYS, thus revealing the mechanism of JPYS in correcting anemia in CKD. Experimental CKD rats with anemia were induced by 5/6 nephrectomy. Rats were administrated orally with high dose (6.0 g/kg/d) and low dose (1.5 g/kg/d) of JPYS for 90 days. Serum hepcidin level was determined to evaluate iron homeostasis. The protein expressions of HIF-2α, erythropoietin (EPO), ferritin, and ferroportin (FPN) and the phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2) were detected by Western blot. The results showed that JPYS treatment significantly ameliorated kidney function by reducing increased levels of blood urea nitrogen (BUN), serum creatinine (Scr), and urine protein (UPRO). Periodic acid-Schiff (PAS) and Masson staining observation showed that the renal pathological damage was restored in JPYS-treated CKD rats. In parallel, JPYS markedly improved CKD anemia through upregulation of red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT). JPYS stimulated EPO and HIF-2α protein expressions in both the kidney and liver of CKD rats. Furthermore, JPYS induced the phosphorylation of ERK1/2 protein. In addition, JPYS regulated protein expression of ferritin and FPN in both the liver and spleen of CKD rats and the serum level of hepcidin. In conclusion, JPYS induces the expression of EPO through ERK-mediated HIF-2α protein accumulation and regulates systemic iron recycling, supporting its role in promoting erythropoiesis and improvement of anemia in CKD.

Chemical characterisation and quantification of the major constituents in the Chinese herbal formula Jian-Pi-Yi-Shen pill by UPLC-Q-TOF-MS/MS and HPLC-QQQ-MS/MS.

INTRODUCTION: Jian-Pi-Yi-Shen pill (JPYSP) is a Chinese medicine formula developed for the treatment of anaemic patients with chronic kidney disease (CKD). OBJECTIVE: To investigate the chemical profile of JPYSP in the treatment of renal anaemia. METHODS: A method coupling ultra-performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) was established to characterise the chemical constituents present in JPYSP. Subsequently, a high-performance liquid chromatography method coupled with triple-quadrupole tandem mass spectrometry (HPLC-QQQ-MS/MS) was developed to quantify the major constituents from the identified compounds related to the treatment of CKD and anaemia. RESULTS: A total of 71 compounds were tentatively identified from JPYSP, including saponins, flavonoids, sesquiterpenoids, coumarins, phenylpropanoids, anthranones, anthraquinones, tannins, phenolic acids and others. Amongst them, 12 compounds (i.e. astragaloside IV, calycosin, calycosin 7-O-glucoside, salvianolic acid A, rosmarinic acid, rhein, liquiritin, formononetin, atractylenolide I, dioscin, tanshinone IIA, and acteoside) were further quantified simultaneously by HPLC-QQQ-MS/MS. CONCLUSION: The newly developed approach is suitable for the chemical profiling analysis and quality control of JPYSP, and could lead to additional pharmacodynamic studies involving the components of JPYSP.

Distinct Responses of Gut Microbiota to Jian-Pi-Yi-Shen Decoction Are Associated With Improved Clinical Outcomes in 5/6 Nephrectomized Rats.

Gut dysbiosis contributes to the development and progression of chronic kidney disease (CKD) and its complications. However, the effect of drugs on the gut microbiota of CKD patients and its influence on treatment outcomes remains to be explored. Here, we assessed whether the response of gut microbiota to the traditional Chinese medicine Jian-Pi-Yi-Shen (JPYS) decoction differed from that to piperazine ferulate (PF), a kidney-targeted drug, by 16S rDNA sequencing, and whether the difference could be linked with drug-specific clinical outcomes. We showed that both JPYS and PF improved renal function, but only JPYS was able to restore the blood reticulocyte counting and serum calcium level in CKD rats. We also found that weighted UniFrac beta-diversity of the gut microbiome of the JPYS treated rats was significantly different from that of PF. Microbiome markers of drug-specific response were identified and subjected to correlation network analysis, together with clinical parameters and KEGG pathways. Among the microbiome markers of CKD, Corynebacterium was found to form a network hub that was closely correlated with the JPYS responder Enterococcus, suggesting a potential indirect impact of JPYS on Corynebacterium via interspecies interactions. We also identified two network hubs of the PF responder Blautia and the JPYS-only marker Coprococcus, which were connected with many genera and clinical parameters. They might serve as keystone taxa driving the response of gut microbiota to the drugs and influence host outcomes. Moreover, the JPYS-only marker Clostridium_XIVb was found to be connected to many pathways that are associated with CKD progression and might account for the improved outcomes in the JPYS treated rats. At last, the identified keystone markers of drug response were validated by qPCR for their differential abundance between CKD and the two drugs. Taken together, our study revealed that the responses of gut microbiota to JPYS were distinct from that to PF, and pinpointed drug-specific keystone microbiome markers closely correlated to clinical parameters, which could serve as candidate microbiome targets for further studies on their roles in medicating the drug efficacy of TCM in CKD.

Involvement of Circulating Exosomal MicroRNAs in Jian-Pi-Yi-Shen Formula Protection Against Adenine-Induced Chronic Kidney Disease.

Jian-Pi-Yi-Shen formula (JPYSF) is a traditional Chinese medicine (TCM) formula used in clinic to treat chronic kidney disease (CKD) for decades. However, the mechanisms of JPYSF in treating CKD have not been fully elucidated. The aim of the present study was to test the renoprotective effect of JPYSF on CKD rat model and investigate the potential mechanism from the perspective of serum exosomal microRNAs (miRNAs). CKD rat model was induced by feeding Sprague-Dawley rats a diet containing 0.75% w/w adenine for four weeks. The rats in the treatment group were given 10.89 g/kg JPYSF by gavage every day, starting from the 3rd week of the adenine-containing diet for six weeks. Serum biochemistry and histopathology were used to evaluate the renoprotective effects of JPYSF. Serum exosomes were isolated by ExoQuick-TC PLUS exosomes extraction kit and were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Exosomal miRNAs profiling was analyzed by small RNA sequencing. The results showed that JPYSF treatment significantly lowered serum creatinine and blood urea nitrogen levels and alleviated renal pathological injury in CKD rats. Furthermore, serum exosomes were successfully isolated and identified. Small RNA sequencing revealed that 4 exosomal miRNAs (miR-192-5p, miR-194-5p, miR-802-5p, and miR-143-3p) were significantly downregulated in the CKD group and were markedly upregulated after JPYSF treatment. At last, miR-192-5p was identified as the most relevant miRNA for CKD diagnosis and JPYSF treatment. In conclusion, JPYSF protects kidney from adenine-induced CKD, which may be associated with modulation of exosomal miRNAs.

Metabolomics Analysis Reveals the Protection Mechanism of Huangqi-Danshen Decoction on Adenine-Induced Chronic Kidney Disease in Rats.

Huangqi-Danshen decoction (HDD) is a commonly used drug pair for clinical treatment of chronic kidney disease (CKD) in traditional Chinese medicine with good efficacy. However, the potential mechanisms of this action have not been well elucidated. The aim of this study was to explore the metabolic profiling variations in response to HDD treatment in a CKD rat model. CKD rat model was induced by adding 0.75% adenine to the diet for 4 weeks. The rats in the treatment group received HDD extract orally at the dose of 4.7 g/kg/day during the experiment. At the end of the experiment, serum and kidney samples were collected for biochemical and pathological examination. Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) was used to analyze metabolic profiling variations in the kidney. The results showed that treatment with HDD markedly attenuated kidney injury and improved renal function. A total of 28 metabolites contributing to CKD phenotype were found and identified in the kidney samples. The primary metabolic pathways disordered in the kidney of CKD rats were glycerophospholipid metabolism, glycosylphosphatidylinositol-anchor biosynthesis, and citrate cycle. Partial least squares discriminant analysis (PLS-DA) score plot showed that the three groups of renal samples were obviously divided into three categories, and the metabolic trajectory of the HDD treatment group moved to the control group. (E)-Piperolein A, phosphatidylcholines (PC) (18:1/22:6), phosphatidylinositols (PI) (13:0/18:1), PI (15:0/20:3), phosphatidylserines (PS) (O-20:0/12:0), and triglyceride (TG) (22:4/24:0/O-18:0) represented potential biomarkers of the renoprotective effects of HDD against CKD. In conclusion, HDD has renoprotective effect against adenine-induced CKD, which may be mediated via partially restoration of perturbed metabolism in the kidney.

Metabolomics Reveals Effect of Zishen Jiangtang Pill, a Chinese Herbal Product on High-Fat Diet-Induced Type 2 Diabetes Mellitus in Mice.

A Chinese herbal decoction, Zishen Jiangtang Pill (ZJP), has been clinically prescribed to diabetic patients to prevent excessive blood sugar levels for decades. However, the potential mechanisms of this action have not been well investigated. The purpose of this study was to explore the metabolic variations in response to ZJP treatment for an animal model of obese type 2 diabetes. An UHPLC-Orbitrap/MS-based metabolomics tool was conducted to reveal the potential mechanisms of ZJP on diabetic mice. The treatment with ZJP significantly restored the increased levels of insulin, glucose and total cholesterol in high-fat diet mice. A total of 26 potential biomarkers were found and identified in serum samples, amongst which 24 metabolites were robustly affected and driven back to the control-like levels after ZJP treatment. By analyzing the metabolic pathways, glutathione metabolism, steroid hormone biosynthesis and glycerophospholipid metabolism were suggested to be closely involved in diabetes disease. From the above outcomes, it can be concluded that ZJP exhibits a promising anti-diabetic activity, largely due to the regulation of phospholipid metabolism, including phosphatidylcholines, lysophosphatidylcholines, and phosphatidylinositol.

Jian-Pi-Yi-Shen Decoction Relieves Renal Anemia in 5/6 Nephrectomized Rats: Production of Erythropoietin via Hypoxia Inducible Factor Signaling.

Jian-Pi-Yi-Shen (JPYS) is one of the herbal medicines for treatment of anemic patients with chronic kidney disease (CKD). However, less of scientific evidence to support JPYS involved in treating anemia has been revealed. Here, an animal study was performed to investigate its hematopoietic activities and the underlying mechanism. The 5/6 nephrectomized inductions of CKD anemic rats were randomly divided into two groups: CKD group and JPYS group. Sham-operated rats served as sham group. JPYS (1.36 g/kg/d) was administered orally to CKD rats daily for six consecutive weeks. Results showed that JPYS treatment notably improved renal function and pathological injury in CKD rats. JPYS also restored the hematological parameters, including red blood cells, hemoglobin, and hematocrit. In parallel, the reduction level of EPO was reversed by JPYS. Furthermore, JPYS induced the accumulation of hypoxia inducible factor (HIF)-α protein expression. Collectively, these results provide convincing evidence for JPYS decoction in ameliorating CKD-associated anemia, and its mechanism might be related to regulate EPO production via HIF signaling pathway.

Huangqi-Danshen Decoction Ameliorates Adenine-Induced Chronic Kidney Disease by Modulating Mitochondrial Dynamics.

Chronic kidney disease (CKD) is a leading public health problem with high morbidity and mortality. However, the therapies remain limited. Traditional Chinese medicine (TCM) has been used for treating kidney disease for thousands of years and is an effective alternative treatment for CKD patients in China and other Asian countries. In the present study, we aimed to investigate the effect and mechanism of Huangqi-Danshen decoction (HDD), a TCM herbal decoction, on treating CKD. CKD rat model was induced by adding 0.75% adenine to the diet for 4 weeks. HDD extract was administrated orally to CKD rats at the dose of 4.7 g/kg/d for consecutive 4 weeks in adenine-induced CKD rats. Kidney function was evaluated by the levels of serum creatinine (Scr) and blood urea nitrogen (BUN). The pathological changes of kidney tissues were observed by periodic acid-Schiff (PAS) and Masson's trichrome staining. The proteins expression of renal fibrosis and mitochondrial dynamics were determined and quantified by Western blot analysis. CKD rats showed obvious decline in renal function as evidenced by increased levels of Scr and BUN, which were blunted by HDD treatment. HDD could also improve tubular atrophy and interstitial fibrosis of CKD rats. Moreover, HDD downregulated fibronectin, type IV collagen, and α-smooth muscle actin expression in CKD rats. Furthermore, mitochondrial dynamics was disturbed in CKD rats, which manifested as increased mitochondrial fission and decreased mitochondrial fusion. HDD treatment restored mitochondrial dynamics in CKD rats by repressing dynamin-related protein 1 and Mid 49/51 expression, promoting mitofusin 2 expression, and suppressing optic atrophy 1 proteolysis. In conclusion, HDD could significantly retard CKD progression through modulating mitochondrial dynamics.

Apoptosis induced by 9,11­dehydroergosterol peroxide from Ganoderma Lucidum mycelium in human malignant melanoma cells is Mcl­1 dependent.

9,11-Dehydroergosterol peroxide [9(11)-DHEP] is an important steroid from medicinal mushroom, which has been reported to exert antitumor activity in several tumor types. However, the role of 9(11)­DHEP toward the malignant melanoma cells has not been investigated. In the present study, the steroid from Ganoderma lucidum was purified on a submerged culture, and its antitumor mechanisms on A375 human malignant melanoma cells was investigated by MTT, flow cytometry and western blotting. The studies demonstrated that apoptotic mechanisms of the steroid were caspase­dependent and mediated via the mitochondrial pathway. The steroid did not cause significant changes in the expression levels of B­cell lymphoma 2 (Bcl­2) family proteins, Bcl­2­like protein 11, p53 upregulated modulator of apoptosis, Bcl­2­associated X protein, BH3 interacting­domain death agonist, Bcl­2­associated death promoter and Bcl­2, but it significantly downregulated induced myeloid leukemia cell differentiation protein Mcl­1 (Mcl­1) in melanoma cells, suggesting the key role of Mcl­1 in regulating apoptosis of melanoma cells induced by the steroid. These properties of 9(11)­DHEP advocate its usage as supplements in human malignant melanoma chemoprevention. The present study also suggests that mycelium of G. lucidum has a potential for producing bioactive substances and extracts with applications in medicine.