Tangshen Formula Research Articles

Network Pharmacology, Molecular Docking, and Experimental Verification to Reveal the Mitophagy-Associated Mechanism of Tangshen Formula in the Treatment of Diabetic Nephropathy [Retraction

Network Pharmacology, Molecular Docking, and Experimental Verification to Reveal the Mitophagy-Associated Mechanism of Tangshen Formula in the Treatment of Diabetic Nephropathy [Retraction

Tangshen formula targets the gut microbiota to treat non-alcoholic fatty liver disease in HFD mice: A 16S rRNA and non-targeted metabolomics analyses

BackgroundTangshen formula (TSF) has an ameliorative effect on hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD), but the role played by the gut microbiota in this process is unknown. MethodWe conducted three batches of experiments to explore the role played by the gut microbiota: TSF administration, antibiotic treatment, and fecal microbial transplantation. NAFLD mice were induced with a high-fat diet to investigate the ameliorative effects of TSF on NAFLD features and intestinal barrier function. 16S rRNA sequencing and serum untargeted metabolomics were performed to further investigate the modulatory effects of TSF on the gut microbiota and metabolic dysregulation in the body. ResultsTSF ameliorated insulin resistance, hypercholesterolemia, lipid metabolism disorders, inflammation, and impairment of intestinal barrier function. 16S rRNA sequencing analysis revealed that TSF regulated the composition of the gut microbiota and increased the abundance of beneficial bacteria. Antibiotic treatment and fecal microbiota transplantation confirmed the importance of the gut microbiota in the treatment of NAFLD with TSF. Subsequently, untargeted metabolomics identified 172 differential metabolites due to the treatment of TSF. Functional predictions suggest that metabolisms of choline, glycerophospholipid, linoleic acid, alpha-linolenic acid, and arachidonic acid are the key metabolic pathways by which TSF ameliorates NAFLD and this may be influenced by the gut microbiota. ConclusionTSF treats the NAFLD phenotype by remodeling the gut microbiota and improving metabolic profile, suggesting that TSF is a functional gut microbial and metabolic modulator for the treatment of NAFLD.

Tangshen Formula alleviates inflammatory injury against aged diabetic kidney disease through modulating gut microbiota composition and related amino acid metabolism

Diabetic kidney disease (DKD) is leading causes and one of the fastest growing causes of chronic kidney disease worldwide, and leads to high morbidity and mortality. Emerging evidences have revealed gut microbiota dysbiosis and related metabolism dysfunction play a dominant role in DKD progression and treatment through modulating inflammation. Our previous studies showed that Tangshen Formula (TSF), a Chinese herbal prescription, exhibited anti-inflammatory effect on DKD, but underlying mechanism that involved gut microbiota and related metabolism in aged model remained obscure. Here, BTBR ob/ob mice were used to establish aged DKD model, and 16S rRNA sequence and untargeted metabolomic analyses were employed to investigate the correlation between colonic microbiota and serum metabolism. The aged ob/ob mice exhibited obvious glomerular and renal tubule injury and kidney function decline in kidney, while TSF treatment significantly attenuated these abnormalities. TSF also exhibited potent anti-inflammatory effect in aged ob/ob mice indicating by reduced proinflammatory factor IL-6 and TNF-α, MCP-1 and COX-2 in serum, kidney and intestine, which suggested the involvement of gut microbiota with TSF effect. The 16S rDNA sequencing of the colonic microbiome and untargeted serum metabolomics analysis revealed significant differences in gut microbiota structure and serum metabolomic profiles between WT and ob/ob mice. Notably, TSF treatment reshaped the structure of gut microbiota and corrected the disorder of metabolism especially tryptophan metabolism and arginine biosynthesis. TSF increased Anaeroplasma and Barnesiella genera and decreased Romboutsia, Akkermansia, and Collinsella genera, and further elevated tryptophan, 5-hydroxyindoleacetate, glutamic acid, aspartate and reduced 4-hydroxy-2-quinolinecarboxylic acid, indole-3-acetic acid, xanthurenic acid, glutamine. Further correlation analysis indicated that disturbed gut microbiota was linked to tryptophan metabolism and arginine biosynthesis to regulate inflammation in aged DKD. Our data revealed that TSF attenuated renal inflammation by modulating gut microbiota and related amino acid metabolism in aged DKD model, highlighting gut microbiota and related metabolism functioned as potential therapeutic target for DKD in elderly patients.

Network Pharmacology, Molecular Docking, and Experimental Verification to Reveal the Mitophagy-Associated Mechanism of Tangshen Formula in the Treatment of Diabetic Nephropathy.

This study investigated the mechanism of TSF in treating DN through network pharmacology, molecular docking, and experimental validation. To identify critical active ingredients, targets, and DN genes in TSF, multiple databases were utilized for screening purposes. The drug-compound-target network was constructed using Cytoscape 3.9.1 software for network topological analysis. The protein interaction relationship was analyzed using the String database platform. Metascape database conducted enrichment analysis on the key targets using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. The renoprotective effect was evaluated using a mouse model of diabetic nephropathy (db/db mice) that occurred spontaneously. Validation of the associated targets and pathways was performed using Western Blot (WB), Polymerase Chain Reaction (PCR), and Immunohistochemical methods (IHC). The network analysis showed that the TSF pathway network targeted 24 important targets and 149 significant pathways. TSF might have an impact by focusing on essential objectives such as TP53, PTEN, AKT1, BCL2, BCL2L1, PINK-1, PARKIN, LC3B, and NFE2L2, along with various growth-inducing routes. Our findings demonstrated that TSF effectively repaired the structure of mitochondria in db/db mice. TSF greatly enhanced the mRNA levels of PINK-1. WB and IHC findings indicated that TSF had a notable impact on activating the PINK-1/PARKIN signaling pathway in db/db mice, significantly increasing LC3 and NRF2 expression. Our results indicate that TSF effectively addresses DN by activating the PINK-1/PARKIN signaling pathway and enhancing Mitochondrion structure in experimental diabetic nephropathy.

Tangshen formula protects against podocyte apoptosis via enhancing the TFEB-mediated autophagy-lysosome pathway in diabetic nephropathy

Ethnopharmacological relevanceDiabetic nephropathy (DN) is the leading cause of end-stage kidney disease and currently there are no specific and effective drugs for its treatment. Podocyte injury is a detrimental feature and the major cause of albuminuria in DN. We previously reported Tangshen Formula (TSF), a Chinese herbal medicine, has shown therapeutic effects on DN. However, the underlying mechanisms remain obscure. Aim of the studyThis study aimed to explore the protective effect of TSF on podocyte apoptosis in DN and elucidate the potential mechanism. Materials and methodsThe effects of TSF were assessed in a murine model using male KKAy diabetic mice, as well as in advanced glycation end products-stimulated primary mice podocytes. Transcription factor EB (TFEB) knockdown primary podocytes were employed for mechanistic studies. In vivo and in vitro studies were performed and results assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. ResultsTSF treatment alleviated podocyte apoptosis and structural impairment, decreased albuminuria, and mitigated renal dysfunction in KKAy mice. Notably, TSF extracted twice showed a more significant reduction in proteinuria than TSF extracted three times. Accumulation of autophagic biomarkers p62 and LC3, and aberrant autophagic flux in podocytes of DN mice were significantly altered by TSF therapy. Consistent with the in vivo results, TSF prevented the apoptosis of primary podocytes exposed to AGEs and activated autophagy. However, the anti-apoptosis capacity of TSF was countered by the autophagy–lysosome inhibitor chloroquine. We found that TSF increased the nuclear translocation of TFEB in diabetic podocytes, and thus upregulated transcription of its several autophagic target genes. Pharmacological activation of TFEB by TSF accelerated the conversion of autophagosome to autolysosome and lysosomal biogenesis, further augmented autophagic flux. Conversely, TFEB knockdown negated the favorable effects of TSF on autophagy in AGEs-stimulated primary podocytes. ConclusionsThese findings indicate TSF appears to attenuate podocyte apoptosis and promote autophagy in DN via the TFEB-mediated autophagy-lysosome system. Thus, TSF may be a therapeutic candidate for DN.

Tangshen formula improves diabetic nephropathy in STZ-induced diabetes rats fed with hyper-methionine by regulating the methylation status of kidney

BackgroundThe objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250–300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing.ResultsBoth the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes.ConclusionWhile HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation.

Effect of Tangshen formula on the remodeling of small intestine and colon in Zucker diabetic fatty rats

Background and aimPrevious study have demonstrated that Tangshen Formula (TSF) could attenuate colonic histomorphological remodeling in the diabetic rat model induced by high fat diet plus low dosage streptozotocin (STZ). However, it is not clear whether TSF has same effect on small intestine and the effect on biomechanical properties of bowel. The aim of this study is to investigate the effect of TSF on histomorphological and biomechanical remodeling of small intestine and colon by using Zucker Diabetic Fatty (ZDF) Rat model. Materials and methodsZDF rats (obese fa/fa) with blood glucose higher than 11.7 mmol/L were divided into ZDF group (diabetic control group) and ZDF + TSF group (TSF treatment group), the later were intragastrically administered TSF. The ZDF rats (lean fa/+) were served as normal control (ZL) group. The rats in the ZL and ZDF groups were administered with saline. The experimental period covered from 8 weeks to 24 weeks. At the end of experiment, the ileal and colonic segments were studied in vitro. The histomorphometry and biomechanical parameters were measured. ResultsCompared with ZL group histomorphologically, the wet weight per unit length, wall thickness, wall area and fractions of total and type I and type III collagen in different layers for both ileum and colon increased in ZDF group. Those increasing parameters were partially inhibited in ZDF + TSF group. Compared with ZL group biomechanically, ZDF and ZDF + TSF groups had smaller opening angle and residual strain in ileum, and bigger opening angle and residual strain in colon. Whereas the wall became softer in circumferential direction and stiffer in longitudinal direction for both ileum and colon. However, no difference of biomechanical parameters was found between ZDF and ZDF + TSF groups. ConclusionThe histomorphological and biomechanical remodeling of ileum and colon were happened in ZDF rats （obese fa/fa）. TSF could partly attenuate ileal and colonic histomorphological remodeling rather than biomechanical remodeling.

The mechanisms of Qizhu Tangshen formula in the treatment of diabetic kidney disease: Network pharmacology, machine learning, molecular docking and experimental assessment

BackgroundQizhu Tangshen Formula (QZTS) has been shown therapeutic effects on diabetic kidney disease (DKD). However, to date, the pharmacological mechanisms remain vague. MethodsTo explore the underlying mechanisms of QZTS in treating DKD using network pharmacology, machine learning, molecular docking and experimental assessment. ResultsFirst, we found that QZTS improved glycolipid metabolism disorder, decreased proteinuria and alleviated kidney tissue injury in DKD model KKAy mice. Then, by integrating multiple databases, a total of 96 targets of 74 active compounds in QZTS and 759 DKD-related genes were acquired. Next, we identified 13 hub targets of QZTS in DKD by three rank algorithms, including functional similarity, topological similarity and shortest path. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that the pathways mainly centered on the processes of glycolipid metabolism disorder, inflammation and angiogenesis. Among them, VEGF signaling pathway was significantly enriched. Molecular docking showed that key active compounds of QZTS all had relatively good binding affinity with predicted hub targets. Finally, animal experiments found that QZTS significantly inhibited the secretion of plasma VEGF and downregulated the protein and mRNA expression levels of AKT, p38MAPK and VEGFR2. ConclusionOur results indicated that QZTS treated DKD via multiple targets and pathways and the VEGF signaling pathway may be highly involved in this process.

Effect of Tangshen formula on alleviating oxidative renal injury in UUO mice by downregulated expression of NADPH oxidase

ObjectiveTo explore the effect of Tangshen formula on the oxidative renal injury in UUO mice. MethodSeven-week-old male mice C57BL/6N were selected and randomly divided into sham-surgery group, UUO model group (UUO-7d and UUO-14d), Tangshen formula (TF) group (UUO + TF-7d and UUO + TF-14d) and fosinopril group (UUO + fosinopril-7d, UUO + fosinopril-14d). The model were established after 5d medication, and continuous postoperative medications for 7d or 14d were also carried out. The renal pathology was observed, the MDA concentration and SOD activity of renal tissue and serum were evaluated, along with the expression of NADPH oxidase in renal tissue. ResultCompared with that of sham-surgery group, the inflammatory cells in renal interstitium of UUO mice is characterized by diffuse infiltration and dramatical proliferation of collagen fibers, while the introduction of Tangshen formula helps to alleviate this situation, which decreases MDA concentration (all P < 0.001 for renal tissue and serum) but increases SOD activity (P < 0.001 in renal tissue, P < 0.05 in serum), as well as downregulates the expression of Nox4 (P < 0.001) and phosphorylated p47phox (P < 0.001) in renal tissue. ConclusionTangshen formula alleviates the pathological injury of renal tissue in UUO mice and fibrosis of renal interstitium, the underlying mechanism is relative to the downregulated expression of NADPH oxidase, upon which the oxidative stress level of kidney and serum in UUO mice is decreased.

Characteristics of Serum Metabolites and Gut Microbiota in Diabetic Kidney Disease.

Disturbance of circulating metabolites and disorders of the gut microbiota are involved in the progression of diabetic kidney disease (DKD). However, there is limited research on the relationship between serum metabolites and gut microbiota, and their involvement in DKD. In this study, using an experimental DKD rat model induced by combining streptozotocin injection and unilateral nephrectomy, we employed untargeted metabolomics and 16S rRNA gene sequencing to explore the relationship between the metabolic profile and the structure and function of gut microbiota. Striking alterations took place in 140 serum metabolites, as well as in the composition and function of rat gut microbiota. These changes were mainly associated with carbohydrate, lipid, and amino acid metabolism. In these pathways, isomaltose, D-mannose, galactonic acid, citramalic acid, and prostaglandin B2 were significantly upregulated. 3-(2-Hydroxyethyl)indole, 3-methylindole, and indoleacrylic acid were downregulated and were the critical metabolites in the DKD model. Furthermore, the levels of these three indoles were restored after treatment with the traditional Chinese herbal medicine Tangshen Formula. At the genera level, g_Eubacterium_nodatum_group, g_Lactobacillus, and g_Faecalibaculum were most involved in metabolic disorders in the progression of DKD. Notably, the circulating lipid metabolites had a strong relationship with DKD-related parameters and were especially negatively related to the mesangial matrix area. Serum lipid indices (TG and TC) and UACR were directly associated with certain microbial genera. In conclusion, the present research verified the anomalous circulating metabolites and gut microbiota in DKD progression. We also identified the potential metabolic and microbial targets for the treatment of DKD.

TGF-β signaling in diabetic nephropathy: An update

Abstract Diabetic nephropathy (DN) is a common complication in patients with diabetes and the leading cause of end-stage renal disease. Accumulating evidence shows that transforming growth factor beta-1 (TGF-β1) is a key mediator in the pathogenesis of DN. TGF-β1 binds to its receptors to activate canonical and noncanonical downstream signaling pathways to exert its biological activities. Among them, canonical Smad signaling is the major pathway responsible for the development of DN. In addition to TGF-β1, many stress molecules, such as advanced glycation end products (AGEs), angiotensin II (Ang II), and C-reactive protein (CRP), can also activate Mothers against decapentaplegic homologs (Smads) via the extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) cross talk mechanism. Furthermore, TGF-β/Smad signaling can also cross talk with nuclear factor kappa B (NF-κB) signaling to regulate renal inflammation via the induction of IκBα by Smad7. In the context of renal fibrosis, Smad3 is pathogenic, while Smad2 and Smad7 are protective. TGF-β signaling also upregulates the pathogenic microRNAs (miRNAs) (namely, miR-21, miR-192, and miR-377) and long noncoding RNAs (lncRNAs) (namely, Erbb4-IR (intron region, IR), LncRNA9884, and Arid2-IR) but downregulates the protective miRNAs (namely, miR-29a/b and miR-200a) to mediate DN. Thus, targeting TGF-β signaling, either by blocking its ligand, its receptor (i.e., TGF-β receptor-2 [TGFBR2]), Smad3, and downstream miRNAs/lncRNAs or by overexpressing Smad7, has been shown to improve DN. In addition, pharmaceutically targeting TGF-β signaling using chemical inhibitors and traditional Chinese medicine (TCM), including Tangshen formula, Chaihuang-Yishen granule, and herbal extracts (berberine, asiatic acid, and naringenin), also shows renoprotective effect in diabetes. In summary, TGF-β signaling is a critical pathway leading to DN and may be a therapeutic target for combating DN.

UPLC-MS/MS method for the determination of the herb composition of Tangshen formula and the in vivo pharmacokinetics of its metabolites in rat plasma.

Tangshen formula (TSF) is a traditional Chinese medicine composed of seven medicinal herbs including Astragalus membranaceus, Rehmannia glutinosa Libosch, Citrus aurantium L., etc. which is used to treat diabetic nephropathy III, IV qi and yin deficiency and stasis syndrome. Most of the studies on TSF are pharmacological and pharmacodynamic experiments. There are few basic studies on its chemical substances, and the effective constituents are not clear. To analyse the main chemical components of TSF and the absorbed components in rat plasma following oral administration based on liquid chromatography tandem mass spectrometry (LC-MS/MS). Moreover, providing a rapid and valid analytical strategy for simultaneous determination of six components in rat plasma and use it in pharmacokinetic studies. A total of 132 components were identified in TSF, and 44 components were identified in rat plasma after oral TSF, 35 of which were prototype components and nine were metabolic components. A sensitive and reliable LC-MS/MS method was developed for simultaneous determination of six components in rat plasma. The intra-day and inter-day precision relative standard deviation (RSD) was lower than 15%; the accuracy of low, medium and high concentrations ranged from 80% to 120%. The recovery met the requirements and the RSD of the recoveries was less than 15%. A total of 132 components were identified in TSF. The LC-MS/MS quantitative method for the simultaneous determination of morroniside, loganin, notoginsenoside R1 , ginsenoside Re, ginsenoside Rb1 and astragaloside IV in rat plasma was established for the first time. The pharmacokinetic parameters are clarified, which can guide the clinical medication of TSF.

Tangshen Formula Improves Diabetes-Associated Myocardial Fibrosis by Inhibiting TGF-β/Smads and Wnt/β-Catenin Pathways.

Cardiovascular disease has become the main cause of death among complications of diabetes. Myocardial fibrosis is a crucial pathological change of cardiovascular disease. Tangshen Formula (TSF) shows a good clinical effect in the treatment of diabetic kidney disease (DKD). However, whether TSF alleviates diabetes-associated myocardial fibrosis is still unknown. In the present research, we studied the effect and mechanism of TSF in the treatment of myocardial fibrosis in vivo and in vitro. We found that TSF treatment significantly downregulates myocardial fibrosis-related markers, including collagens I and III, and α-SMA. TSF also protects primary mouse cardiac fibroblast (CF) from transforming growth factor-β1- (TGF-β1-) induced damage. Moreover, TSF decreased the expression levels of TGF-β/Smad-related genes (α-SMA, collagens I and III, TGF-β1, and pSmad2/3), and increased Smad7 gene expression. Finally, TSF decreased the expressions of wnt1, active-β-catenin, FN, and MMP7 to regulate the Wnt/β-catenin pathway. Taken together, TSF seems to attenuate myocardial fibrosis in KKAy mice by inhibiting TGF-β/Smad2/3 and Wnt/β-catenin signaling pathways.

Tangshen Formula Attenuates Renal Fibrosis by Downregulating Transforming Growth Factor β1/Smad3 and LncRNA-MEG3 in Rats with Diabetic Kidney Disease

Abstract Background and Objective: The traditional Chinese Tangshen formula (TSF) has been reported to ameliorate diabetic kidney disease (DKD) in humans and animals. However, the effect of TSF on renal fibrosis remains unclear. Transforming growth factor-β1 (TGF-β1)/Smad3 signaling and lncRNA MEG3 are important in renal fibrosis. In this study, we examined the therapeutic effect of TSF on renal fibrosis and explored whether it was related to the modulation of TGFβ1/Smad3 signaling and lncRNA MEG3 expression. Materials and Methods: Experiments were performed in rats in vivo and in the HK2 cells in vitro. DKD was induced in rats by uninephrectomy combined with a single streptozotocin injection. The HK2 cells were stimulated by high glucose (HG) to explore the mechanism of TSF effects in vitro. Results: TSF significantly attenuated renal injury by lowering proteinuria and renal histological damage in DKD rats. TSF reduced collagen deposition by decreasing the expression of the fibrotic indicators collagen I, collagen IV, and fibronectin at the protein and mRNA levels, which suggested that TSF ameliorated DKD by decreasing renal fibrosis. Furthermore, TSF decreased TGF-β1 expression and suppressed the levels of phosphorylated Smad3 and Smad2/3 in vivo. Moreover, TSF downregulated the lncRNA MEG3 level in DKD rats. TSF reversed the upregulation of collagen I and fibronectin expression and downregulated Smad2/3 phosphorylation in the HK2 cells stimulated with HG. Conclusions: TSF ameliorates renal fibrosis in rats with DKD by suppressing TGF-β1/Smad3 signaling and lncRNA MEG3 expression.

Protective Effect of Tangshen Formula () on Interstitial Cells of Cajal in Colon of Diabetic Rats.

To explore the effect of Tangshen Formula (, TSF), a Chinese herbal medicine, on interstitial cells of Cajal (ICC) in the colon of diabetic rats. Fifty-four male Wistar rats were randomly divided into normal control (NC, n=14) and high-fat diet (HFD) groups (n=40). After 6 weeks, the rats in the HFD group were injected intraperitoneally streptozotocin once (30 mg/kg). Thirty rats with fasting blood glucose higher than 11.7 mmol/L were randomly divided into diabetes (DM) and TSF groups, 15 rats in each group. Rats in the NC and DM groups were intragastrically administered with saline, and those in the TSF group were given with TSF (2.4 g/kg) once daily for 20 weeks. Expression levels of Bax, Bcl-2, and caspase-3 in colonic smooth muscle layer were measured by Western blotting and immunohistochemical staining. The number of ICC was determined by immunohistochemical staining. Immunofluorescence was used for analyzing the ratio of classically activated macrophages (M1) and alternatively activated macrophages (M2) to total macrophages. Electron microscopy was used to observe the epithelial ultrastructure and junctions. TSF appeared to partially prevented loss of ICC in DM rats (P<0.05). Compared with the NC group, expression levels of Bcl-2, Bax, caspase-3, and TNF-α as well as the ratio of M1 to total macrophages increased in DM rats (all P<0.05), and the ratio of M2 to total macrophages decreased (P<0.05 or P<0.01). Compared with the DM group, TSF decreased the expression levels of abovementioned proteins and restore M2 to total macrophages ratio (P<0.05 or P<0.01). TSF appeared to attenuate the ultrastructural changes of epithelia and improve the tight and desmosome junctions between epithelia reduced in the DM rats. Reduced number of ICC in DM rats may be associated with damage of the intestinal barrier. The protective effects of TSF on ICC may be through repair of the epithelial junctions, which attenuates inflammation and inflammation-initiated apoptosis in colon of DM rats.

Mechanism of tangshen formula in treating diabetic nephropathy revealed by network pharmacology approach.

Mechanism of tangshen formula in treating diabetic nephropathy revealed by network pharmacology approach.

A novel and comprehensive strategy for quality control in complex Chinese medicine formula using UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS combined with network pharmacology analysis: Take Tangshen formula as an example

The quality control of Chinese herbal medicines (CHM) is a key concern on the modernization and globalization. However, it is still a difficult task due to its multi-component, multi-target, multi-pathways. This study aims to provide a novel and comprehensive strategy for quality control in complex Chinese medicines (CHM) formulas by UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS combined with network pharmacology analysis. Tangshen formula (TSF) was used as an example for complex CHM formulas. The UHPLC-Q-Orbitrap HRMS was firstly applied to identify or tentatively assign 85 compounds in TSF. Subsequently, key active compounds for TSF treating diabetic nephropathy (DN) were chose by chemical-target-pathways network in network pharmacology. The results showed that 13 key bioactive compounds against DN including naringin, daidzein, genistein, formononetin, chlorogenic acid, aloe-emodin, nobiletin, tangeritin, ginsenoside Rg1, hesperetin, hesperidin, rhein, and limonin with three high topological features in chemical-target-pathways network were selected as Q-markers for quality control of TSF. Finally, the UHPLC-MS/MS was performed to simultaneously determine the concentrations of 13 Q-markers. And their concentrations were ranged from 11.57 to 3 788 µg·g−1. It suggested that many key bioactive compounds not only have high contents but also have wide range contents for the quality of complex CHM formulas. This study should be helpful to guide the selection of the Q-markers and provide new strategy for quality control of complex CHM formulas.

A Network Pharmacology-Based Approach for Exploring Key Active Compounds and Pharmacological Mechanisms of Tangshen Formula for Treatment of Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the common and severe microvascular complications of diabetes mellitus (DM). The occurrence and development of DN are related to multiple factors in the human body, which makes DN a complex disease, and the pathogeneses of DN have not yet been fully illustrated. Furthermore, DN lacks effective drugs for treatment nowadays. Chinese herbal medicine (CHM) often shows the feature of multicomponents, multitargets, multipathways, and synergistic effects and shows a promising source of new therapeutic drugs for DN. As a CHM, Tangshen Formula (TSF) was used to treat DN patients in China. However, its bioactive compounds and holistic pharmacological mechanisms on DN are both unclear. A network pharmacology approach was firstly applied to explore multiple active compounds and multiple key pharmacological mechanisms for TSF treating DN by drug-targeted interaction databases, herb-compound-target network, protein-protein interaction network, compound-target-pathway network, and analysis methods. And the results showed that TSF have the characteristic of multicomponents, multitargets, multipathways, and synergistic effects for treating DN. The quercetin, naringenin, kaempferol, and isorhamnetin as key active compounds and the PI3K-Akt signaling pathway, TNF signaling pathway, nonalcoholic fatty liver disease (NAFLD), focal adhesion, rap1 signaling pathway, T cell receptor signaling pathway, MAPK signaling pathway, and insulin resistance as the key molecular mechanisms play important roles in TSF treating DN. Moreover, quercetin, naringenin, kaempferol, and isorhamnetin were successfully detected in TSF by the UHPLC-MS/MS analysis method. And their concentrations were 0.224, 8.295, 0.0564, and 0.0879 mg·kg-1, respectively. The present findings not only provide new insights for a deeper understanding of the constituent basis and pharmacology of TSF but also provide guidance for further pharmacological studies on TSF.

Tangshen Formula Attenuates Diabetic Kidney Injury by Imparting Anti-pyroptotic Effects via the TXNIP-NLRP3-GSDMD Axis.

We previously reported that Tangshen formula (TSF), a Chinese herbal medicine for diabetic kidney disease (DKD) therapy, imparts renal protective effects. However, the underlying mechanisms of these effects remain unclear. Pyroptosis is a form of programmed cell death that can be triggered by the NLRP3 inflammasome. Recently, the association between the pyroptosis of renal resident cells and DKD was established, but with limited evidence. This study aimed to investigate whether the renal protective effects of TSF are related to its anti-pyroptotic effect. DKD rats established by right uninephrectomy plus a single intraperitoneal injection of STZ and HK-2 cells stimulated by AGEs were used. In vivo, TSF reduced the 24 h urine protein (24 h UP) of DKD rats and alleviated renal pathological changes. Meanwhile, the increased expression of pyroptotic executor (GSDMD) and NLRP3 inflammasome pathway molecules (NLRP3, caspase-1, and IL-1β) located in the tubules of DKD rats were downregulated with TSF treatment. In vitro, we confirmed the existence of pyroptosis in AGE-stimulated HK-2 cells and the activation of the NLRP3 inflammasome. TSF reduced pyroptosis and NLRP3 inflammasome activation in a dosage-dependent manner. Then, we used nigericin to determine that TSF imparts anti-pyroptotic effects by inhibiting the NLRP3 inflammasome. Finally, we found that TSF reduces reactive oxygen species (ROS) production and thioredoxin-interacting protein (TXNIP) expression in AGE-stimulated HK-2 cells. More importantly, TSF decreased the colocalization of TXNIP and NLRP3, indicating that ROS-TXNIP may be the target of TSF. In summary, the anti-pyroptotic effect via the TXNIP-NLRP3-GSDMD axis may be an important mechanism of TSF for DKD therapy.

Protective Role of Tangshen Formula on the Progression of Renal Damage in db/db Mice by TRPC6/Talin1 Pathway in Podocytes.

Tangshen Formula (TSF) is a Chinese Medicine formula that has been reported to alleviate proteinuria and protect renal function in humans and animals with diabetic kidney disease (DKD). However, little is known about its mechanism in improving proteinuria. The dysregulation of podocyte cell-matrix adhesion has been demonstrated to play an important role in the pathogenesis and progression of proteinuric kidney diseases including DKD. In the present study, the underlying protective mechanism of TSF on podocytes was investigated using the murine model of type 2 DKD db/db mice in vivo and advanced glycation end products (AGEs)-stimulated primary mice podocytes in vitro. Results revealed that TSF treatment could significantly mitigate reduction of podocyte numbers and foot process effacement, reduce proteinuria, and protect renal function in db/db mice. There was a significant increase in expression of transient receptor potential canonical channel 6 (TRPC6) and a decrease in expression of talin1 in podocytes of db/db mice. The results of AGEs-stimulated primary mice podocytes showed increased cell migration and actin-cytoskeleton rearrangement. Moreover, primary mice podocytes stimulated by AGEs displayed an increase in TRPC6-dependent Ca2+ influx, a loss of talin1, and translocation of nuclear factor of activated T cell (NFATC) 2. These dysregulations in mice primary podocytes stimulated by AGEs could be significantly attenuated after TSF treatment. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC6 agonist, blocked the protective role of TSF on podocyte cell-matrix adherence. In conclusion, TSF could protect podocytes from injury and reduce proteinuria in DKD, which may be mediated by the regulation of the TRPC6/Talin1 pathway in podocytes.