This study aimed to investigate the effects and mechanisms of the Supplemented Shenqi Dihuang Decoction Formula(SSDDF) in improving podocyte pyroptosis in diabetic kidney disease(DKD) by regulating dyslipidemia-mediated lipid droplet accumulation and the Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. First, a modified DKD rat model was established. After successful modeling, the DKD rats were administered low-dose SSDDF(SSDDF-L), high-dose SSDDF(SSDDF-H), dapagliflozin(DAPA), or saline(vehicle) by gavage for six consecutive weeks. In the in vivo study, the general condition of the rats, blood glucose(BG), urinary albumin(UAlb), liver and renal function indexes, blood lipid profiles, and serum short-chain fatty acid(SCFA) levels were examined. Glomerular histopathological features, podocyte foot process morphology, and glomerular basement membrane(GBM) ultrastructure were compared. The protein expression levels of podocyte slit diaphragm structural molecules(nephrin and podocin) in renal tissue, key signaling molecules of the TLR4/MyD88/NF-κB pathway [TLR4, MyD88, phosphorylated inhibitor of NF-κBα(p-IκBα), and phosphorylated NF-κB p65(p-p65)],key molecules of the classical pyroptosis pathway [NOD-like receptor thermal protein domain-associated protein 3(NLRP3), apoptosis-associated speck-like protein containing a CARD(ASC), cleaved-caspase-1, and gasdermin D-N(GSDMD-N)], and effector inflammatory factors [interleukin-1β(IL-1β) and interleukin-18(IL-18)] were analyzed. Second, in the in vitro study, a lipotoxic podocyte injury model was constructed by treating MPC-5 cells with high glucose(HG) and palmitic acid(PA). The injured cells were then treated with either SSDDF-H-containing serum or the TLR4 inhibitor TAK-242. The lipid droplet characteristics and the expression levels of proteins associated with the TLR4/MyD88/NF-κB pathway and pyroptosis in podocytes were observed and analyzed. Additionally, in the network pharmacology study, potential targets of SSDDF and DKD were predicted and screened using the HERB and SwissTargetPrediction databases, and a "drug-component-target network" of SSDDF was constructed. DKD-related targets were integrated from the OMIM, GeneCards, and TTD databases. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses for the intersecting targets were performed using the DAVID database. Molecular docking was performed between the core components of SSDDF and key targets. The network pharmacology results suggested that the therapeutic effects of SSDDF on DKD were likely related to the regulation of lipid metabolism and inflammation-related signaling pathways. The in vivo results showed that SSDDF-L and SSDDF-H improved, to varying degrees, the general condition and the levels of BG, UAlb, serum creatinine(Scr), blood urea nitrogen(BUN), total cholesterol(TC), triglyceride(TG), the ratio of low-density lipoprotein cholesterol(LDL-C) to high-density lipoprotein cholesterol(HDL-C), and SCFA in DKD rats, ameliorated glomerular histopathological changes and podocyte ultrastructure, and improved renal protein expression levels of nephrin, podocin, TLR4, MyD88, p-IκBα, p-p65, NLRP3, ASC, cleaved caspase-1, GSDMD-N, IL-1β, and IL-18. Notably, regarding the improvement of proteinuria in the modified DKD rats, SSDDF was less effective than DAPA. However, for renal function and SCFA levels, SSDDF-H was superior to DAPA, and for blood lipid regulation, glomerulosclerosis, and podocyte injury, SSDDF showed similar efficacy to DAPA. The in vitro results showed that both SSDDF-H-containing serum and the TLR4 inhibitor reduced lipid droplet accumulation, inhibited TLR4/MyD88/NF-κB pathway activation, and alleviated podocyte pyroptosis, with comparable effects. In conclusion, SSDDF ameliorates podocyte pyroptosis in DKD by regulating dyslipidemia-mediated lipid droplet accumulation and the TLR4/MyD88/NF-κB signaling pathway. These findings provide pharmacological evidence for precisely elucidating the scientific connotation of the Jinling school's therapeutic approach to "Shenxiao disease".

Background:Therapeutic heterogeneity limits the efficacy of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC). Shuyu Wan (SYW), a classic TCM formula, has shown potential in modulating gut microbiota (GM) and enhancing immunotherapy, yet its synergistic mechanism with PD-1 inhibitors remains unclear. Materials and Methods:SYW components were identified by UPLC-MS. NSCLC-related targets were integrated with SYW targets for pathway enrichment, and molecular docking validated component-target binding. NSCLC syngeneic mice were treated with SYW and/or PD-1 inhibitor (RMP1-14). Tumor growth, histopathology, serum cytokines, tumor-infiltrating CD8+T cell subsets (flow cytometry), PD-1/PD-L1 expression and co-localization (immunofluorescence), GM composition (16S rRNA), and metabolomics were assessed. FMT verified the role of GM-TME crosstalk. Results:SYW monotherapy showed no significant tumor inhibition, whereas SYW combined with PD-1 inhibitor dose-dependently suppressed NSCLC growth. The combination reduced PD-1 expression and PD-1/PD-L1 co-localization, elevated serum IL-12, IFN-γ, and TNF-α, increased total tumor-infiltrating CD8+ T cells, decreased PD-1+ and TIM-3+ exhausted subsets, and expanded IFN-γ+ and Granzyme B+ effector subsets. Concurrently, it reshaped GM (increased Bacillota, decreased Patescibacteria) and altered metabolites (L-glycine, L-proline). These effects were abolished in antibiotic treated mice and restored by FMT, suggesting GM-TME crosstalk as essential. Conclusion:SYW acts as a microbiota-dependent immune sensitizer that potentiates PD-1 inhibitor efficacy in NSCLC by remodeling GM and enhancing effector CD8+ T cell infiltration while reducing exhaustion. GM-TME crosstalk is the potential mechanism, supporting SYW as an adjunct to PD-1 blockade in NSCLC therapy.

Introduction:Rhein, an anthraquinone derived from rhubarb, exhibits renoprotective effects in aging and kidney injury; however, the mechanistic interplay with TNF-α-mediated cell death pathways remains undefined. Methods:Using D-galactose (D-gal)-treated NRK-52E cells and aged rats, we assessed rhein's effects with/without mTOR regulators (rapamycin/MHY1485) or etanercept (TNF-α inhibitor). Protein levels of klotho, phosphorylated (p)-mTOR, p-p62, caspase-8, beclin1, light chain 3 II, receptor-interacting protein kinase (RIPK)1, RIPK3, and p-mixed lineage kinase-like (MLKL) were measured. The concentration of reactive oxygen species (ROS) and the staining for senescence-associated-β-galactosidase (SA-β-gal) were also assessed. The network framework of "rhein-target-pathways" was identified. Additionally, serum untargeted metabolomics and KEGG pathway analysis were performed to identify altered metabolites and underlying metabolic pathways. Results:The results indicated that rhein increased the protein levels of klotho, p-mTOR, p-p62, and caspase-8, as well as decreased the concentration of ROS, the staining for SA-β-gal, and the protein levels of beclin1, light chain 3 II, RIPK1, RIPK3, and p-MLKL in NRK-52E cells exposed to D-gal. Compared to mTOR regulators (rapamycin or MHY1485) alone, the co-treatment of rhein and mTOR regulators decreased mTOR-mediated autophagy signaling in NRK-52E cells treated by D-gal. In addition, rhein decreased tumor necrosis factor (TNF)-α and TNF-α receptor1 protein levels. Interestingly, the effects of etanercept in TNF-α-mediated necroptosis and autophagy were similar to those of rhein. Consistent with the in vitro findings, rhein alleviated oxidative stress and exerted renal protective effects in aged model rats. Serum untargeted metabolomic analysis revealed that rhein treatment significantly altered 85 metabolites compared to the model group rats, with 35 metabolites upregulated and 50 downregulated. KEGG pathway analysis identified the TNF-α signaling pathway as a key metabolic pathway involved. Discussion:In conclusion, rhein protected the kidneys by activating p-mTOR and downregulating TNF-α, necroptosis and autophagy. Rhein mitigates renal aging and fibrotic injury by targeting TNF-α-mediated autophagy-necroptosis crosstalk, positioning it as a novel multi-target therapeutic agent for age-related kidney injury.

This study aims to explore the effects and mechanisms of the core formula(TCF)improving glomerulosclerosis(GS)in diabetic kidney disease(DKD)by Jinling medical school based on reticulophagy(ER-phagy)-pyroptosis-apoptosis-necroptosis(PANoptosis)signaling pathway in podocytes.Firstly,the modified DKD rat models were established.After modeling successfully,the DKD rat models received either TCF,empagliflozin(EMPA),or saline by gavage for 8 weeks,respectively.Secondly,MPC5 cells were subjected to treatment under high-glucose(HG)conditions alone,or HG combined individually with drug-containing serum of the low-dose of TCF(L-TCF)and the high-dose of TCF(H-TCF),EMPA,or Torin 1(autophagy agonist).In the in vivo studies,the changes of general conditions and renal injury indicators,pathological characteristics of GS,and the protein expression levels of podocyte marker molecules were observed,respectively.Furthermore,the changes of the level of reactive oxygen species(ROS)in the kidneys,the expression levels of the core receptor and marker molecules of ER-phagy,and the expression degree of Z-DNA binding protein 1(ZBP1)were also observed.In the in vivo and in vitro studies,we analyzed changes in the expression levels of ZBP1 protein in the kidneys and podocytes,as well as the protein expression levels of key signaling molecules in the cellular PANoptosis were investigated,respectively.The in vivo study results showed that,for the DKD model rats,general conditions and renal injury indicators,pathological characteristics of GS,and the protein expression levels of podocyte marker molecules were improved,respectively.Moreover,the level of ROS in the kidneys,the expression level of the core receptor and marker molecules of ER-phagy,and the expression degree and protein expression level of ZBP 1,as well as the protein expression levels of key signaling molecules in the cellular PANoptosis were ameliorated,respectively.Notably,the improvement effects of TCF and EMPA were basically similar,but the beneficial effect of the protein expression level of interleukin-18(IL-18)in the kidneys,as an inflammatory factor,was superior to EMPA.The in vitro study results showed that,high and low doses of TCF and EMPA both could ameliorate the protein expression levels of key signaling molecules in the PANoptosis pathway in podocytes treated with HG,and the improvement effect of H-TCF was superior to that of L-TCF or EMPA.In conclusion,the study clarifies that TCF improves GS in DKD by regulating ER-phagy-PANoptosis signaling pathway in podocytes in vivo and in vitro,and has laid the groundwork for exploring mechanisms and scientific implications of treating diabetic kidney disease by Jinling medical school.

This study aims to explore the medication rules and mechanisms of treating chronic renal failure(CRF)by Jinling medical school based on data mining,network pharmacology,and experimental validation systematically and deeply.Firstly,the study selected the papers published by the inherited clinicians in Jinling medical school in Chinese journals using the subject headings named"traditional Chinese medicine(TCM)+chronic renal failure","TCM+chronic renal inefficiency",or"TCM+consumptive disease"in China National Knowledge Infrastructure,Wanfang,and VIP Chinese Science and Technology Periodical Database and screened TCM formulas for treating CRF according to inclusion and exclusion criteria.The study analyzed the frequency of use of single TCM and the four properties,five tastes,channel tropism,and efficacy of TCM used with high frequency and performed association rule and clustering analysis,respectively.As a result,a total of 215 TCM formulas and 235 different single TCM were screened,respectively.The TCM used with high frequency included Astragali Radix,Rhei Radix et Rhizoma,Salviae Miltiorrhizae Radix et Rhizoma,Poria,and Atractylodis Macrocephalae Rhizoma(top 5).The single TCM characterized by"cold properties,sweet flavor,and restoring spleen channel"and the TCM with the efficacy of tonifying deficiency had the highest frequency of use,respectively.Then,the TCM with the rules of"blood-activating and stasis-removing"and"diuretic and dampness-penetrating"appeared.In addition,the core combination of TCM[(Hexin Formula,HXF)]included"Astragali Radix,Rhei Radix et Rhizoma,Poria,Salviae Miltiorrhizae Radix,and Angelicae Sinensis Radix".The network pharmacology analysis showed that HXF had 91 active compounds and 250 corresponding protein targets including prostaglandin-endoperoxide synthase 2(PTGS2),PTGS1,sodium voltage-gated channel alpha subunit 5(SCN5A),cholinergic receptor muscarinic 1(CHRM1),and heat shock protein 90 alpha family class A member 1(HSP90AA1)(top 5).Gene Ontology(GO)function analysis revealed that the core targets of HXF predominantly affected biological processes,cellular components,and molecular functions such as positive regulation of transcription by ribonucleic acid polymerase Ⅱ and DNA template transcription,formation of cytosol,nucleus,and plasma membrane,and identical protein binding and enzyme binding.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed that CRF-related genes were involved in a variety of signaling pathways and cellular metabolic pathways,primarily involving"phosphatidylinositol 3-kinase(PI3K)-protein kinase B(Akt)pathway"and"advanced glycation end products-receptor for advanced glycation end products".Molecular docking results showed that the active components in HXF such as isomucronulatol 7-O-glucoside,betulinic acid,sitosterol,and przewaquinone B might be crucial in the treatment of CRF.Finally,a modified rat model with renal failure induced by adenine was used,and the in vivo experimental confirmation was performed based on the above-mentioned predictions.The results verify that HXF can regulate mitochondrial autophagy in the kidneys and the PI3K-Akt-mammalian target of rapamycin(mTOR)signaling pathway activation at upstream,so as to alleviate renal tubulointerstitial fibrosis and then delay the progression of CRF.

This study aimed to explore the mechanisms and molecular targets of total flavones of Abelmoschus manihot(TFA) plus empagliflozin(EM) in attenuating diabetic tubulopathy(DT) by targeting mitochondrial homeostasis and pyroptosis-apoptosis-necroptosis(PANoptosis). In the in vivo study, the authors established the DT rat models through a combination of uninephrectomy, administration of streptozotocin via intraperitoneal injections, and exposure to a high-fat diet. Following modeling successfully, the DT rat models received either TFA, EM, TFA+EM, or saline(as a vehicle) by gavage for eight weeks, respectively. In the in vitro study, the authors subjected the NRK52E cells with or without knock-down Z-DNA binding protein 1(ZBP1) to a high-glucose(HG) environment and various treatments including TFA, EM, and TFA+EM. In the in vivo and in vitro studies, The authors investigated the relative characteristics of renal tubular injury and renal tubular epithelial cells damage induced by reactive oxygen species(ROS), analyzed the relative characteristics of renal tubular PANoptosis and ZBP1-mediatted PANoptosis in renal tubular epithelial cells, and compared the relative characteristics of the protein expression levels of marked molecules of mitochondrial fission in the kidneys and mitochondrial homeostasis in renal tubular epithelial cells, respectively. Furthermore, in the network pharmacology study, the authors predicted and screened targets of TFA and EM using HERB and SwissTargetPrediction databases; The screened chemical constituents and targets of TFA and EM were constructed the relative network using Cytoscape 3.7.2 network graphics software; The relative targets of DT were integrated using OMIM and GeneCards databases; The intersecting targets of TFA, EM, and DT were enriched and analyzed signaling pathways by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG) software using DAVID database. In vivo study results showed that TFA+EM could improve renal tubular injury, the protein expression levels and characteristics of key signaling molecules in PANoptosis pathway in the kidneys, and the protein expression levels of marked molecules of mitochondrial fission in the kidneys. And that, the ameliorative effects in vivo of TFA+EM were both superior to TFA or EM. Network pharmacology study results showed that TFA+EM treated DT by regulating the PANoptosis signaling pathway. In vitro study results showed that TFA+EM could improve ROS-induced cell injury, ZBP1-mediatted PANoptosis, and mitochondrial homeostasis in renal tubular epithelial cells under a state of HG, including the protein expression levels of marked molecules of mitochondrial fission, mitochondrial ultrastructure, and membrane potential level. And that, the ameliorative effects in vitro of TFA+EM were both superior to TFA or EM. More importantly, using the NRK52E cells with knock-down ZBP1, the authors found that, indeed, ZBP1 was mediated PANoptosis in renal tubular epithelial cells as an upstream factor. In addition, TFA+EM could regulate the protein expression levels of marked signaling molecules of PANoptosis by targeting ZBP1. In summary, this study clarified that TFA+EM, different from TFA or EM, could attenuate DT with multiple targets by ameliorating mitochondrial homeostasis and inhibiting ZBP1-mediated PANoptosis. These findings provide the clear pharmacological evidence for the clinical treatment of DT with a novel strategy of TFA+EM, which is named "coordinated traditional Chinese and western medicine".

This study explores the role and underlying molecular mechanisms of fucoidan sulfate(FPS)in regulating heme oxygenase-1(Hmox1)-mediated ferroptosis to ameliorate myocardial injury in diabetic cardiomyopathy(DCM)through in vivo and in vitro experiments and network pharmacology analysis.In vivo,a DCM rat model was established using a combination of"high-fat diet feeding+two low-dose streptozotocin(STZ)intraperitoneal injections".The rats were randomly divided into four groups:normal,model,FPS,and dapagliflozin(Dapa)groups.In vitro,a cellular model was created by inducing rat cardiomyocytes(H9c2 cells)with high glucose(HG),using zinc protoporphyrin(ZnPP),an Hmox1 inhibitor,as the positive control.An automatic biochemical analyzer was used to measure blood glucose(BG),serum aspartate aminotransferase(AST),serum lactate dehydrogenase(LDH),and serum creatine kinase-MB(CK-MB)levels.Echocardiography was used to assess rat cardiac function,including ejection fraction(EF)and fractional shortening(FS).Pathological staining was performed to observe myocardial morphology and fibrotic characteristics.DCFH-DA fluorescence probe was used to detect reactive oxygen species(ROS)levels in myocardial tissue.Specific assay kits were used to measure serum brain natriuretic peptide(BNP),myocardial Fe2+,and malondialdehyde(MDA)levels.Western blot(WB)was used to detect the expression levels of myosin heavy chain 7B(MYH7B),natriuretic peptide A(NPPA),collagens type Ⅰ(Col-Ⅰ),α-smooth muscle actin(α-SMA),ferritin heavy chain 1(FTH1),solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4),4-hydroxy-2-nonenal(4-HNE),and Hmox1.Immunohistochemistry(IHC)was used to examine Hmox1 protein expression patterns.FerroOrange and Highly Sensitive DCFH-DA fluorescence probes were used to detect intracellular Fe2+and ROS levels.Transmission electron microscopy was used to observe changes in mitochondrial morphology.In network pharmacology,FPS targets were identified through the PubChem database and PharmMapper platform.DCM-related targets were integrated from OMIM,GeneCards,and DisGeNET databases,while ferroptosis-related targets were obtained from the FerrDb database.A protein-protein interaction(PPI)network was constructed for the intersection of these targets using STRING 11.0,and core targets were screened with Cytoscape 3.9.0.Molecular docking analysis was conducted using AutoDock and PyMOL 2.5.In vivo results showed that FPS significantly reduced AST,LDH,CK-MB,and BNP levels in DCM model rats,improved cardiac function,decreased the expression of myocardial injury proteins(MYH7B,NPPA,Col-Ⅰ,and α-SMA),alleviated myocardial hypertrophy and fibrosis,and reduced Fe2+,ROS,and MDA levels in myocardial tissue.Furthermore,FPS regulated the expression of ferroptosis-related markers(Hmox1,FTH1,SLC7A11,GPX4,and 4-HNE)to varying degrees.Network pharmacology results revealed 313 potential targets for FPS,1 125 targets for DCM,and 14 common targets among FPS,DCM,and FerrDb.Hmox1 was identified as a key target,with FPS showing high docking activity with Hmox1.In vitro results demonstrated that FPS restored the expression levels of ferroptosis-related proteins,reduced intracellular Fe2+and ROS levels,and alleviated mitochondrial structural damage in cardiomyocytes.In conclusion,FPS improves myocardial injury in DCM,with its underlying mechanism potentially involving the regulation of Hmox1 to inhibit ferroptosis.This study provides pharmacological evidence supporting the therapeutic potential of FPS for DCM-induced myocardial injury.

This study aims to observe the effects of the traditional Chinese medicine prescription Dahuang Zhechong Pills(DHZCP)on renal aging and explore its potential multi-target effects.Rats were assigned into the normal,model,DHZCP,and vitamin E(VE)groups.Firstly,the rat model of D-galactose(D-gal)-induced renal aging was established.During the modeling period,the rats in the 4 groups were administrated with double distilled water,double distilled water,DHZCP suspension,and VE suspension,respectively,by gavage every day.On day 60 of intervention,the indicators of renal aging and injury in rats were measured,including the function,histopathological characteristics,senescence-associated β-galactosidase(SA-β-gal)staining,and expression levels of Klotho and proteins associated with cell cycle arrest and senescence-associated secretory phenotype(SASP)in the renal tissue.Moreover,non-targeted metabolomic analysis of the renal tissue was performed for the 4 groups of rats to screen out the potential biomarkers and metabolic pathways.Finally,the signaling pathways of key targets were preliminarily validated.The results showed that DHZCP and VE significantly improved the renal function,histopathological features of renal tubular/interstitial tissue,and degree of SA-β-gal staining,up-regulated the expression level of Klotho,and down-regulated the expression levels of proteins associated with cell cycle arrest and SASP in the renal tissue of the aging rats.In addition,DHZCP and VE regulated the metabolites in the renal tissue of the aging rats.There were 21 common differential metabolites.Among them,5 differential metabolites were significantly increased in the aging rats and recovered after DHZCP or VE treatment,and they were involved in the lipid metabolism and energy metabolism pathways.The areas under the curves of the groups in comparison varied within the range of 0.88-1.DHZCP regulated multiple signaling pathways,such as the adenosine monophosphate-activated protein kinase(AMPK),cyclic guanosine monophosphate-protein kinase G(cGMP-PKG),cyclic adenylic acid(cAMP),phosphatidylinositol-3-kinase-protein kinase B(PI3K-Akt),mammalian target of rapamycin(mTOR),and autophagy signaling pathways.In addition,it affected the multiple metabolic pathways,such as renin secretion,longevity regulation pathway,diabetic cardiomyopathy,and niacin and nicotinamide metabolism.DHZCP and VE significantly up-regulated the expression level of the key proteins in the AMPK signaling pathway in the renal tissue of the aging rats.In all,DHZCP and VE could mitigate renal aging and injury.DHZCP exerted multi-target effects via multiple signaling pathways and metabolic pathways in the kidney,in which the AMPK signaling pathway may be one of the key targets for action.

Rhein as an anthraquinone compound isolated from rhubarb is considered effective in treating kidney diseases and renal aging. However, the molecular mechanisms underlying the protective effects of rhein in aged kidney are not fully elucidated. In this study, we observed the renal protective effects of rhein and investigated the therapeutic mechanisms of rhein in oxidative stress-induced renal aging and injury. The results indicated that rhein increased the protein levels of klotho, phosphorylated (p)-mTOR, p-p62, and caspase-8, as well as decreased the concentration of reactive oxygen species (ROS), the staining for senescence-associated-β-galactosidase (SA-β-gal) and the protein levels of beclin1, light chain 3 II, receptor-interacting protein kinase (RIPK)1, RIPK3, and p-mixed lineage kinase-like (MLKL) in tubular cells (NRK-52E cells) exposed to D-galactose (D-gal). Compared with mTOR regulators (rapamycin or MHY1485) alone, the co-treatment of rhein and mTOR regulators decreased mTOR-mediated autophagy signaling in NRK-52E cells treated by D-gal. In addition, the research identified that tumor necrosis factor (TNF)-α signaling plays a pivotal role in the development of the “rhein–target–pathways” network framework. Rhein decreased TNF-α and TNF-α receptor1 protein levels. Interestingly, the changes in the effect of etanercept in TNF-α-mediated necroptosis and autophagy were similar to those of rhein. Accordingly, in D-gal-induced aged model rats, rhein alleviated aged kidneys by activating p-mTOR and downregulating autophagy signaling. Our results suggested that rhein exerted reducing oxidative stress level and renoprotective effects on D-gal-induced renal aging and injury by mTOR-mediated autophagy and RIPK1/RIPK3/MLKL-dependent necroptosis. Further, rhein may regulate TNF-α-mediated autophagy and necroptosis in aged tubular cells. Thus, rhein may be a potential therapeutic drug against renal aging and injury.

Ethnopharmacological relevance Recently, podocyte mitochondrial dysfunction and necroptosis have been shown to play critical roles in renal fibrosis (RF) in diabetic kidney disease (DKD); however, these conditions lack effective treatment. In China, the supplemented Gegen Qinlian Decoction Formula (SGQDF), which originates from the classical prescription Gegen Qinlian Decoction, has been widely used to treat patients with DKD. However, it remains unclear whether SGQDF alleviates podocyte injury-associated RF in patients with DKD. Aim of study This study aimed to clarify the therapeutic effects of SGQDF compared with those of empagliflozin (EMPA) on podocyte mitochondrial fission and RF in DKD and its necroptosis-related mechanisms. Materials and methods Modified DKD rat models were developed through a combination of uninephrectomy, streptozotocin administration through intraperitoneal injection, and exposure to a high-fat diet. Following RF formation, the DKD rat models received either a high dose of SGQDF (H-SGQDF), a low dose of SGQDF (L-SGQDF), EMPA, or vehicle for 4 weeks. In our in vitro study, we subjected cultured murine podocytes to a high-glucose environment and various treatments including Mdivi-1, adalimumab, and necrostatin-1, with or without H-SGQDF or EMPA. SGQDF target prediction and molecular docking verification were performed. For the in vivo study, we focused on examining changes in the parameters associated with renal injury, RF, and oxidative stress (OS)-induced injuries in podocytes. Both in vivo and in vitro studies included an analysis of changes in podocyte mitochondrial fission, TNF-α-induced podocyte necroptosis, and the RIPK1/RIPK3/MLKL signaling pathway activation. Results SGQDF improved renal injury markers, including body weight, blood glucose, serum creatinine, blood urea nitrogen, and urinary albumin, in a dose-dependent manner. The beneficial effects of H-SGQDF in vivo were greater than those of L-SGQDF alone in vivo. Interestingly, similar to EMPA, H-SGQDF ameliorated RF and reduced OS-induced podocyte injury in diabetic kidneys. Furthermore, TNF-α signaling was shown to be important in the network construction of “the SGQDF-component-target.” Based on this, we also showed that the beneficial effects in vivo and in vitro of H-SGQDF were closely related to the improvement in mitochondrial dysfunction and the inhibition of TNF-α-induced necroptosis in podocytes. Conclusion In the present study, we showed that H-SGQDF, similar to EMPA, attenuates podocyte mitochondrial fission and RF, and that the underlying therapeutic mechanisms are closely related to inhibiting the activation of the RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. Our findings provide new pharmacological evidence for the application of H-SGQDF in the RF treatment of DKD.

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.

Recent studies have shown that the occurrence and development of common liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and liver cancer, are related to liver aging(LA). Therefore, to explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a traditional classic prescription in improving LA with multiple targets, the present study randomly divided 24 rats into a normal group, a model group, a DHZCP group, and a vitamin E(VE) group, with six rats in each group. The LA model was induced by continuous intraperitoneal injection of D-galactose(D-gal) in rats. For the LA model rats, the general situation was evaluated by aging phenotype and body weight(BW). LA was assessed by the pathological characteristics of hepatocyte senescence, hepatic function indexes, the staining characteristics of phosphorylated histone family 2A variant(γ-H2AX), and the expression levels of cell cycle arrest proteins(P21, P53, P16) and senescence-associated secretory phenotype(SASP) in the liver. The activation of the reactive oxygen species(ROS)-mediated phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/forkhead box protein O4(FoxO4) signaling pathway was estimated by hepatic ROS expression feature and the protein expression levels of the key signaling molecules in the PI3K/Akt/FoxO4 signaling pathway. The results showed that after the treatment with DHZCP or VE for 12 weeks, for the DHZCP and VE groups, the characterized aging phenotype, BW, pathological characteristics of hepatocyte senescence, hepatic function indexes, relative expression of ROS in the liver, protein expression levels of key signaling molecules including p-PI3K, p-Akt, and FoxO4 in the liver, staining characteristics of γ-H2AX, and the protein expression levels of P16, P21, P53, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the liver were improved, and the effects of DHZCP and VE were similar. Based on the D-gal-induced LA model in rats, this study demonstrates that DHZCP can ameliorate LA with multiple targets in vivo, and its effects and mechanism are related to regulating the activation of the ROS-mediated PI3K/Akt/FoxO4 signaling pathway in the liver. These findings are expected to provide new pharmacological evidence for the treatment of DHZCP in aging-related liver diseases.

This study aimed to explore the effects and mechanisms of total flavones of Abelmoschus manihot(TFA), the extracts from traditional Chinese medicine indicated for kidney diseases, on insulin resistance(IR) and podocyte epithelial-mesenchymal transition(EMT) in diabetic kidney disease(DKD), and further to reveal the scientific connotation. Thirty-two rats were randomly divided into a normal group, a model group, a TFA group, and a rosiglitazone(ROS) group. The modified DKD model was induced in rats by methods including high-fat diet feeding, unilateral nephrectomy, and streptozotocin(STZ) intraperitoneal injection. After modeling, the rats in the four groups were given double-distilled water, TFA suspension, and ROS suspension correspondingly by gavage every day. At the end of the 8th week of drug administration, all rats were sacrificed, and the samples of urine, blood, and kidney tissues were collected. The parameters and indicators related to IR and podocyte EMT in the DKD model rats were examined and observed, including the general condition, body weight(BW) and kidney weight(KW), the biochemical parameters and IR indicators, the protein expression levels of the key signaling molecules and structural molecules of slit diaphragm in the renal insulin receptor substrate(IRS) 1/phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt) pathway, foot process form and glomerular basement membrane(GBM) thickness, the expression of the marked molecules and structural molecules of slit diaphragm in podocyte EMT, and glomerular histomorphological characteristics. The results showed that for the DKD model rats, both TFA and ROS could improve the general condition, some biochemical parameters, renal appearance, and KW. The ameliorative effects of TFA and ROS were equivalent on BW, urinary albumin(UAlb)/urinary creatinine(UCr), serum creatinine(Scr), triglyceride(TG), and KW. Secondly, they could both improve IR indicators, and ROS was superior to TFA in improving fast insulin(FIN) and homeostasis model assessment of insulin resistance(HOMA-IR). Thirdly, they could both improve the protein expression levels of the key signaling molecules in the IRS1/PI3K/Akt pathway and glomerulosclerosis in varying degrees, and their ameliorative effects were similar. Finally, both could improve podocyte injury and EMT, and TFA was superior to ROS. In conclusion, this study suggested that podocyte EMT and glomerulosclerosis could be induced by IR and the decreased activation of the IRS1/PI3K/Akt pathway in the kidney in DKD. Similar to ROS, the effects of TFA in inhibiting podocyte EMT in DKD were related to inducing the activation of the IRS1/PI3K/Akt pathway and improving IR, which could be one of the scientific connotations of TFA against DKD. This study provides preliminary pharmacological evidence for the development and application of TFA in the field of diabetic complications.

Ferroptosis-related renal tubular lesions play important roles in diabetic kidney disease (DKD) progression, and these pathophysiological responses are collectively described as diabetic tubulopathy (DT), which lacks an effective treatment. Total flavones from Abelmoschus manihot (TFA), a natural extract that extensively used in patients with chronic kidney disease, has been used for treatment of renal tubular injury in DKD; however, whether TFA alleviates DT and its potential mechanisms remain unclear. Hence, we investigated the effects of TFA, compared to dapagliflozin, in DT management both in vivo and in vitro, using a DKD rat model and the NRK-52 E cells. Following modeling, the DKD rats received TFA, dapagliflozin, or vehicle for 6 weeks. For the in vitro research, the NRK-52 E cells were exposed to advanced glycation end products (AGEs) plus ferrostatin-1 (Fer-1), dapagliflozin, or TFA. Changes in biochemical parameters and renal tubular injury were analyzed in vivo, while changes in ferroptosis of renal tubular cells and the ferroptosis-related proteins expression were analyzed both in vivo and in vitro. We found that TFA and dapagliflozin improved biochemical parameters, renal tubular injury, and ferroptosis in the DKD rats. Moreover, TFA and dapagliflozin inhibited ferroptosis by ameliorating iron deposition, lipid peroxidation capacity, and ferroptosis-related proteins expression in vitro, which was similar to the effects of Fer-1. Collectively, this study demonstrated that TFA treated DT in a manner similar to dapagliflozin by inhibiting ferroptosis of renal tubular cells via improving iron deposition and antioxidant capacity. Our findings provide new pharmacological evidence for TFA application in DT treatment.

目的:探究黄蜀葵花有效成分黄蜀葵花总黄酮(TFA)在体内抑制糖尿病肾脏疾病(DKD)肾组织周细胞-肌成纤维细胞转分化(PMT)的作用和机制.方法:将30只大鼠随机分为假手术组、模型组、TFA组和伊马替尼(IMA)组.采用"高脂饲料喂养、单侧肾切除、链脲佐菌素(STZ)腹腔注射"3种措施联合建立改良型DKD大鼠模型.成模后,4组大鼠分别给予其双蒸水、TFA混悬液以及IMA混悬液灌胃,连续4周.在药物干预的第4周末,处死全部大鼠,收集尿液、血液和肾组织,采用肾组织病理染色、蛋白质分析和透射电子显微镜等技术进行检测和观察肾组织PMT相关指标.结果:与模型组比较,TFA组和IMA组UNAG与TFA组UACR水平显著降低(P＜0.01);细胞外基质和肾间质胶原相对面积显著降低(P＜0.01);TFA组和IMA组显著改善周细胞超微结构特征、周细胞和肌成纤维细胞标志分子蛋白表达强度和表达水平(P＜0.01,P＜0.05);TFA和IMA可以抑制肾组织血管内皮生长因子受体(PDGFR)、血小板源性生长因子受体(VEGFR)信号通路关键信号分子蛋白表达水平(P＜0.05,P＜0.01).结论:TFA与IMA体内能改善DKD大鼠RIF,其机制可能是通过调控周细胞-内皮细胞相关PDGFR、VEGFR信号通路活性而抑制肾组织PMT.该研究为黄蜀葵花治疗DKD患者RIF提供了可靠的药理学证据.

To explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a classical prescription, in improving testicular aging(TA) in vivo, the authors randomly divided 24 male rats into four groups: the normal, model, DHZCP and vitamin E(VE) groups. The TA rat model was established by continuous gavage of D-galactose(D-gal). During the experiment, the rats in the DHZCP and VE groups were given DHZCP suspension and VE suspension, respectively by gavage, while those in the normal and model groups were gavaged saline separately every day. After the co-administration of D-gal and various drugs for 60 days, all rats were sacrificed, and their blood and testis were collected. Further, various indexes related to TA and necroptosis of testicular cells in the model rats were examined and investigated, which included the aging phenotype, total testicular weight, testicular index, histopathological features of testis, number of spermatogenic cells, sex hormone level, expression characteristics of reactive oxygen species(ROS) in testis, expression levels and characteristics of cyclins in testis, and protein expression levels of the key molecules in receptor-interacting serine/threonine-protein kinase 1(RIPK1)/receptor-interacting serine/threonine-protein kinase 3(RIPK3)/mixed lineage kinase domain like pseudokinase(MLKL) signaling pathway in each group. The results showed that, for the TA model rats, both DHZCP and VE improved their aging phenotype, total testicular weight, testicular index, pathological features of testis, number of spermatogenic cells, serum testosterone and follicle stimulating hormone levels, expression characteristics of ROS and protein expression levels and characteristics of P21 and P53 in testis. In addition, DHZCP and VE improved the protein expression levels of the key molecules in RIPK1/RIPK3/MLKL signaling pathway in testis of the model rats. Specifically, DHZCP was better than VE in the improvement of RIPK3. In conclusion, in this study, the authors found that DHZCP, similar to VE, ameliorated D-gal-induced TA in model rats in vivo, and its mechanism was related to reducing necroptosis of testicular cells by inhibiting the activation of RIPK1/RIPK3/MLKL signaling pathway. This study provided preliminary pharmacological evidence for the development and application of classical prescriptions in the field of men's health.