Dual Role of Motherwort in Renal Health: Protective and Detrimental Effects

Dual Role of Motherwort in Renal Health: Protective and Detrimental Effects | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dual Role of Motherwort in Renal Health: Protective and Detrimental Effects Hongmin Yu, Tao Wang, Xiaomei Chen, Cheng Zhang, Qing Xu, Meixia Huang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8740889/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Motherwort (MW) is known for its renoprotective effects, but it has also been reported to potentially cause kidney damage. This study aimed to explore the distinct mechanisms underlying the protective and detrimental effects of MW on renal health, providing a theoretical basis for its safe clinical application. We explored the effects of MW in cisplatin-induced acute kidney injury (AKI) in BALB/c mice. We found that in AKI mice administration of the MW at doses of 10 and 40 g/kg for 4 days alleviated the loss of body weight, increased the renal index, reduced pathological kidney damage, and lowered the levels of blood urea nitrogen (BUN), serum creatinine (Scr), kidney injury molecule‑1 (KIM‑1), and neutrophil gelatinase‑associated lipocalin (NGAL). Proteomic analysis and subsequent validation indicated that the mechanisms were associated with ferroptosis and autophagy pathways. In healthy mice, short‑term MW treatment at the same doses (10 and 40 g/kg) did not affect renal function. However, when healthy mice were administered a higher dose of MW (80 g/kg) by gavage for an extended period (14 days), it induced body weight loss, triggered renal damage, and elevated BUN, Scr, KIM‑1, and NGAL levels. At this dosage, MW also increased oxidative stress, promoted apoptosis, and upregulated the expression of epidermal growth factor receptor (EGFR) pathway‑related proteins. Our findings demonstrate that MW plays a dual role in kidney protection and injury through distinct mechanisms, offering important guidance for its clinical application. Health sciences/Diseases Health sciences/Nephrology motherwort ferritinophagy EGFR pathway oxidative stress apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Leonurus japonicus Houtt (common name: motherwort, MW) is a plant in the Labiaceae family, which is widely used as a traditional medicine in China, Korea, and Japan. Traditionally, MW is administered to promote blood circulation, regulate menstruation, and induce diuresis, detumescence, and detoxification 1 .It is also commonly used for several symptoms and diseases, such as edema and gynecological diseases 2 . Research shows that it exhibits a protective effect on kidney damage by playing an antioxidant role and inhibiting reactive oxygen species 3 . However, modern toxicology studies have indicated it has a dual effect on the kidneys, especially nephrotoxicity associated with long-term and high-dose administration 4 . Animal experiments have confirmed that the toxic target organs of MW extract primarily include the kidneys and liver 5 . Kidney toxicity is more severe than liver toxicity, leading to a series of abnormalities in renal function index along with pathological changes in renal tissue, resulting in substantial damage to the organ 6 . In this study, we investigated the dual mechanisms underlying the effects of MW on renal function, elucidating the renoprotective effects of MW and the mechanisms responsible for renal dysfunction induced by prolonged high-dose administration is essential for a comprehensive evaluation of the safety profile of MW and its formulations in clinical practice. Cisplatin, a broad-spectrum anti-cancer drug 7 . However, acute kidney injury (AKI) remains one of its most prevalent side effects 8 . Cisplatin-induced AKI is a classical model commonly used to study AKI. Current evidences showed that oxidative stress and ferroptosis play vital roles in the pathogenesis of cisplatin-induced nephrotoxicity 9 . Given the notable antioxidant properties of MW, this study employed a cisplatin-induced renal injury model to investigate the potential renoprotective effects of MWagainst cisplatin-induced nephrotoxicity 10 . In the present study, we initially discovered the predominant mechanisms for MW on ferritinophagy and the ferroptosis signaling pathway through proteomics study. Further studies showed that autophagy activates nuclear receptor coactivator 4 (NCOA4) and mediates ferritinophagy, and ferritin is degraded in lysosomes, releasing a large amount of free Fe2 + and aggravating ferroptosis 11 . Moreover, we found that MW effectively inhibited ferroptosis and lysosome degradation. Therefore, we proposed that MW effectively interferes with ferroptosis by blocking ferritinophagy mediated by NCOA4. In recent years, increasing attention has been directed toward the toxicity associated with Chinese herbs. Consequently, this study aimed to examine the adverse reactions of MW. The administration of a wide dosage of MW for 14 days was observed to have an impact on renal function and renal histomorphology. The Nrf2/HO-1 signaling axis plays a crucial role in the regulation of anti-inflammatory and antioxidant responses, mitigation of mitochondrial damage, and control of cell death 12 . The findings of our study demonstrated that MW significantly affected the expression of Nrf2, HO-1, and SOD2 proteins. MW also affected apoptosis and epithelial growth factor receptor (EGFR) pathway-related proteins. Therefore, the study revealed that varying doses and durations of MW administration could elicit dual effects via diverse mechanisms. Materials and Methods Preparation of MW MW was purchased from Beijing Ben Cao Fang Yuan Pharmaceutical and Technology Co., Ltd. (Anhui, China); it is identified as the dry aboveground part of Leonurus japonicas Houtt by Professor Yang Chengzi of Fujian University of Traditional Chinese Medicine. The MW was extracted with methanol according to a previous method 13 . Chemical analysis by UHPLC-QTOF-MS/MS led to the identification of 33 compounds, by matching HR-MS data against the Natural Products MS/MS Library (Supplementary Fig. 1), such as eight marker compounds, including leonurine, 4′,5-dihydroxy-7-methoxyfavone, rutin, hyperoside, apigenin, quercetin, kaempferol, and salicylic acid, were quantified (Supplementary Table 1). Animals Male BALB/c mice (18–22 g), 6 weeks old, were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd. (China Shanghai, SCXK(HU)2017-0012), with a total of 66 individuals. All experimental protocols were authorized by the Laboratory Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (FJTCM IACUC 2022250). Animals were handled under the International Guiding Principles for Biomedical Research Involving Animals, issued by the Council for the International Organizations of Medical Sciences. The mice were housed in the Laboratory Animal Center of Fujian University of Traditional Chinese Medicine. The environmental temperature was maintained at 22 ± 2 ℃ with a 12 h light/dark cycle and humidity of 50%. Establishment of Animal Models and Drug Administration In the first part, the investigation on the improvement effect of Leonurus japonicus on acute kidney injury, all animals were divided into six groups (n = 6): control (Ctrl), cisplatin (Cis), cisplatin + 10 g/kg MW (Cis+MW10), cisplatin + 40 g/kg MW (Cis+MW40), 10 g/kg MW (MW10), and 40 g/kg MW (MW40). Cisplatin was administered as a single intraperitoneal dose of 20 mg/kg per mouse. The MW intervention group received daily oral administration of motherwort solution for 4 consecutive days(10 g/kg or 40 g/kg). Kidney and blood samples were collected for analysis. In the second part, the investigation on the damage effect of Leonurus japonicus on normal kidneys under long-term and high-dose exposure, all animals were divided into five groups (n = 6): control (Ctrl), 10, 20, 40 and 80 g/kg MW (MW10, MW20, MW40, MW80). Intervention group received daily oral administration of motherwort solution for 14 consecutive days. Kidney and blood samples were collected for analysis. Renal histological studies After conducting the clinical score analysis, the same groups of mice were used for harvesting kidney tissues and fixing them in 4% paraformaldehyde. The kidney tissue was then dehydrated, aparaffin-embedded, and cut into 5 µm slices. Subsequently, dimethyl benzene dewaxing and wood staining were performed for 8 to 10 min each, followed by a series of alcohol dehydration steps (2% hydrochloric acid, 2% eosin stain) lasting 1 to 2 min each at concentrations of 80%, 90%, and finally 100%. This was followed by three washes with xylene at a concentration of 100%, each lasting for 5 min. The sections were then cleaned with xylene and sealed with neutral gum. Renal pathological alterations was observed using an optical microscope, photographed, and recorded within the field of vision. Finally, the score for every histopathologic feature was calculated for each animal. Biochemical measurements The levels of blood urea nitrogen (BUN), serum creatinine (Scr) and Malondialdehyde (MDA) in mouse plasma were determined using a urea assay kit, creatinine assay kit, and MDA assay kit, respectively. Kidney tissues were rinsed in normal saline, and then weighed and homogenized. LPO in the supernatant of homogenized renal tissues from rats was measured using an LPO assay kit. All kits were purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. and used according to the manufacturer’s protocols. Data independent acquisition (DIA) analysis and proteomics analysis Kidneys were homogenized and resuspended in lysis buffer (6 M urea,1% Protease Inhibitor, 1% phosphatase inhibitor), and equal amounts were purified using SDS-PAGE. The gel pieces stained with Coomassie brilliant blue were excised and subjected to in-gel digestion using trypsin. Extracted peptides were desalted using StrataXSPE and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS analyses were performed on an QExactiveTMHF-X (Thermo Fisher Scientific), coupled online to an ultraperformance liquid chromatography system. The resulting MS/MS data were processed using MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched on Mus_musculus_10090_SP_20200509. fasta against the SwissProt database (17045 entries) concatenated with reverse decoy database. When the differentially expressed proteins (DEPs) were obtained, the proteins were annotated by Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. GO identifies three aspects of biology: cellular component (CC), biological process (BP), and molecular function (MF). The dataset was extracted from KEGG database, and the gene-coding proteins were obtained by STRING ( https://cn.string-db.org/ ). The intersection analysis of DEPs and pathway-related proteins was carried out. Real-time quantitative polymerase chain reaction PCR (qRT-PCR) Tissue RNA was extracted from renal tissues using Trizol reagent according to the standard protocol. After reverse-transcription, RT-qPCR was performed on the QuantStudio 3 system (Applied Biosystems, USA). The mRNA levels of target genes were normalized and analyzed by the 2-∆∆Ct method. The following primers were used for RT-qPCR:β-actin forward 5′-TTGTCCACCTTCCAGCAGATGT-3′ and reverse 5′-AGCTCAGTAACAGTCCGCCTAG-3′, PTGS2 forward 5′-TGAGTGGGGTGATGAGCAAC-3′ and reverse 5′-TTCAGAGGGCAATGCGGTTCT-3′, GPX4 forward 5′-AATCAAGGAGTTTGCAGCCG-3′ and reverse 5′-CCACGCAGCCGTTCTTATCA-3′, Ceruloplasmin forward 5′-CGGATCACTACACAGGTGGC-3′ and reverse 5′-CCATTCCACCTCTACGGCTG-3′, Transferrin forward 5′-AGACTTCGAGTTGCTCTGCC-3′ and reverse 5′-CAGAAATTGCCGGTGCAGTC-3′, ATG7 forward 5′-CACGGTTCGATAATGTTCTTCC-3′ and reverse 5′-GTCTCCTCGTCACTCATGTCCC-3′, ATG5 forward, 5′-GGCCATCAACCGGAAACTCA-3′ and reverse 5′-CGCTCCGTCGTGGTCTGATAT-3′, NCOA4 forward 5′-GAGGTGTAGTGATGCACGGA-3′ and reverse 5′-GACGGCTTATGCAACTGTGAA-3′, FTH-1 forward 5′-GCCGAGAAACTGATGAAGCTGC-3′ and reverse 5′-GCACACTCCATTGCATTCAGCC-3′, Man2b1 forward 5′-GGTGGTAGCAGTCCCTATCA-3′ and reverse 5′-CTCAGGTTGCGATCCGAATC-3′, Neu1 forward 5′-GCCCTACGAGCTTCCAGATG-3′ and reverse 5′-CAGGGTCGAAGGTCACATCC-3′, LIPA forward 5′-TCACAGATGCCTGAGTTGGC-3′ and reverse 5′-GGCAAGCGTCCCAATTGAAG-3′, NGAL forward 5′-ACAGAAGGCAGCTTTACGATGT-3′ and reverse 5′-ACTGGTTGTAGTCCGTGGTGG-3′, and KIM-1 forward 5′-GGAGATACCTGGAGTAATCACACT-3′ and reverse, 5′-CACGCTTAGAGATGCTGACTTC-3′. Western blot analysis Renal tissues were lysed in RIPA buffer containing protease inhibitors (Thermo Scientific) for protein extraction 14 . Total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with one of the primary antibodies: KIM-1 (1:1000, Novus Biologicals), NGAL (1:1000, Bio-Techne Corporation), GPX4 (1:1000, Abcam), PTGS2 (1:1000, Abcam), Man2b1 (1:1000, Novus Biologicals), Neu1 (1:1000, Thermo Fisher Scientific Inc.), LIPA (1:1000, Proteintech Group Inc.), ATG5 (1:1000, Abcam), ATG7 (1:1000, Cell Signaling Technology), LC3I (1:1000, Proteintech Group Inc.), LC3II (1:1000, Proteintech Group Inc.), Ceruloplasmin (1:1000, Abcam), Transferrin (1:10000, Abcam), NCOA4 (1:2000, Thermo Fisher Scientific Inc.), FTH-1 (1:1000, Cell Signaling Technology), Nrf2(1:1000, Cell Signaling Technology), Bcl-2(1:1000, Proteintech Group Inc.), Bax(1:1000, Proteintech Group Inc.), SOD2(1:1000, Cell Signaling Technology), HO-1(1:10000, Abcam), EGFR(1:10000, Abcam),Grb2(1:1000, Proteintech Group Inc.), p-raf(1:10000, Abcam), raf(1:1000, Zen BioScience), p-MEK(1:10000, Abcam), MEK(1:10000, Abcam), p-ERK(1:1000, Zen BioScience), ERK(1:1000, Proteintech Group Inc.), β-actin(1:10000, Abcam) and GAPDH (1:2000, Proteintech Group Inc.). The blots were washed and probed with one of the secondary antibody goat anti-mouse IgG(H + L) (1:3000, Thermo Scientific) and goat anti-rabbit or IgG(H + L) (1:3000, Thermo Scientific). The protein signals were detected by enhanced chemiluminescence (Beyotime Biotechnology), and images of the blots were obtained using a ChemiDoc XRS+ imaging system (Bio-Rad) and analyzed with ImageJ software. Results are expressed as fold changes after normalization to GAPDH or β-actin. Prussian blue staining Relevant literature should be consulted for the experimental procedures 15 , The tissues were fixed in a solution of paraformaldehyde and subsequently subjected to embedding. Following sectioning, the tissue underwent a conventional dewaxing process, wherein the section was immersed in Perls' stain for 15 to 30 min. Subsequently, the sections were immersed in a nuclear solid red staining solution for 5 to 10 min. This was followed by standard dehydration and clearing procedures, culminating in sealing with neutral gum to safeguard the sections and preserve the staining outcomes. TUNEL staining A TUNEL apoptosis kit (B0013, LABELAD) was used according to the manufacturer’s instructions to detect the apoptosis of kidney tissue. After dewaxing, 20 µg/mL proteinase K solution (100 µL) was dropped to cover the tissue, and the sections were incubated at room temperature for 20 min. After washing with PBS for 5 min, 100 µL of TUNEL equilibration buffer was dropped and incubated for 5 min. About 50 µL of TUNEL reaction mixture was added, incubated for 2 h, and washed with PBS for 5 min. Thereafter, 0.1% Triton X-100 (containing 5 mg/mL BSA) buffer was applied three times, followed by the addition of 50µL of DAPI-containing anti-fluorescence sealing solution to the sections, which were incubated for 10 min. They were then observed and photographed under a fluorescence inverted microscope. Statistical analysis Data are presented as mean ± standard deviation (SD). All statistical analyses were conducted using SPSS software (version 26.0). The normality of continuous variables was assessed using the Shapiro-Wilk test. Since all variables demonstrated normality (P > 0.05), one-way analysis of variance (ANOVA) was employed to determine significant differences among groups. For data that did not follow a normal distribution, the non-parametric Kruskal-Wallis test was used. Values at p < 0.05 were considered significant. Results MW attenuates cisplatin induced AKI in mice Cisplatin resulted in a significant weight loss and increased renal index (kidney weight/body weight), Scr levels, and BUN levels. 10 and 40 g/kg MW efficiently reduced these changes. In addition, effects of MW40 were superior to those of MW10 on renal index, Scr, and BUN. However, MW control administration did not exhibit any effect (Fig. 1 A-D). KIM-1and NGAL are biomarkers of AKI, representing renal function 16 , 17 . The mRNA and protein expression levels of KIM-1 and NGAL in kidney tissue from the Cis + MW (10 and 40) group were significantly lower than those in the Cis group, but MW control administration did not exhibit any effect (Fig. 1 E–H). The HE staining results of renal tissue showed that compared with the Ctrl group, the epithelial cells exhibited cellular edema, the renal glomeruli demonstrated atrophy, and some cells displayed necrosis and disorganized arrangement. Moreover, MW10 and MW40 provided effective protection, and no changes were observed in the control administration of MW10 and MW40 (Fig. 1 I). Proteomics analysis of renal tissue and determination of ferroptosis and lysosomes as the key drivers The proteomics analysis was conducted to investigate the renal tissues in the cisplatin-induced AKI model treated by MW. The proteomics analysis identified 4150 proteins. When p-value < 0.05, a change in differential expression level exceeding 1.5 was used as a significant upregulation threshold, and a decrease of less than 1/1.5 was used as a significant downregulation threshold. A total of 55 proteins had common changes, including ceruloplasmin and transferrin related to iron metabolism (Fig. 2 A). GO analysis showed that the effect of MW on cisplatin-induced AKI was mainly related to cell composition, biological process molecular function, and lysosomes and iron–ion binding (Figs. 2 B–G). KEGG pathway enrichment analysis also showed that ferroptosis and lysosomes were key pathways (Fig. 2 H). In summary, the proteomic analysis illustrated that ferroptosis and lysosomes might be the key drivers of MW treatment of cisplatin-induced AKI. MW attenuated ferroptosis in cisplatin-induced mice Ferroptosis frequently shows an increased level of LPO 18 . Cisplatin induced the increase in serum MDA and tissue LPO levels (Fig. 3 A-B). MW administration could significantly reduce the levels of MDA and LPO, and the effects of MW40 were superior to those of MW10. Prostaglandin endoperoxide synthase 2 (PTGS2) and Glutathione peroxidase 4 (GPX4) are markers of ferroptosis. After modeling with cisplatin, the gene and protein expression levels of PTGS2 increased and those of GPX4 decreased. However, MW administration effectively reduced these changes (Fig. 3 C-F). MW reduced lysosomes-related hydrolase proteins in cisplatin-induced mice KEGG proteomics results showed that MW protecting AKI was closely related to lysosomes. lysosomes-related hydrolase proteins were detected to evaluate the effects of MW on lysosomes in cisplatin-induced mice. The mRNA and protein expression of α-mannosidase(Man2b1), Neuraminidase 1(NEU-1), and Lipase(LIPA) decreased in the cisplatin group mice, whereas a substantial increase in mRNA and protein expression of Man2b1, NEU-1, and LIPA was observed in MW-treated mice (Fig. 4 ). MW-attenuated ferritinophagy in cisplatin-induced mice Ferritinophagy is a type of selective autophagy, which increases the degradation of ferritin in lysosomes 12 , 13 . Ceruloplasmin converts Fe 2+ to Fe 3+ and Promote ferritin transport of excessive iron into cells and stored in ferritin. In this experiment, we observed that MW inhibited the increase in ceruloplasmin (Figs. 5 A–B) and transferrin (Figs. 5 C–D), as well as autophagy key proteins LC3II/LC3Ⅰ (microtubule- associated protein1-light chain-3, LC3 ) (Fig. 5 E) and Autophagy-related gene 5(ATG5) (Figs. 5 F–G), Autophagy-related protein 7(ATG7) (Figs. 5 H–I). Nuclear receptor coactivator (NCOA4) mediates ferritin autophagy by promoting ferritin degradation into lysosomes, increasing free iron and aggravating subsequent ferroptosis 19 . Interestingly, we observed that MW inhibited the activation of NCOA4 (Figs. 5 J–K) and promoted the elevation of Ferritin heavy chain 1(FTH-1) (Figs. 5 L–M). We evaluated the level of iron–ion in renal tissue by Prussian blue staining. Compared with the control group, the iron–ion staining in the kidney tissue of Cis. group mice increased significantly, and MW remarkably reduced the staining area (Fig. 5 N). These results showed that MW reduced ferritinophagy and free iron. High-dose MW damaged renal function of mice In the previous experiment, we observed that administering 10 and 40g/kg for 4 days did not damage the kidneys of mice. However, when the dosage was increased and the administration time was prolonged, we observed kidney damage caused by 80g/kg MW after administering for 14 days. We observed significant weight loss and increased renal index, BUN, Cre, MDA, KIM-1 and NGAL levels(Figs. 6 A– 6 H). The HE staining results of renal tissue showed that compared with the Ctrl group, 80g/kg MW provided ob-vious side effects on kidney with the epithelial cells exhibiting cellular edema, the renal glomeruli demonstrated atrophy, and some cells displayed necrosis and disorganized arrangement (Fig. 6 I). High-dose MW promoted oxidative stress and apoptosis in renal tissue The level of apoptosis was enhanced by MW, as indicated through the TUNEL staining of renal tissue (Figs. 7 A-B). In addition, we observed that MW inhibited apoptotic protein Bcl-2 promoted the content of pro-apoptotic protein Bax (Figs. 7 C-D).The promotion of kidney injury is significantly influenced by oxidative stress, and apoptosis is intricately linked to the pathophysiology of kidney injury 20 . In this experiment, we observed that MW inhibited the expression of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2), Heme Oxygenase-1 (HO-1), and Superoxide dismutase 2 (SOD2) proteins (Figs. 7 E– 7 G). These results implied that 4 g/mL MW promoted oxidative stress and apoptosis in renal tissues. MW may induced renal injury through the EGFR pathway The activation of EGFR can induce cell cycle entry and promote cell proliferation, regulate transcription factors in the nucleus, and inhibit certain apoptosis-inducing signals or molecules 21 . In this experiment, we observed that MW increased the expression of EGFR and Grb2 proteins (Figs. 8 A-B). In addition, we observed that MW facilitated the phosphorylation of raf, MEK, and ERK proteins, which play crucial roles in the EGFR signaling pathway (Figs. 8 C-E). These results implied that MW may induce renal injury via the EGFR signaling pathway. Discussion MW, as a traditional herbal medicine, is widely used for several diseases in China, Korea, and Japan. MW promotes blood circulation, regulates menstruation, promotes water retention, introduces detumescence 20 , exhibits antipyretic properties, and detoxifies. The study also found that MW exerted a protective effect on kidney damage, which is why it is frequently employed in the treatment of kidney disease. However, in long-term clinical practice, MW also has adverse reactions, which may affect kidney function and cause kidney tissue damage 21 , 22 . The dual role of MW in kidney protection and injury and its mechanism have attracted research attention. In this study, a cisplatin-induced mouse kidney injury model was used. The pathogenesis of cisplatin-induced AKI is complex and multifactorial, involving DNA damage, energy consumption, oxidative stress, endoplasmic reticulum stress, inflammation, vascular dysfunction, and mitochondrial damage 23 , 24 . This study found that 4 days of administration of MW extract could reduce the kidney index, renal index (kidney weight/body weight), serum Scr levels, and BUN levels. Previous literature also reported that the AKI caused by doxorubicin, lipopolysaccharide, and sepsis has a significant protective effect of MW 25, 26 . Subsequently, we used proteomics to analyze the mechanism by which MW protects kidney injury. Proteomics was used to identify the therapeutic targets by comparing the differential changes in the proteome in animal tissues after MW treatment on cisplatin-induced AKI. Ferroptosis pathogenesis encompasses diverse mechanisms, including ROS-driven iron-dependent non-apoptotic cell death 27 and PD-L1-mediated immune regulation (affecting T cell activation, proliferation, and cytotoxic factor secretion) 28 . Notably, our proteomic analysis demonstrates that the treatment of AKI with MW might be closely related to lysosome, iron metabolism, and ferroptosis.. Specifically, MW effectively inhibited the increase in MDA, LPO, and GPX4 expression but increased the decrease in PTGS2 expression. In addition, MW increased the reduction of related active enzymes of lysosome. Subsequently, we analyzed the upstream mechanism of MW regulating ferroptosis. Ceruloplasmin and transferrin are differential proteins in proteomics. Experiments verified that MW inhibited the increase in ceruloplasmin and transferrin. Ferritin can alter sequestered iron via autophagy (ferritinophagy) 29 . Ferritinophagy is a kind of selective autophagy mediated by NCOA4 30 . NCOA4, which is activated by the autophagy-related gene (ATG), is a cargo receptor for ferritinophagy that interacts with FTH1 and promotes the transport of ferritin to the lysosome for degradation, thereby causing iron release. Iron accumulation induces ferroptosis 31 , 32 . We observed that MW inhibited the expression of autophagy key proteins ATG5, ATG7, and LC3II/LC3I and reduced the activation of NCOA4 and increased the expression of FTH1 in renal issue of AKI mice. Thus, MW attenuated ferritinophagy, reduced the degradation of ferritin and the release of free iron ions, and inhibited ferroptosis, protecting kidney damage caused by cisplatin. The protective effect of MW on cisplatin-induced AKI was mainly related to the inhibition of ferroptosis. The above results showed that MW had a protective effect on kidney injury. In recent years, More and more studies are paying attention to the damaging effects of motherwort on the kidneys Many studies have demonstrated that 30-120g/kg MW may cause kidney function injury in rats 20 , so we conducted further research to explore the relevant mechanism.After 14 days of continuous administration of 80 g/kg MW in normal mice, we found that the weight of mice decreased significantly, whereas the kidney index, BUN, Scr, and MDA increased. Animal experiments revealed that 80 g/kg MW promoted the expression of KIM-1 and NGAL genes and proteins, and HE results showed that a large dose of MW could significantly damage kidney tissue.Then we conducted further exploration into its damage mechanism. We found that the expression levels of Nrf2 and HO-1 proteins in kidney tissue decreased, whereas the expression level of SOD2 was significantly reduced. Furthermore, MW exhibited a significant inhibitory effect on the expression of anti-apoptotic protein Bcl2 and induced an increase in the expression of pro-apoptotic protein Bax. These findings implied a correlation between MW-induced kidney injury and oxidative stress, as well as cellular apoptosis. EGFR belongs to the family of transmembrane receptor tyrosine protein kinases, playing a crucial role not only in fine-tuning cellular signal transduction and promoting tumor cell survival but also in regulating cellular metabolism, proliferation, migration, and differentiation 33 . The activation of EGFR can lead to kidney damage, and preclinical studies have shown its potential as a therapeutic target for chronic kidney disease 34 We observed a correlation between MW-induced kidney injury and the EGFR signaling pathway. We analyzed the expression levels of EGFR, Grb2, p-raf/raf, p-MEK/MEK, and p-ERK/ERK and discovered that using MW significantly enhanced the expression of proteins associated with the EGFR signaling pathway. These findings indicated that MW-induced kidney injury was not only linked to oxidative stress and apoptosis but also via activating the EGFR signaling pathway. Thus, long-term high-dose MW may be related to EGFR signaling and downstream oxidative stress and apoptosis pathways in kidney injury. Conclusions Our findings indicated that MW played a dual role in kidney protection and damage, exerting its effects through distinct mechanisms. MW mitigated cisplatin-induced kidney damage by inhibiting ferroptosis. However, prolonged administration of high doses of MW induced oxidative stress, apoptosis and the EGFR signaling pathway in kidney, ultimately causing kidney injury. Declarations Author Contributions Hongmin Yu: Investigation, Validation, Formal Analysis. Tao Wang: Investigation, Validation, Formal Analysis. Xiaomei Chen: Data Curation, Visualization, Writing – Original Draft. Cheng Zhang: Investigation, Resources. Qing Xu: Investigation, Resources. Meixia Huang: Methodology, Conceptualization. Yingzheng Wang: Writing – Original Draft, Supervision. Jie Xu: Writing – Review & Editing. Yinghao Wang: Conceptualization, Funding Acquisition, Supervision, Writing – Review & Editing. Acknowledgements Not applicable. Competing interests The authors declare that they have no competing interests, and all authors should confirmits accuracy. Conflict of Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding information This work was supported by the [the National Natural Science Foundation of China #1] under Grant [number 82173996]; [National Natural Science Foundation of Fujian province #2] under Grant [number 2021J01920]. Availability of data and material All data generated or analyzed during this study are included in this publishedarticle. Ethical review approval and consent to participate in research All animal experiments were conducted in accordance with the relevant guidelines and regulations and were approved by the Laboratory Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (approval number FJTCM IACUC 2022250). Publishing consent Not applicable. References Miao, L. L. et,al. Leonurus japonicus (Chinese motherwort), an excellent traditional medicine for obstetrical and gynecological diseases: A comprehensive overview. Biomedecine & pharmacotherapie . 117 , 109060 (2019). Wang, C. et,al. 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Ferritinophagy/ferroptosis: Iron-related newcomers in human diseases. Journal of Cellular Physiology . 233(12) , 9179–9190 (2018). Xinyu X. et al. Motherwort Injection for Preventing Uterine Hemorrhage in Women With Induced Abortion: A Systematic Review and Meta-Analysis of Randomized Evidence. Frontiers in pharmacology . 13 , 916665 (2022). de Boer, H. J., & Cotingting, C.. Medicinal plants for women’s healthcare in southeast Asia: A meta-analysis of their traditional use, chemical constituents, and pharmacology. Journal of Ethnopharmacology . 151(2) , 747–767 (2014). Han, S. R. et al.. Toxicity assessment of Leonuri Herba aqueous extract orally administered to rats for 13 consecutive weeks. Journal of Ethnopharmacology . 149(1) , 371–376 (2013). Zhu, S. et al. DNA damage response in cisplatin-induced nephrotoxicity. Archives of Toxicology . 89(12) , 2197–2205 (2015). Yu, B. et al. TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy. Theranostics. 13(13) , 4356–4375 (2023). Han, L. et al. Leonurine preconditioning attenuates ischemic acute kidney injury in rats by promoting Nrf2 nuclear translocation and suppressing TLR4/NF-κB pathway. Chemical & pharmaceutical bulletin . 70(1) , 66–73 (2022). Cheng, H. et al. Leonurine ameliorates kidney fibrosis via suppressing TGF-β and NF-κB signaling pathway in UUO mice. International Immunopharmacology . 25(2) , 406–415 (2015). Jiang, X., Stockwell, B. R., & Conrad, M.. Ferroptosis: Mechanisms, biology and role in disease. Nature Reviews Molecular Cell Biology . 22(4) , 266–282 (2021). Wang, M. et al. Therapeutic induction of ferroptosis in tumors using PD-L1 targeting antibody nanogel conjugates. Cell Chemical Biology . 31(12) , 2039–2051.e6 (2024). Wang, Y. et al. Lipocalin-2 promotes CKD vascular calcification by aggravating VSMCs ferroptosis through NCOA4/FTH1-mediated ferritinophagy. Cell Death & Disease . 15(11) , 865 (2024). Jiang, Y. Y. et al. KA-mediated excitotoxicity induces neuronal ferroptosis through activation of ferritinophagy. CNS Neuroscience & Therapeutics . 30(9) , e70054 (2024). Zhang, T. et al. Iron regulatory protein two facilitates ferritinophagy and DNA damage/repair through guiding ATG9A trafficking. Journal of Biological Chemistry , 300(10) , 107767 (2024). Yang, B. et al. Inhibition of JNK signaling attenuates photoreceptor ferroptosis caused by all-trans-retinal. Free Radical Biology and Medicine . 227 , 179–189 (2025). Nie, H. et al. Synthesis of novel deuterated EGFR/ALK dual-target inhibitors and their activity against non-small cell lung cancer. European Journal of Medicinal Chemistry . 283 , 117146 (2025). Rayego-Mateos S. et al. Role of Epidermal Growth Factor Receptor (EGFR) and Its Ligands in Kidney Inflammation and Damage. Mediators of inflammation . 2018 , 8739473 (2018). Additional Declarations No competing interests reported. Supplementary Files Supplementmaterials.docx GraphicalAbstrac1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 May, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers invited by journal 15 Apr, 2026 Editor assigned by journal 08 Apr, 2026 Editor invited by journal 13 Feb, 2026 Submission checks completed at journal 10 Feb, 2026 First submitted to journal 10 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8740889","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":626321232,"identity":"d40fa44e-dbfe-40ab-b99b-cd917909c378","order_by":0,"name":"Hongmin Yu","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hongmin","middleName":"","lastName":"Yu","suffix":""},{"id":626321233,"identity":"f134fa47-2b30-4af0-8abb-a6e138b27101","order_by":1,"name":"Tao Wang","email":"","orcid":"","institution":"Rehabilitation Hospital affiliated to Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Wang","suffix":""},{"id":626321234,"identity":"f9449ebe-3bcf-498e-97a0-5d561d9209ab","order_by":2,"name":"Xiaomei Chen","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaomei","middleName":"","lastName":"Chen","suffix":""},{"id":626321235,"identity":"249f2f32-8d62-465c-8bb6-8b8332045051","order_by":3,"name":"Cheng Zhang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zhang","suffix":""},{"id":626321239,"identity":"b5bee651-1cfb-48c2-ba56-0803dcabee65","order_by":4,"name":"Qing Xu","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Xu","suffix":""},{"id":626321241,"identity":"a5eb42f4-0281-4e02-b070-7b3073f7940d","order_by":5,"name":"Meixia Huang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Meixia","middleName":"","lastName":"Huang","suffix":""},{"id":626321242,"identity":"3e9085de-30c0-4020-84bd-9270ac76c004","order_by":6,"name":"Yingzheng Wang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yingzheng","middleName":"","lastName":"Wang","suffix":""},{"id":626321244,"identity":"041d64c6-e432-4876-ab0e-7810848d252a","order_by":7,"name":"Jie Xu","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Xu","suffix":""},{"id":626321245,"identity":"800bd096-9f32-4710-8e7a-16d0e6768450","order_by":8,"name":"Yinghao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYFACHhBhw8PP30C8Fkag2jQZyRkHSNNy2MagIYFIDQY3co8/+JlznseA4QDjh485RGnJS2zs3Xabx5y5gVly5jYitJjdyDFsZgRqsWw4wMbMS4KWczwGBxJI03KABC32Z94YzuzdlswjOeNgM3F+kWzPMfjwc5udPT9/88EPH4nRwiCQAGOB4ocowH+ASIWjYBSMglEwcgEAa9w46jBoBvcAAAAASUVORK5CYII=","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yinghao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-30 12:10:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8740889/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8740889/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107621274,"identity":"7f6abcd1-1505-484b-9997-270c34ccd141","added_by":"auto","created_at":"2026-04-23 09:41:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":468581,"visible":true,"origin":"","legend":"\u003cp\u003eMW attenuates cisplatin-induced AKI in mice. (\u003cstrong\u003eA\u003c/strong\u003e) renal index(n=6), (\u003cstrong\u003eB\u003c/strong\u003e)Weight changes(n=6), (\u003cstrong\u003eC\u003c/strong\u003e)Serum creatinine(n=6), (\u003cstrong\u003eD\u003c/strong\u003e) Serum BUN(n=6), (\u003cstrong\u003eE-F\u003c/strong\u003e) mRNA and protein expression of KIM-1(n=3), (\u003cstrong\u003eG-H\u003c/strong\u003e) mRNA and protein expression of NGAL(n=3), (\u003cstrong\u003eI\u003c/strong\u003e) Representative images of H\u0026amp;E staining of renal tissues (original magnification × 200). All data are expressed as mean±S.D. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Ctrl group, #\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Cis group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/44ce08efebd9be5a70771f33.png"},{"id":107621529,"identity":"3b4f2eb1-f209-481b-8717-f0fea2af37ce","added_by":"auto","created_at":"2026-04-23 09:41:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214599,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic analysis of renal tissues in cisplatin-induced mice. (\u003cstrong\u003eA\u003c/strong\u003e) Volcano diagram and Venn diagram of differentially expressed proteins (DEPs). Bubble map of GO analysis: (\u003cstrong\u003eB-C\u003c/strong\u003e) Cellular Components, (\u003cstrong\u003eD-E\u003c/strong\u003e) Molecular Function, (\u003cstrong\u003eF-G\u003c/strong\u003e) Biological Process. (\u003cstrong\u003eH\u003c/strong\u003e) Cluster Analysis of KEGG Pathways.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/bc68501b25c4d30cce2c1c52.png"},{"id":107621371,"identity":"bfa1b5d9-0978-419c-b58f-fe03d653fb2c","added_by":"auto","created_at":"2026-04-23 09:41:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":283698,"visible":true,"origin":"","legend":"\u003cp\u003eMW attenuated ferroptosis in cisplatin-induced mice. (\u003cstrong\u003eA\u003c/strong\u003e) Serum MDA (n=6), (\u003cstrong\u003eB\u003c/strong\u003e)LPO of renal tissue (n=6), mRNA and protein expression of GPX4(\u003cstrong\u003eC-D\u003c/strong\u003e), PTGS2(\u003cstrong\u003eE-F\u003c/strong\u003e) (n=3). All data are expressed as mean±S.D. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 vs.Ctrl group, #\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 vs. Cis group, \u0026amp;\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/399bae19fd05e732ddeeae50.png"},{"id":107621361,"identity":"2822d220-acfe-4ec6-a070-025605f85f90","added_by":"auto","created_at":"2026-04-23 09:41:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":303493,"visible":true,"origin":"","legend":"\u003cp\u003eMW reduced lysosome related hydrolase proteins in cisplatin-induced mice. mRNA and protein expression of Man2b1(\u003cstrong\u003eA-B\u003c/strong\u003e), NEU1 (\u003cstrong\u003eC-D\u003c/strong\u003e), LIPA(\u003cstrong\u003eE-F\u003c/strong\u003e). All data are expressed as mean±S.D. (n=3). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Ctrl group, #\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Cis group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/434753b9f6b38c7edc1cca12.png"},{"id":107621421,"identity":"710cab28-bd41-42d5-b7d9-06c69a98a6a4","added_by":"auto","created_at":"2026-04-23 09:41:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":315412,"visible":true,"origin":"","legend":"\u003cp\u003eMW attenuated ferritinophagy in cisplatin-induced mice. mRNA and protein expression of ceruloplasmin(\u003cstrong\u003eA-B\u003c/strong\u003e), transferrin(\u003cstrong\u003eC-D\u003c/strong\u003e), ATG5(\u003cstrong\u003eF-G\u003c/strong\u003e), ATG7(\u003cstrong\u003eH-I\u003c/strong\u003e), NCOA4(\u003cstrong\u003eJ-K\u003c/strong\u003e), FTH-1(\u003cstrong\u003eL-M\u003c/strong\u003e), (\u003cstrong\u003eE\u003c/strong\u003e) protein expression ratio of LC3II/LC3I, (\u003cstrong\u003eN\u003c/strong\u003e) Prussian blue staining of detection of iron ion level. All data are expressed as mean±S.D. (n=3). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Ctrl group, #\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Cis group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/69eebb0c9b3909f336b5de32.png"},{"id":107621369,"identity":"2776ce86-7962-4427-b503-10b06ac683b9","added_by":"auto","created_at":"2026-04-23 09:41:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":476589,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-dose MW damaged renal function of mice. (\u003cstrong\u003eA\u003c/strong\u003e) Weight changes (n=6), (\u003cstrong\u003eB\u003c/strong\u003e) renal index (n=6), (\u003cstrong\u003eC\u003c/strong\u003e) serum BUN (n=6), (\u003cstrong\u003eD\u003c/strong\u003e) serum creatinine (n=6), (\u003cstrong\u003eE\u003c/strong\u003e) serum MDA (n=6), (\u003cstrong\u003eF and G\u003c/strong\u003e) mRNA expression of KIM-1 and NGAL (n=3), (\u003cstrong\u003eH\u003c/strong\u003e) protein expression of KIM-1 (n=3), and (\u003cstrong\u003eI\u003c/strong\u003e) representative images of H\u0026amp;E staining of renal tissues (original magnification × 200). All data are expressed as mean±S.D. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Ctrl group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/1c11260170e686787bf22e29.png"},{"id":107621385,"identity":"213f8bd4-ee66-499c-89aa-b0440a11f59b","added_by":"auto","created_at":"2026-04-23 09:41:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":437578,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-dose MW promoted oxidative stress and apoptosis in renal tissue. Tunel staining of renal tissue (\u003cstrong\u003eA and B\u003c/strong\u003e), protein expression of Bcl-2 (\u003cstrong\u003eC\u003c/strong\u003e), protein expression of Bax (\u003cstrong\u003eD\u003c/strong\u003e), protein expression of SOD2 (\u003cstrong\u003eE\u003c/strong\u003e), protein expression of Nrf2 (\u003cstrong\u003eF\u003c/strong\u003e), and protein expression of HO-1 (\u003cstrong\u003eG\u003c/strong\u003e). All data are expressed as mean±S.D. (n=3). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Ctrl group.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/600bd204d43cea2ecab6984e.png"},{"id":107621495,"identity":"6852a7ce-5787-4d23-ac40-aefffdfc3e3b","added_by":"auto","created_at":"2026-04-23 09:41:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":194898,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-dose MW may induce kidney injury through the EGFR pathway. (\u003cstrong\u003eA\u003c/strong\u003e) Protein expression of EGFR, (\u003cstrong\u003eB\u003c/strong\u003e) protein expression of Grb2, (\u003cstrong\u003eC\u003c/strong\u003e) protein expression ratio of P-raf/raf, (\u003cstrong\u003eD\u003c/strong\u003e) protein expression ratio of P-MEK/MEK, and (\u003cstrong\u003eE\u003c/strong\u003e) protein expression ratio of P-ERK/ERK. All data are expressed as the mean±S.D. (n=3). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 vs. Ctrl group.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/aa0eeb70d70bc02fa8c3e1c5.png"},{"id":107621698,"identity":"57ff52c0-7290-4498-a0b0-a2986b6cb1c7","added_by":"auto","created_at":"2026-04-23 09:42:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2794746,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/3605e756-27a4-4ada-a9eb-61993a84cea6.pdf"},{"id":107621577,"identity":"26882374-e80d-4393-9fdc-a47471a087da","added_by":"auto","created_at":"2026-04-23 09:42:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":74513,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/890604018e7e20614befd392.docx"},{"id":107621368,"identity":"d50e1251-96fb-4242-b5f7-247d0950247b","added_by":"auto","created_at":"2026-04-23 09:41:18","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":360448,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstrac1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8740889/v1/7bd22d7fee71f23ab4bbdba1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual Role of Motherwort in Renal Health: Protective and Detrimental Effects","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eLeonurus japonicus\u003c/em\u003e Houtt (common name: motherwort, MW) is a plant in the Labiaceae family, which is widely used as a traditional medicine in China, Korea, and Japan. Traditionally, MW is administered to promote blood circulation, regulate menstruation, and induce diuresis, detumescence, and detoxification\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.It is also commonly used for several symptoms and diseases, such as edema and gynecological diseases\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Research shows that it exhibits a protective effect on kidney damage by playing an antioxidant role and inhibiting reactive oxygen species\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, modern toxicology studies have indicated it has a dual effect on the kidneys, especially nephrotoxicity associated with long-term and high-dose administration\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Animal experiments have confirmed that the toxic target organs of MW extract primarily include the kidneys and liver\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Kidney toxicity is more severe than liver toxicity, leading to a series of abnormalities in renal function index along with pathological changes in renal tissue, resulting in substantial damage to the organ\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In this study, we investigated the dual mechanisms underlying the effects of MW on renal function, elucidating the renoprotective effects of MW and the mechanisms responsible for renal dysfunction induced by prolonged high-dose administration is essential for a comprehensive evaluation of the safety profile of MW and its formulations in clinical practice.\u003c/p\u003e \u003cp\u003eCisplatin, a broad-spectrum anti-cancer drug\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, acute kidney injury (AKI) remains one of its most prevalent side effects\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Cisplatin-induced AKI is a classical model commonly used to study AKI. Current evidences showed that oxidative stress and ferroptosis play vital roles in the pathogenesis of cisplatin-induced nephrotoxicity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Given the notable antioxidant properties of MW, this study employed a cisplatin-induced renal injury model to investigate the potential renoprotective effects of MWagainst cisplatin-induced nephrotoxicity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the present study, we initially discovered the predominant mechanisms for MW on ferritinophagy and the ferroptosis signaling pathway through proteomics study. Further studies showed that autophagy activates nuclear receptor coactivator 4 (NCOA4) and mediates ferritinophagy, and ferritin is degraded in lysosomes, releasing a large amount of free Fe2\u0026thinsp;+\u0026thinsp;and aggravating ferroptosis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Moreover, we found that MW effectively inhibited ferroptosis and lysosome degradation. Therefore, we proposed that MW effectively interferes with ferroptosis by blocking ferritinophagy mediated by NCOA4.\u003c/p\u003e \u003cp\u003eIn recent years, increasing attention has been directed toward the toxicity associated with Chinese herbs. Consequently, this study aimed to examine the adverse reactions of MW. The administration of a wide dosage of MW for 14 days was observed to have an impact on renal function and renal histomorphology. The Nrf2/HO-1 signaling axis plays a crucial role in the regulation of anti-inflammatory and antioxidant responses, mitigation of mitochondrial damage, and control of cell death\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The findings of our study demonstrated that MW significantly affected the expression of Nrf2, HO-1, and SOD2 proteins. MW also affected apoptosis and epithelial growth factor receptor (EGFR) pathway-related proteins. Therefore, the study revealed that varying doses and durations of MW administration could elicit dual effects via diverse mechanisms.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of MW\u003c/h2\u003e \u003cp\u003eMW was purchased from Beijing Ben Cao Fang Yuan Pharmaceutical and Technology Co., Ltd. (Anhui, China); it is identified as the dry aboveground part of Leonurus japonicas Houtt by Professor Yang Chengzi of Fujian University of Traditional Chinese Medicine. The MW was extracted with methanol according to a previous method\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Chemical analysis by UHPLC-QTOF-MS/MS led to the identification of 33 compounds, by matching HR-MS data against the Natural Products MS/MS Library (Supplementary Fig.\u0026nbsp;1), such as eight marker compounds, including leonurine, 4\u0026prime;,5-dihydroxy-7-methoxyfavone, rutin, hyperoside, apigenin, quercetin, kaempferol, and salicylic acid, were quantified (Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eMale BALB/c mice (18\u0026ndash;22 g), 6 weeks old, were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd. (China Shanghai, SCXK(HU)2017-0012), with a total of 66 individuals. All experimental protocols were authorized by the Laboratory Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (FJTCM IACUC 2022250). Animals were handled under the International Guiding Principles for Biomedical Research Involving Animals, issued by the Council for the International Organizations of Medical Sciences. The mice were housed in the Laboratory Animal Center of Fujian University of Traditional Chinese Medicine. The environmental temperature was maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ℃ with a 12 h light/dark cycle and humidity of 50%.\u003c/p\u003e\n\u003ch3\u003eEstablishment of Animal Models and Drug Administration\u003c/h3\u003e\n\u003cp\u003eIn the first part, the investigation on the improvement effect of Leonurus japonicus on acute kidney injury, all animals were divided into six groups (n\u0026thinsp;=\u0026thinsp;6): control (Ctrl), cisplatin (Cis), cisplatin\u0026thinsp;+\u0026thinsp;10 g/kg MW (Cis+MW10), cisplatin\u0026thinsp;+\u0026thinsp;40 g/kg MW (Cis+MW40), 10 g/kg MW (MW10), and 40 g/kg MW (MW40). Cisplatin was administered as a single intraperitoneal dose of 20 mg/kg per mouse. The MW intervention group received daily oral administration of motherwort solution for 4 consecutive days(10 g/kg or 40 g/kg). Kidney and blood samples were collected for analysis.\u003c/p\u003e \u003cp\u003eIn the second part, the investigation on the damage effect of Leonurus japonicus on normal kidneys under long-term and high-dose exposure, all animals were divided into five groups (n\u0026thinsp;=\u0026thinsp;6): control (Ctrl), 10, 20, 40 and 80 g/kg MW (MW10, MW20, MW40, MW80). Intervention group received daily oral administration of motherwort solution for 14 consecutive days. Kidney and blood samples were collected for analysis.\u003c/p\u003e\n\u003ch3\u003eRenal histological studies\u003c/h3\u003e\n\u003cp\u003eAfter conducting the clinical score analysis, the same groups of mice were used for harvesting kidney tissues and fixing them in 4% paraformaldehyde. The kidney tissue was then dehydrated, aparaffin-embedded, and cut into 5 \u0026micro;m slices. Subsequently, dimethyl benzene dewaxing and wood staining were performed for 8 to 10 min each, followed by a series of alcohol dehydration steps (2% hydrochloric acid, 2% eosin stain) lasting 1 to 2 min each at concentrations of 80%, 90%, and finally 100%. This was followed by three washes with xylene at a concentration of 100%, each lasting for 5 min. The sections were then cleaned with xylene and sealed with neutral gum. Renal pathological alterations was observed using an optical microscope, photographed, and recorded within the field of vision. Finally, the score for every histopathologic feature was calculated for each animal.\u003c/p\u003e\n\u003ch3\u003eBiochemical measurements\u003c/h3\u003e\n\u003cp\u003eThe levels of blood urea nitrogen (BUN), serum creatinine (Scr) and Malondialdehyde (MDA) in mouse plasma were determined using a urea assay kit, creatinine assay kit, and MDA assay kit, respectively. Kidney tissues were rinsed in normal saline, and then weighed and homogenized. LPO in the supernatant of homogenized renal tissues from rats was measured using an LPO assay kit. All kits were purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. and used according to the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData independent acquisition (DIA) analysis and proteomics analysis\u003c/h2\u003e \u003cp\u003eKidneys were homogenized and resuspended in lysis buffer (6 M urea,1% Protease Inhibitor, 1% phosphatase inhibitor), and equal amounts were purified using SDS-PAGE. The gel pieces stained with Coomassie brilliant blue were excised and subjected to in-gel digestion using trypsin. Extracted peptides were desalted using StrataXSPE and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS analyses were performed on an QExactiveTMHF-X (Thermo Fisher Scientific), coupled online to an ultraperformance liquid chromatography system. The resulting MS/MS data were processed using MaxQuant search engine (v.1.6.15.0). Tandem mass spectra were searched on Mus_musculus_10090_SP_20200509. fasta against the SwissProt database (17045 entries) concatenated with reverse decoy database. When the differentially expressed proteins (DEPs) were obtained, the proteins were annotated by Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. GO identifies three aspects of biology: cellular component (CC), biological process (BP), and molecular function (MF). The dataset was extracted from KEGG database, and the gene-coding proteins were obtained by STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The intersection analysis of DEPs and pathway-related proteins was carried out.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReal-time quantitative polymerase chain reaction PCR (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eTissue RNA was extracted from renal tissues using Trizol reagent according to the standard protocol. After reverse-transcription, RT-qPCR was performed on the QuantStudio 3 system (Applied Biosystems, USA). The mRNA levels of target genes were normalized and analyzed by the 2-∆∆Ct method. The following primers were used for RT-qPCR:β-actin forward 5\u0026prime;-TTGTCCACCTTCCAGCAGATGT-3\u0026prime; and reverse 5\u0026prime;-AGCTCAGTAACAGTCCGCCTAG-3\u0026prime;, PTGS2 forward 5\u0026prime;-TGAGTGGGGTGATGAGCAAC-3\u0026prime; and reverse 5\u0026prime;-TTCAGAGGGCAATGCGGTTCT-3\u0026prime;, GPX4 forward 5\u0026prime;-AATCAAGGAGTTTGCAGCCG-3\u0026prime; and reverse 5\u0026prime;-CCACGCAGCCGTTCTTATCA-3\u0026prime;, Ceruloplasmin forward 5\u0026prime;-CGGATCACTACACAGGTGGC-3\u0026prime; and reverse 5\u0026prime;-CCATTCCACCTCTACGGCTG-3\u0026prime;, Transferrin forward 5\u0026prime;-AGACTTCGAGTTGCTCTGCC-3\u0026prime; and reverse 5\u0026prime;-CAGAAATTGCCGGTGCAGTC-3\u0026prime;, ATG7 forward 5\u0026prime;-CACGGTTCGATAATGTTCTTCC-3\u0026prime; and reverse 5\u0026prime;-GTCTCCTCGTCACTCATGTCCC-3\u0026prime;, ATG5 forward, 5\u0026prime;-GGCCATCAACCGGAAACTCA-3\u0026prime; and reverse 5\u0026prime;-CGCTCCGTCGTGGTCTGATAT-3\u0026prime;, NCOA4 forward 5\u0026prime;-GAGGTGTAGTGATGCACGGA-3\u0026prime; and reverse 5\u0026prime;-GACGGCTTATGCAACTGTGAA-3\u0026prime;, FTH-1 forward 5\u0026prime;-GCCGAGAAACTGATGAAGCTGC-3\u0026prime; and reverse 5\u0026prime;-GCACACTCCATTGCATTCAGCC-3\u0026prime;, Man2b1 forward 5\u0026prime;-GGTGGTAGCAGTCCCTATCA-3\u0026prime; and reverse 5\u0026prime;-CTCAGGTTGCGATCCGAATC-3\u0026prime;, Neu1 forward 5\u0026prime;-GCCCTACGAGCTTCCAGATG-3\u0026prime; and reverse 5\u0026prime;-CAGGGTCGAAGGTCACATCC-3\u0026prime;, LIPA forward 5\u0026prime;-TCACAGATGCCTGAGTTGGC-3\u0026prime; and reverse 5\u0026prime;-GGCAAGCGTCCCAATTGAAG-3\u0026prime;, NGAL forward 5\u0026prime;-ACAGAAGGCAGCTTTACGATGT-3\u0026prime; and reverse 5\u0026prime;-ACTGGTTGTAGTCCGTGGTGG-3\u0026prime;, and KIM-1 forward 5\u0026prime;-GGAGATACCTGGAGTAATCACACT-3\u0026prime; and reverse, 5\u0026prime;-CACGCTTAGAGATGCTGACTTC-3\u0026prime;.\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eRenal tissues were lysed in RIPA buffer containing protease inhibitors (Thermo Scientific) for protein extraction\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with one of the primary antibodies: KIM-1 (1:1000, Novus Biologicals), NGAL (1:1000, Bio-Techne Corporation), GPX4 (1:1000, Abcam), PTGS2 (1:1000, Abcam), Man2b1 (1:1000, Novus Biologicals), Neu1 (1:1000, Thermo Fisher Scientific Inc.), LIPA (1:1000, Proteintech Group Inc.), ATG5 (1:1000, Abcam), ATG7 (1:1000, Cell Signaling Technology), LC3I (1:1000, Proteintech Group Inc.), LC3II (1:1000, Proteintech Group Inc.), Ceruloplasmin (1:1000, Abcam), Transferrin (1:10000, Abcam), NCOA4 (1:2000, Thermo Fisher Scientific Inc.), FTH-1 (1:1000, Cell Signaling Technology), Nrf2(1:1000, Cell Signaling Technology), Bcl-2(1:1000, Proteintech Group Inc.), Bax(1:1000, Proteintech Group Inc.), SOD2(1:1000, Cell Signaling Technology), HO-1(1:10000, Abcam), EGFR(1:10000, Abcam),Grb2(1:1000, Proteintech Group Inc.), p-raf(1:10000, Abcam), raf(1:1000, Zen BioScience), p-MEK(1:10000, Abcam), MEK(1:10000, Abcam), p-ERK(1:1000, Zen BioScience), ERK(1:1000, Proteintech Group Inc.), β-actin(1:10000, Abcam) and GAPDH (1:2000, Proteintech Group Inc.). The blots were washed and probed with one of the secondary antibody goat anti-mouse IgG(H\u0026thinsp;+\u0026thinsp;L) (1:3000, Thermo Scientific) and goat anti-rabbit or IgG(H\u0026thinsp;+\u0026thinsp;L) (1:3000, Thermo Scientific). The protein signals were detected by enhanced chemiluminescence (Beyotime Biotechnology), and images of the blots were obtained using a ChemiDoc XRS+ imaging system (Bio-Rad) and analyzed with ImageJ software. Results are expressed as fold changes after normalization to GAPDH or β-actin.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePrussian blue staining\u003c/h2\u003e \u003cp\u003eRelevant literature should be consulted for the experimental procedures\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, The tissues were fixed in a solution of paraformaldehyde and subsequently subjected to embedding. Following sectioning, the tissue underwent a conventional dewaxing process, wherein the section was immersed in Perls' stain for 15 to 30 min. Subsequently, the sections were immersed in a nuclear solid red staining solution for 5 to 10 min. This was followed by standard dehydration and clearing procedures, culminating in sealing with neutral gum to safeguard the sections and preserve the staining outcomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL staining\u003c/h2\u003e \u003cp\u003eA TUNEL apoptosis kit (B0013, LABELAD) was used according to the manufacturer\u0026rsquo;s instructions to detect the apoptosis of kidney tissue. After dewaxing, 20 \u0026micro;g/mL proteinase K solution (100 \u0026micro;L) was dropped to cover the tissue, and the sections were incubated at room temperature for 20 min. After washing with PBS for 5 min, 100 \u0026micro;L of TUNEL equilibration buffer was dropped and incubated for 5 min. About 50 \u0026micro;L of TUNEL reaction mixture was added, incubated for 2 h, and washed with PBS for 5 min. Thereafter, 0.1% Triton X-100 (containing 5 mg/mL BSA) buffer was applied three times, followed by the addition of 50\u0026micro;L of DAPI-containing anti-fluorescence sealing solution to the sections, which were incubated for 10 min. They were then observed and photographed under a fluorescence inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). All statistical analyses were conducted using SPSS software (version 26.0). The normality of continuous variables was assessed using the Shapiro-Wilk test. Since all variables demonstrated normality (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), one-way analysis of variance (ANOVA) was employed to determine significant differences among groups. For data that did not follow a normal distribution, the non-parametric Kruskal-Wallis test was used. Values at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eMW attenuates cisplatin induced AKI in mice\u003c/p\u003e \u003cp\u003eCisplatin resulted in a significant weight loss and increased renal index (kidney weight/body weight), Scr levels, and BUN levels. 10 and 40 g/kg MW efficiently reduced these changes. In addition, effects of MW40 were superior to those of MW10 on renal index, Scr, and BUN. However, MW control administration did not exhibit any effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). KIM-1and NGAL are biomarkers of AKI, representing renal function\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The mRNA and protein expression levels of KIM-1 and NGAL in kidney tissue from the Cis\u0026thinsp;+\u0026thinsp;MW (10 and 40) group were significantly lower than those in the Cis group, but MW control administration did not exhibit any effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;H). The HE staining results of renal tissue showed that compared with the Ctrl group, the epithelial cells exhibited cellular edema, the renal glomeruli demonstrated atrophy, and some cells displayed necrosis and disorganized arrangement. Moreover, MW10 and MW40 provided effective protection, and no changes were observed in the control administration of MW10 and MW40 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProteomics analysis of renal tissue and determination of ferroptosis and lysosomes as the key drivers\u003c/p\u003e \u003cp\u003eThe proteomics analysis was conducted to investigate the renal tissues in the cisplatin-induced AKI model treated by MW. The proteomics analysis identified 4150 proteins. When p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, a change in differential expression level exceeding 1.5 was used as a significant upregulation threshold, and a decrease of less than 1/1.5 was used as a significant downregulation threshold. A total of 55 proteins had common changes, including ceruloplasmin and transferrin related to iron metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). GO analysis showed that the effect of MW on cisplatin-induced AKI was mainly related to cell composition, biological process molecular function, and lysosomes and iron\u0026ndash;ion binding (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;G). KEGG pathway enrichment analysis also showed that ferroptosis and lysosomes were key pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). In summary, the proteomic analysis illustrated that ferroptosis and lysosomes might be the key drivers of MW treatment of cisplatin-induced AKI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMW attenuated ferroptosis in cisplatin-induced mice\u003c/p\u003e \u003cp\u003eFerroptosis frequently shows an increased level of LPO\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Cisplatin induced the increase in serum MDA and tissue LPO levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). MW administration could significantly reduce the levels of MDA and LPO, and the effects of MW40 were superior to those of MW10. Prostaglandin endoperoxide synthase 2 (PTGS2) and Glutathione peroxidase 4 (GPX4) are markers of ferroptosis. After modeling with cisplatin, the gene and protein expression levels of PTGS2 increased and those of GPX4 decreased. However, MW administration effectively reduced these changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMW reduced lysosomes-related hydrolase proteins in cisplatin-induced mice\u003c/p\u003e \u003cp\u003eKEGG proteomics results showed that MW protecting AKI was closely related to lysosomes. lysosomes-related hydrolase proteins were detected to evaluate the effects of MW on lysosomes in cisplatin-induced mice. The mRNA and protein expression of α-mannosidase(Man2b1), Neuraminidase 1(NEU-1), and Lipase(LIPA) decreased in the cisplatin group mice, whereas a substantial increase in mRNA and protein expression of Man2b1, NEU-1, and LIPA was observed in MW-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMW-attenuated ferritinophagy in cisplatin-induced mice\u003c/p\u003e \u003cp\u003eFerritinophagy is a type of selective autophagy, which increases the degradation of ferritin in lysosomes\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Ceruloplasmin converts Fe\u003csup\u003e2+\u003c/sup\u003e to Fe\u003csup\u003e3+\u003c/sup\u003e and Promote ferritin transport of excessive iron into cells and stored in ferritin. In this experiment, we observed that MW inhibited the increase in ceruloplasmin (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B) and transferrin (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;D), as well as autophagy key proteins LC3II/LC3Ⅰ (microtubule- associated protein1-light chain-3, LC3 ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and Autophagy-related gene 5(ATG5) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF\u0026ndash;G), Autophagy-related protein 7(ATG7) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u0026ndash;I). Nuclear receptor coactivator (NCOA4) mediates ferritin autophagy by promoting ferritin degradation into lysosomes, increasing free iron and aggravating subsequent ferroptosis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Interestingly, we observed that MW inhibited the activation of NCOA4 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ\u0026ndash;K) and promoted the elevation of Ferritin heavy chain 1(FTH-1) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL\u0026ndash;M). We evaluated the level of iron\u0026ndash;ion in renal tissue by Prussian blue staining. Compared with the control group, the iron\u0026ndash;ion staining in the kidney tissue of Cis. group mice increased significantly, and MW remarkably reduced the staining area (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). These results showed that MW reduced ferritinophagy and free iron.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHigh-dose MW damaged renal function of mice\u003c/p\u003e \u003cp\u003eIn the previous experiment, we observed that administering 10 and 40g/kg for 4 days did not damage the kidneys of mice. However, when the dosage was increased and the administration time was prolonged, we observed kidney damage caused by 80g/kg MW after administering for 14 days. We observed significant weight loss and increased renal index, BUN, Cre, MDA, KIM-1 and NGAL levels(Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). The HE staining results of renal tissue showed that compared with the Ctrl group, 80g/kg MW provided ob-vious side effects on kidney with the epithelial cells exhibiting cellular edema, the renal glomeruli demonstrated atrophy, and some cells displayed necrosis and disorganized arrangement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHigh-dose MW promoted oxidative stress and apoptosis in renal tissue\u003c/p\u003e \u003cp\u003eThe level of apoptosis was enhanced by MW, as indicated through the TUNEL staining of renal tissue (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). In addition, we observed that MW inhibited apoptotic protein Bcl-2 promoted the content of pro-apoptotic protein Bax (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D).The promotion of kidney injury is significantly influenced by oxidative stress, and apoptosis is intricately linked to the pathophysiology of kidney injury\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In this experiment, we observed that MW inhibited the expression of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2), Heme Oxygenase-1 (HO-1), and Superoxide dismutase 2 (SOD2) proteins (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). These results implied that 4 g/mL MW promoted oxidative stress and apoptosis in renal tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMW may induced renal injury through the EGFR pathway\u003c/p\u003e \u003cp\u003eThe activation of EGFR can induce cell cycle entry and promote cell proliferation, regulate transcription factors in the nucleus, and inhibit certain apoptosis-inducing signals or molecules\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In this experiment, we observed that MW increased the expression of EGFR and Grb2 proteins (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B). In addition, we observed that MW facilitated the phosphorylation of raf, MEK, and ERK proteins, which play crucial roles in the EGFR signaling pathway (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-E). These results implied that MW may induce renal injury via the EGFR signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMW, as a traditional herbal medicine, is widely used for several diseases in China, Korea, and Japan. MW promotes blood circulation, regulates menstruation, promotes water retention, introduces detumescence\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, exhibits antipyretic properties, and detoxifies. The study also found that MW exerted a protective effect on kidney damage, which is why it is frequently employed in the treatment of kidney disease. However, in long-term clinical practice, MW also has adverse reactions, which may affect kidney function and cause kidney tissue damage\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The dual role of MW in kidney protection and injury and its mechanism have attracted research attention. In this study, a cisplatin-induced mouse kidney injury model was used. The pathogenesis of cisplatin-induced AKI is complex and multifactorial, involving DNA damage, energy consumption, oxidative stress, endoplasmic reticulum stress, inflammation, vascular dysfunction, and mitochondrial damage\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This study found that 4 days of administration of MW extract could reduce the kidney index, renal index (kidney weight/body weight), serum Scr levels, and BUN levels. Previous literature also reported that the AKI caused by doxorubicin, lipopolysaccharide, and sepsis has a significant protective effect of MW \u003csup\u003e25, 26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSubsequently, we used proteomics to analyze the mechanism by which MW protects kidney injury. Proteomics was used to identify the therapeutic targets by comparing the differential changes in the proteome in animal tissues after MW treatment on cisplatin-induced AKI. Ferroptosis pathogenesis encompasses diverse mechanisms, including ROS-driven iron-dependent non-apoptotic cell death\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and PD-L1-mediated immune regulation (affecting T cell activation, proliferation, and cytotoxic factor secretion)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Notably, our proteomic analysis demonstrates that the treatment of AKI with MW might be closely related to lysosome, iron metabolism, and ferroptosis.. Specifically, MW effectively inhibited the increase in MDA, LPO, and GPX4 expression but increased the decrease in PTGS2 expression. In addition, MW increased the reduction of related active enzymes of lysosome. Subsequently, we analyzed the upstream mechanism of MW regulating ferroptosis.\u003c/p\u003e \u003cp\u003eCeruloplasmin and transferrin are differential proteins in proteomics. Experiments verified that MW inhibited the increase in ceruloplasmin and transferrin. Ferritin can alter sequestered iron via autophagy (ferritinophagy)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Ferritinophagy is a kind of selective autophagy mediated by NCOA4\u003csup\u003e30\u003c/sup\u003e. NCOA4, which is activated by the autophagy-related gene (ATG), is a cargo receptor for ferritinophagy that interacts with FTH1 and promotes the transport of ferritin to the lysosome for degradation, thereby causing iron release. Iron accumulation induces ferroptosis\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. We observed that MW inhibited the expression of autophagy key proteins ATG5, ATG7, and LC3II/LC3I and reduced the activation of NCOA4 and increased the expression of FTH1 in renal issue of AKI mice. Thus, MW attenuated ferritinophagy, reduced the degradation of ferritin and the release of free iron ions, and inhibited ferroptosis, protecting kidney damage caused by cisplatin. The protective effect of MW on cisplatin-induced AKI was mainly related to the inhibition of ferroptosis.\u003c/p\u003e \u003cp\u003eThe above results showed that MW had a protective effect on kidney injury. In recent years, More and more studies are paying attention to the damaging effects of motherwort on the kidneys Many studies have demonstrated that 30-120g/kg MW may cause kidney function injury in rats\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, so we conducted further research to explore the relevant mechanism.After 14 days of continuous administration of 80 g/kg MW in normal mice, we found that the weight of mice decreased significantly, whereas the kidney index, BUN, Scr, and MDA increased. Animal experiments revealed that 80 g/kg MW promoted the expression of KIM-1 and NGAL genes and proteins, and HE results showed that a large dose of MW could significantly damage kidney tissue.Then we conducted further exploration into its damage mechanism. We found that the expression levels of Nrf2 and HO-1 proteins in kidney tissue decreased, whereas the expression level of SOD2 was significantly reduced. Furthermore, MW exhibited a significant inhibitory effect on the expression of anti-apoptotic protein Bcl2 and induced an increase in the expression of pro-apoptotic protein Bax. These findings implied a correlation between MW-induced kidney injury and oxidative stress, as well as cellular apoptosis.\u003c/p\u003e \u003cp\u003eEGFR belongs to the family of transmembrane receptor tyrosine protein kinases, playing a crucial role not only in fine-tuning cellular signal transduction and promoting tumor cell survival but also in regulating cellular metabolism, proliferation, migration, and differentiation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The activation of EGFR can lead to kidney damage, and preclinical studies have shown its potential as a therapeutic target for chronic kidney disease\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003eWe observed a correlation between MW-induced kidney injury and the EGFR signaling pathway. We analyzed the expression levels of EGFR, Grb2, p-raf/raf, p-MEK/MEK, and p-ERK/ERK and discovered that using MW significantly enhanced the expression of proteins associated with the EGFR signaling pathway. These findings indicated that MW-induced kidney injury was not only linked to oxidative stress and apoptosis but also via activating the EGFR signaling pathway. Thus, long-term high-dose MW may be related to EGFR signaling and downstream oxidative stress and apoptosis pathways in kidney injury.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings indicated that MW played a dual role in kidney protection and damage, exerting its effects through distinct mechanisms. MW mitigated cisplatin-induced kidney damage by inhibiting ferroptosis. However, prolonged administration of high doses of MW induced oxidative stress, apoptosis and the EGFR signaling pathway in kidney, ultimately causing kidney injury.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHongmin Yu: Investigation, Validation, Formal Analysis. Tao Wang:\u0026nbsp;Investigation, Validation, Formal Analysis. Xiaomei Chen: Data Curation, Visualization, Writing – Original Draft. Cheng Zhang: Investigation, Resources. Qing Xu: Investigation, Resources. Meixia Huang: Methodology, Conceptualization. Yingzheng Wang: Writing – Original Draft, Supervision. Jie Xu: Writing – Review \u0026amp; Editing. Yinghao Wang: Conceptualization, Funding Acquisition, Supervision, Writing – Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests, and all authors should confirmits accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the [the National Natural Science Foundation of China #1] under Grant [number 82173996]; [National Natural Science Foundation of Fujian province #2] under Grant [number 2021J01920].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this publishedarticle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical review approval and consent to participate in research\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the relevant guidelines and regulations and were approved by the Laboratory Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (approval number FJTCM IACUC 2022250).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublishing consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMiao, L. 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Role of Epidermal Growth Factor Receptor (EGFR) and Its Ligands in Kidney Inflammation and Damage. \u003cem\u003eMediators of inflammation\u003c/em\u003e. \u003cstrong\u003e2018\u003c/strong\u003e, 8739473 (2018). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"motherwort, ferritinophagy, EGFR pathway, oxidative stress, apoptosis ","lastPublishedDoi":"10.21203/rs.3.rs-8740889/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8740889/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMotherwort (MW) is known for its renoprotective effects, but it has also been reported to potentially cause kidney damage. This study aimed to explore the distinct mechanisms underlying the protective and detrimental effects of MW on renal health, providing a theoretical basis for its safe clinical application. We explored the effects of MW in cisplatin-induced acute kidney injury (AKI) in BALB/c mice. We found that in AKI mice administration of the MW at doses of 10 and 40 g/kg for 4 days alleviated the loss of body weight, increased the renal index, reduced pathological kidney damage, and lowered the levels of blood urea nitrogen (BUN), serum creatinine (Scr), kidney injury molecule‑1 (KIM‑1), and neutrophil gelatinase‑associated lipocalin (NGAL). Proteomic analysis and subsequent validation indicated that the mechanisms were associated with ferroptosis and autophagy pathways. In healthy mice, short‑term MW treatment at the same doses (10 and 40 g/kg) did not affect renal function. However, when healthy mice were administered a higher dose of MW (80 g/kg) by gavage for an extended period (14 days), it induced body weight loss, triggered renal damage, and elevated BUN, Scr, KIM‑1, and NGAL levels. At this dosage, MW also increased oxidative stress, promoted apoptosis, and upregulated the expression of epidermal growth factor receptor (EGFR) pathway‑related proteins. Our findings demonstrate that MW plays a dual role in kidney protection and injury through distinct mechanisms, offering important guidance for its clinical application.\u003c/p\u003e","manuscriptTitle":"Dual Role of Motherwort in Renal Health: Protective and Detrimental Effects","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 09:40:04","doi":"10.21203/rs.3.rs-8740889/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:03:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7943667625053615351314818967482316146","date":"2026-04-19T10:49:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-15T11:19:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T05:43:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-13T06:37:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-10T15:18:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-10T14:11:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f1f4ab49-ea84-4777-8a43-1d7c1d7d62b3","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-03T00:03:39+00:00","index":91,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66658039,"name":"Health sciences/Diseases"},{"id":66658040,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2026-04-23T09:40:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 09:40:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8740889","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8740889","identity":"rs-8740889","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}