Celastrol improves kidney damage in spontaneous hypertensive rats by regulating the Nrf2/Ho-1 pathway

Celastrol improves kidney damage in spontaneous hypertensive rats by regulating the Nrf2/Ho-1 pathway | 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 Celastrol improves kidney damage in spontaneous hypertensive rats by regulating the Nrf2/Ho-1 pathway Yijie Deng, JiChun Wang, Xiping Liu, Yue Chen, Min Wang, Xiuwen Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6914458/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The natural triterpenoid celastrol, which comes from Tripterygium wilfordii, has a variety of biological effects. We investigated Celastrol improves kidney damage in spontaneous hypertensive rats by regulating the Nrf2/Ho-1 pathway. A total of 24 12-week-old male spontaneous hypertensive rats (SHR) were randomly allotted to four groups [control group, SHR group, L-CSL + SHR group (0.02 mg/kg/d) and H-CSL + SHR group (0.04 mg/kg/d)]. The results showed that CSL group significantly decrease levels REN, Angiotensin, ACE and ALD and decrease expression levels of TNF- α , IL-1 β and increase expression levels of IL-6 in serum compared with SHR group. Kidney functions, CSL group significantly decrease level of MDA and increase SOD, GSH-Px and CAT compared with SHR group. CSL had a significant inhibitory effect on the increase in the relative expression abundance of Keap1. The Nrf2, Nqo1 and Ho-1 mRNAs were found to be significantly lower in the CSL compared with SHR group. The results show that CSL significantly reduces the pathology of kidney damage in spontaneous hypertensive rats by activating Nrf2/Ho-1, and provides treatment strategies for the kidney damage. Biological sciences/Biochemistry Biological sciences/Biological techniques Celastrol Leigong vine spontaneous hypertension kidney injury Nrf2/Ho-1 pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pharmacological bioactive compounds derived from medicinal plants have gained a lot of attention recently because of their strong, distinctive, and varied activities. The chemical structure of celastrol (CSL), a pentacyclic triterpene that was isolated from Tripterygium wilfordi 1 . Among the many pharmacological effects of CSL are anti-inflammatory and antioxidant 2 . CSL has been shown to inhibit several proinflammatory cytokines, such as nuclear factor-kappa B (NFκB), nitric oxide synthase, adhesion molecules, proinflammatory enzymes, interleukins (IL-2, IL-8), interferon-gamma (IFN-γ), and tumor necrosis alpha (TNF- α ) 3 . Therefore, it shown encouraging effects in a variety of inflammatory conditions, including asthma, ulcerative colitis, Crohn's disease, and rheumatoid arthritis 4 , 5 . CSL has also been shown to have anti-diabetic, neuroprotective, and anti-obesity properties 6 , 7 . Furthermore, several investigations have proven CSL anticancer potential by encouraging apoptosis and preventing cell invasion, angiogenesis, and proliferation 8 , 9 . Tripterygium wilfordis plant extract has been used in a number of clinical investigations because of its wide range of pharmacological activities. Furthermore, in an effort to assess CSL therapeutically for a more thorough examination of Castrol's effects on the kidney, randomised clinical trials were conducted on patients with a variety of conditions, including renal transplantation, rheumatoid arthritis, solid tumours, and diabetic nephropathy 10 , 11 . CSL has been shown to be an antioxidant that can increase the expression and activity of heme oxygenase-1 (HO-1) and reduce the production of reactive oxygen species (ROS) in vascular smooth muscle cells in hypertensive rats 12 . Zhang et al. reported a CSL can improve the kidney injury induced by high-fat diet in obese mice by regulating kidney Keap1/Nrf2 pathway to increase antioxidant level 13 . Additionally, mechanistic investigations shown that CSL inhibited several processes in the production of oxidative stress and inflammation, such as the NF-κB signalling pathway 14 . It has been proposed that NF-κB, a pleiotropic transcription factor, is crucial for gene regulation during oxidative stress and inflammation, which lead to atherosclerosis 15 . This study used SHR kidney injury as a model to explore whether CSL alleviates and improves the pathology of SHR kidney injury through the pathway, providing theoretical basis for the prevention and treatment of SHR kidney injury. Materials and methods Ethics approval The protocol was approved by the Committee on the Ethics of Animal Experiments of Jilin Normal University (Siping, Jilin, China) (Approval number: KJLL20250301). Animal and Experimental design A total of 24 12-week-old male SHRs and 8 12 12-week-old male Wistar-Kyoto (WKY) rats with weights ranging from 180 ± 20 g [License Nomber SCXK (Beijing) 2016-0006] were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co.Ltd, were kept under controlled conditions for temperature, humidity and light, with unrestricted access to food and water available throughout all experimental stages. The SHRs were randomly divided into three groups (n = 8). model group, L-CSL + SHR 0.02 mg/kg/d and H-CSL + SHR 0.04 mg/kg/d. The WKY rats were used for the control group. CSL drug injection solutions were prepared with physiological saline. The CON and SHR groups were given equal-volume physiological saline by intraperitoneal injection the L-CSL + SHR and H-CSL + SHR groups were given drug injections by intraperitoneal injection (drug concentrations were 0.02 mg/kg/d and 0.04 mg/kg/d, respectively). All rats were given intraperitoneal injections daily for 6 consecutive weeks. Twenty-four hours after renal reperfusion, animals were sacrificed by decapitation under anesthesia, and blood samples were collected from different experimental groups. Serum samples were obtained via 15 min of blood centrifugation at 5000 rpm and were stored at − 20°C for subsequent measurements. The right kidney was rapidly removed and separated into two parts; the first part was homogenized and kept at − 80°C for biochemical analyses. The second part was kept in 10% formalin for histopathological examination. Intraperitoneal injection of α-chloroaldose (15879-93-3, Sigma, MH, NE) (80 mg/kg) and ooligosaccharides (51-79-6, Sangon, SH, China) (800 mg/kg) to anesthetize rats. All experiments were carried out according to the guidelines. At the end of each experiment, the rats were euthanized by intravenous injection of 0.5 ml KCl solution (10%) under anesthesia. Blood analysis The serum samples were defrosted at 4 ◦ C and mixed evenly before determination. The levels REN, Angiotensin, ACE and ALD and levels of serum inflammatory factors TNF- α , IL-1 β and IL-6 were detected according to the kit instructions. Antioxidant Indexes The contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) in the kidney were determined by spectrophotometry (PU 8720UV/VIS scanning spectrophotometer) and investigated. Commercial assay kits were provided by the Nanjing Jiancheng Institute of Bioengineering (Nanjing, China), and all procedures were performed following the kit’s protocol. Histology Examination and immunohistochemistry Kidney samples were collected and stored in 4% paraformaldehyde at 4°C overnight. The rest of the procedures followed the standard techniques published in previous papers 16 . For immunohistochemistry, the Kindey tissues were placed in Tissue-Tek OCT and snap-frozen in liquid nitrogen. Thereafter, the samples were cryosectioned (15 µm) and stained for target proteins. Antibodies raised against the following mouse antigens were used: Nrf2 antibody (1:300) and Keap1 antibody (1:200). Accordingly, secondary antibodies were used to amplify signals with AlexaFluor 488 anti-rat, Alexa Fluor 647 anti-guinea pig, and AlexaFluor 488 anti-rabbit IgG. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). Two slides of each rats (n = 6 per group) were imaged using a Zeiss confocal Laser Scanning Microscope 780. Western Blotting Analysis The mixture was used to homogenize kidney tissue after adding phosphatase and pro-tease inhibitors in cell lysis buffer (Biosharp, BL509A Hefei, China). Samples were separated on 10% SDS-PAGE gels after their protein content was equilibrated. After transfer to PVDF membranes (Millipore, IPVH00010, Burlington, CA, USA), samples were blocked for one hour using 5% skim milk. Membranes were first exposed for a 12 h incubation period at 4 ◦ C with some primary antibodies before treatment with HRP-conjugated secondary antibodies. Finally, chemiluminescence was detected using a Dannon 5200 multi-imaging system and a high-sensitivity ECL kit (NCM Biotech, P2300, Suzhou, China). Real-Time PCR The kidney tissues from rats were utilized to isolate using Trizol (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions and stored at -80°C. Then, the RNA was reverse-transcribed into cDNA according to the instructions (Vazyme, Nanjing, China). mRNA expression was determined according to the instructions (Vazyme, Nanjing, China), and its relative expression was calculated using the 2 −ΔΔCT method. Statistical analysis All data are expressed as mean ± standard deviation, and SPSS statistical software was used for single-factor variance analysis (ANOVA). Duncan's multiple-range test was used for comparison. GraphPad Prism software was used for plotting, The standard for statistical significance is p < 0.05. Results Effects of CSL on serum biochemical indexes of SHR rats As shown in Fig. 1 . Compared with CON group, serum levels of REN, AngⅡ, ALD and ACE in SHR group were significantly increased (P < 0.01). Compared with SHR group, low and high dose CSL groups could significantly reduce the levels of these biochemical indexes (P < 0.01). Among them, the content of AngⅡ (P < 0.05) and ACE (P < 0.01) in H-CSL SHR group was significantly different from that in L-CSL SHR group, indicating that CSL could inhibit the changes of plasma related indexes in SHR rats, and it was dose-dependent. Effects of CSL on the levels of inflammatory factors in SHR rats As displayed in Fig. 2 . for serum levels of TNF- α and IL-6 in SHR group were significantly increased (P < 0.01) compared to CON. CSL pretreatment could significantly inhibit the expression levels of TNF- α , IL-1 β and IL-6 (P < 0.01). Compared with low dose, high dose CSL could significantly inhibit IL-1 β level (p < 0.05). It is suggested that CSL can improve the anti-inflammatory level of SHR rats. Effects of CSL on oxidative stress-related indexes in SHR rats As displayed in Fig. 3 , the activities of SOD, GSH-Px and CAT in kidney tissue of SHR group were significantly decreased, and the level of MDA was significantly increased (P < 0.01) compared to CON. CSL pretreatment could significantly increase the activities of SOD, GSH-Px and CAT in kidney tissue of SHR rats, and decrease the level of MDA (P < 0.01), and the activities of SOD, GSH-Px and CAT in kidney of high-dose group were significantly increased compared with those of low-dose group (P < 0.05 or P < 0.01). The results showed that CSL could improve the antioxidant capacity of kidney in SHR rats and had a protective effect on kidney. Effects of CSL on renal pathology in SHR rats H&E staining was used to observe the pathological changes of renal tissue in each treatment group, and the results were shown in Fig. 4 . In CON group, the glomeruli were normal in size and structure, and the basal membrane of renal tubules was normal, no diffuse infiltration and congestion of inflammatory cells were observed. In SHR group, the basal membrane of renal tubules was thickened, the epithelial cells of renal tubules were swollen, and the mesangial cells were hyperplasia. Compared with SHR group, CSL group showed a small amount of inflammatory cell infiltration, a slight thickening of the basal membrane of renal tubules, and a significant improvement in congestion, tubule epithelial cell swelling and mesangial cell proliferation. It is suggested that CSL can reduce the inflammatory cell infiltration and the swelling of renal tubular epithelial cells, and improve the renal inflammatory injury in SHR rats, and the therapeutic effect of high dose is more significant than that of low dose. Effects of CSL on the expression of Nrf2 and Keap1 proteins in kidney of SHR rats The expression levels of Nrf2 and Keap1 proteins in kidney tissues of rats in each group were detected by IHC and WB methods, and the results were shown in Fig. 5 . Compared with CON group, Nrf2 protein expression in kidney tissue of rats in SHR group was extremely significantly decreased (P < 0.01), while Keap1 protein expression was extremely significantly increased (P < 0.01). Compared with SHR group, after CSL pretreatment, the expression of Nrf2 protein in kidney tissue was extremely significantly increased (P < 0.01), and the expression of Keap1 protein was extremely significantly decreased (P < 0.01). The expression levels of Nrf2 and Keap1 protein in kidney tissues of CSL high-dose and low-dose groups were significantly different (P < 0.01). These results indicated that Nrf2 and Keap1 proteins were involved in the process of renal tissue injury in SHR rats, and CSL could regulate Nrf2 and Keap1 proteins. Effects of CSL on mRNA transcription of genes related to Nrf2/Ho-1 signaling pathway in kidney of SHR rats As displayed in Fig. 6 , the mRNA relative expressions of Nrf2, Nqo1 and Ho-1 genes in kidney of SHR group were significantly down-regulated, and the differences were statistically significant (P < 0.01) Compared with CON group. his pathway is activated in the kidneys of hypertensive rats. The mRNA expressions of Nrf2 and Nqo1 in low-dose and high-dose CSL groups were significantly higher than those in SHR group (P < 0.01), and the difference between the two groups was extremely significant (P < 0.01), suggesting that CSL could exert therapeutic effect by regulating the expression of Nrf2/Ho-1 pathway gene in the kidney of SHR rats, and the therapeutic effect was positively correlated with dose. Discussion Through preventing NF-kB activation, CSL has been investigated for its ability to protect rats against renal IRI 17 . However, in this work, we examined CSL antioxidant capacity by examining how it affects the Nrf2/HO-1 pathway. Oxidative stress and inflammation are the key factors leading to the sustained development of hypertensive kidney damage. This study is the first to investigate the molecular mechanism of CSL in improving kidney damage in SHR rats. Studies have shown that hypertensive renal damage mainly includes the damage of various endothelial cells and the infiltration of inflammatory cells. The kidney participates in the formation of blood pressure through the secretion of renin and the regulation of body fluids, and the imbalance of such regulation leads to hypertension 18 . Biological indicators related to renal function include REN, ALD, AngⅡ, and ACE, which are abnormal in SHR rats with kidney injury 19 . In this study, CSL can significantly inhibit the expression levels of renin, ALD, AngⅡ and ACE in SHR rats, and alleviate the pathological changes of kidney injury. Persistent hypertension can cause oxidative stress and inflammation in the kidney, and both play a combined role in hypertension-related kidney damage, exacerbating the pathological development of the kidney 20 . There is growing evidence that the fibrotic kidney is surrounded by a condition of inflammation due to the accumulation of inflammatory cytokines 21 . To slow the advancement of kidney damage in spontaneous hypertensive, it is thus essential to effectively decrease inflammation and ameliorate the inflammatory microenvironment. Anti-inflammatory medications are now extensively utilised in clinical settings to manage kidney inflammation, and in both experimental and clinical studies, tremendous progress has been achieved in the prevention and treatment of kidney fibrosis utilising natural ingredients 22 . A Chinese medicinal herb called Tripterygium wilfordii Hook F has strong anti-inflammatory properties, particularly in rheumatoid arthritis. The active component of Tripterygium wilfordii Hook F, which is utilised in clinical settings to treat immunological disorders, is CSL 23 . After kidney damage, CSL reduced the kidney's inflammatory response by improving the production of IL-10 cytokines and decreasing the release of TNF- α , IL-1 β , and IL-18 inflammatory factors. Wang et al. reported that significantly increase IL-10 and decreased level of TNF‐ α , IL‐1 β and level of IL‐18 in kidney duo to supplementation of CSL 24 . In the current study, CSL treatment a decreased level of TNF- α , IL-1 β and IL-6 in kidney compared to SHR. Parallel to these previous studies, our findings also demonstrated that CSL had a potent anti-inflammatory effect on kidney damage in spontaneous hypertension, both in vitro and in vivo. This suggests that inflammation intervention was a useful tactic for controlling kidney damage in spontaneous hypertension. In the present study, increase the activities of SOD, GSH-Px and CAT and decrease the level of MDA in CSL groups compared with SHR. Younis et al. reported that CSL groups decrease level of MDA and increase content of GSH 17 . Increased ROS generation in SHR rats results in renal cell damage and apoptosis 25 . Nrf2 is a crucial regulator that counteracts oxidative damage by activating genes that produce antioxidants, which helps protect against experimental acute kidney injury. Stress causes Nrf2 to evade its inhibitor (Keap1), enter the nucleus, and increase the production of defense-promoting enzymes such as superoxide dismutase (SOD) and heme oxygenase-1 (HO-1). Kidney damage from ischemia-reperfusion or toxins is lessened by this antioxidant response 26 . Heme oxygenase-1 (HO-1) is an essential cytoprotective mechanism that utilizes enzymatic breakdown to convert pro-oxidant heme into physiologically active chemicals. This catalytic reaction produces three beneficial components biliverdin and its metabolite bilirubin, and carbon monoxide (CO), a gaseous mediator that causes vasodilation and has strong antioxidant properties, with antiapoptotic effects. HO-1 is an essential defense mechanism against oxidative damage and cellular injury, as it simultaneously generates protective molecules and removes a harmful oxidant (heme) 27 . Our results showed an improve in gene expression Nrf2, HO-1 and Nqo1 and decreased Keap1 level in SHR as a defensive response to IR injury, and these results are consistent with a previous study 28 . Meanwhile, mice given CSL showed a significant increase in the gene expression of Nrf2, HO-1, and Nqo1, and a decrease in the level of Keap1 in the kidneys compared to mice treated with SHR. This indicates that CSL protects against SHR by activating Nrf2, which increases HO-1 and Nqo1 levels. In conclusion, CSL can alleviate the pathological changes of kidney injury in SHR rats, possibly by activating the expression of Nrf2/HO-1 pathway gene and protein to improve the level of kidney antioxidant, inhibit the expression of inflammatory factors and thus reduce the inflammatory response. This study provides a new pharmacological and mechanistic understanding of the effect of CSL on renal injury in SHR rats, and helps to better understand the application of CSL in the treatment of hypertensive nephropathy. Declarations Data availability statement The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors. Ethics statement The protocol was approved by the Committee on the Ethics of Animal Experiments of Jilin Normal University (Approval number: KJLL20250301). The studies were conducted in accordance with the local legislation and institutional requirements. AUTHOR CONTRIBUTIONS R.W. and Y.Y. supervised the project and wrote the manuscript; Y.D. and M.Z. designed the experiments and conducted the ELISA and real-time RT-PCR experiments; J.W., Y.C. and M.W. performed a part of the immunoprecipitation and western blot analysis; X.L., X.W. and H.L. designed and conducted the animal studies. X.L., H.L. and B.G. conducted the western blot and immunofluorescent experiments. Funding This study was supported by research grants to Y.Y. from the Program for the Development of Science and Technology of Jilin Province (YDZJ202201ZYTS457);to R.W. from the National Nature Science Foundation of China (31871150) and the Science and Technology Research Project of Jilin Provincial Department of Education (JJKH20210446KJ); to H.L. from the Program for the Development of Science and Technology of Jilin Province (20220101322JC), to M.Z. from the Program for the Development of Science and Technology of Jilin Province (YDZJ202201ZYTS433). Conflict of interest To authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Wang, Y. et al. Celastrol exerts anti-inflammatory effect in liver fibrosis via activation of AMPK‐SIRT3 signalling. J. Cell. Mol. Med. 24 , 941–953 (2020). Younis, N. S. & Ghanim, A. M. H. The Protective Role of Celastrol in Renal Ischemia-Reperfusion Injury by Activating Nrf2/HO-1, PI3K/AKT Signaling Pathways, Modulating NF-κb Signaling Pathways, and Inhibiting ERK Phosphorylation. Cell. Biochem. Biophys. 80 , 191–202 (2022). Jung, H. W., Chung, Y. S., Kim, Y. S. & Park, Y. K. Celastrol inhibits production of nitric oxide and proinflammatory cytokines through MAPK signal transduction and NF-κB in LPS-stimulated BV-2 microglial cells. Exp. Mol. Med. 39 , 715–721 (2007). Pinna, G. F. et al. Celastrol inhibits pro-inflammatory cytokine secretion in Crohn’s disease biopsies. Biochem. 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Free Radic Biol. Med. 69 , 219–228 (2014). Shah, Z. A. et al. The flavanol (-)-epicatechin prevents stroke damage through the Nrf2/HO1 pathway. J. Cereb. Blood Flow. Metab. 30 , 1951–1961 (2010). Shelton, L. M., Park, B. K. & Copple, I. M. Role of Nrf2 in protection against acute kidney injury. Kidney Int. 84 , 1090–1095 (2013). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-6914458","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":479559825,"identity":"1341a00c-82a9-4833-a2c7-c0f91a5399a5","order_by":0,"name":"Yijie Deng","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yijie","middleName":"","lastName":"Deng","suffix":""},{"id":479559826,"identity":"b83f8241-110b-4342-a583-6137b4dd0b22","order_by":1,"name":"JiChun Wang","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"JiChun","middleName":"","lastName":"Wang","suffix":""},{"id":479559827,"identity":"500b9ec9-8c88-40b2-8d5c-f228f844a45f","order_by":2,"name":"Xiping Liu","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiping","middleName":"","lastName":"Liu","suffix":""},{"id":479559828,"identity":"98080ec9-7834-43dc-9352-6cb6249e9442","order_by":3,"name":"Yue Chen","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Chen","suffix":""},{"id":479559829,"identity":"88909409-3d51-40dd-a8b7-f9ad1ede0690","order_by":4,"name":"Min Wang","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Wang","suffix":""},{"id":479559830,"identity":"77c22a2b-93bb-45c7-9e65-fee8c6e53f55","order_by":5,"name":"Xiuwen Wang","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiuwen","middleName":"","lastName":"Wang","suffix":""},{"id":479559831,"identity":"a3fe414c-16cb-4ae9-b53d-ca532bdb3069","order_by":6,"name":"Hua Li","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Li","suffix":""},{"id":479559833,"identity":"a3b12366-4aa5-4e74-99b8-2f70f9eb3da8","order_by":7,"name":"Bo Gu","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Gu","suffix":""},{"id":479559834,"identity":"7408c96b-0b20-435e-bfd1-3528e432c85d","order_by":8,"name":"Min Zhang","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Zhang","suffix":""},{"id":479559836,"identity":"939954ee-118a-446f-8b3f-22e374940f59","order_by":9,"name":"Renjun Wang","email":"","orcid":"","institution":"Jilin Normal University","correspondingAuthor":false,"prefix":"","firstName":"Renjun","middleName":"","lastName":"Wang","suffix":""},{"id":479559838,"identity":"7a744ac3-96f0-4556-a25a-862017f1b8b1","order_by":10,"name":"Yi Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIie2RsUrEQBBAJyyk2ru0GxG8ynpgIQgXsPAzbBaEtYlimeLQyEEsrxb8CD8hx0DS7Jk2nYErTXFiY3GFe2IniVda7OtmmDfDzAA4HP8WFNwHqj62aXy3YIzaPZSTw8ArNXCjvccHX+MeY9I4nJtjGOXkPdd8Igb7Vyt64zeCY2n8VhhikjggzOLzXsVc6ym3u6BZVYjppR/RqGih1FdZn1IkkfxWmhcllJnyiMYKvYz6lbr7UV47FMucCTm34aDSJHK9U8LMROF9fobI/lDCpou8J6sEUF5IMFoJskdWA7uM60Ruuu3tqX3lcm2PrYIFUbuZxb3KpAD/gP9Kq57yHUcZsPfPgQKHw+FwwBd2cF8jOZlbDgAAAABJRU5ErkJggg==","orcid":"","institution":"Jilin Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-06-17 12:38:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6914458/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6914458/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85970560,"identity":"55f624ee-e424-42d7-af49-1601e20e0a02","added_by":"auto","created_at":"2025-07-03 18:25:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55919,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of celastrol treatment on serum levels of (a)REN. (b) AngⅡ. (c) ALD. and (d) ACE. All results were expressed as mean ± SD;n = 6 each. p≤0.05 indicates statistical significance，p≤0.01 indicates high statistical significance，p≤0.001 indicates extreme statistical significance，p≤0.0001 indicates superior statistical significance.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6914458/v1/4383c4d7633cc7f73801ebae.png"},{"id":85970783,"identity":"6aa98f56-958d-4d86-8620-244a15be0ee2","added_by":"auto","created_at":"2025-07-03 18:33:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51440,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of celastrol treatment on serum levels of (a) TNF-\u003cem\u003eα\u003c/em\u003e. (b) IL-1\u003cem\u003eβ\u003c/em\u003e and (c) IL-6. All results were expressed as mean ± SD;\u003cem\u003en\u003c/em\u003e = 8 each. IL-6 interleukin-6, IL-1\u003cem\u003eβ\u003c/em\u003einterleukin-1\u003cem\u003eβ\u003c/em\u003e, TNF \u003cem\u003eα\u003c/em\u003e tumor necrosis factor-alpha. p≤0.05 indicates statistical significance，p≤0.01 indicates high statistical significance，p≤0.001 indicates extreme statistical significance，p≤0.0001 indicates superior statistical significance.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6914458/v1/e0146e158bae891a6f54946d.png"},{"id":85970563,"identity":"4cd4f21d-0209-4ad6-b38b-80c22e2f9264","added_by":"auto","created_at":"2025-07-03 18:25:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72241,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of celastrol treatment on serum levels of (a) SOD. (b) GSH-Px (c) MDA and (d) CAT. All results were expressed as mean ± SD;\u003cem\u003en\u003c/em\u003e = 8 each. SOD superoxide dismutase, GSH-Px glutathione peroxidase, CAT catalase, and MDA malondialdehyde. p≤0.05 indicates statistical significance，p≤0.01 indicates high statistical significance，p≤0.001 indicates extreme statistical significance，p≤0.0001 indicates superior statistical significance.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6914458/v1/b2fd800f9d25605c95b7d971.png"},{"id":85970788,"identity":"b034af82-460d-4bf0-9c8d-e79045325861","added_by":"auto","created_at":"2025-07-03 18:33:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1338686,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CSL on the morphological changes of kidney tissue in SHR rats (400×)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6914458/v1/791cca0b78b117bada241870.png"},{"id":85970786,"identity":"efc510f4-6a26-4b47-8a97-57b3c5cb24a4","added_by":"auto","created_at":"2025-07-03 18:33:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":575107,"visible":true,"origin":"","legend":"\u003cp\u003eThe immunohistochemistry and Western Blotting results. (A) Determination of NLRP3 protein in chicken lung by immunohistochemical method. (B) Representative Western blots showed the protein levels of Nrf2 and Keap1 in the kidney, \u003cem\u003eβ\u003c/em\u003e-actin was used as internal control.n=8 (C) Represents Nrf2 protein expression levels.n=8 (D) Area of Keap1 protein.n=3. All results were expressed as mean ± SD. p≤0.05 indicates statistical significance，p≤0.01 indicates high statistical significance，p≤0.001 indicates extreme statistical significance，p≤0.0001 indicates superior statistical significance.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6914458/v1/2263c3f20b0395a67fafbf7a.png"},{"id":85971192,"identity":"9d6befac-9a00-4601-a85d-477e2c7ab853","added_by":"auto","created_at":"2025-07-03 18:41:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46328,"visible":true,"origin":"","legend":"\u003cp\u003emRNA transcription levels of Nrf2/HO-1 pathway genes in rat kidneys. All results were expressed as mean ± SD;\u003cem\u003en\u003c/em\u003e = 8 in each. p≤0.05 indicates statistical significance，p≤0.01 indicates high statistical significance，p≤0.001 indicates extreme statistical significance，p≤0.0001 indicates superior statistical significance.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6914458/v1/c60b42b5c1fe0805a685758f.png"},{"id":101918398,"identity":"62fbdb72-5baa-42d6-9323-f3b0d142f21b","added_by":"auto","created_at":"2026-02-05 03:40:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2516781,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6914458/v1/b685cfc6-01e5-43f8-8d27-78afba703ccb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Celastrol improves kidney damage in spontaneous hypertensive rats by regulating the Nrf2/Ho-1 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePharmacological bioactive compounds derived from medicinal plants have gained a lot of attention recently because of their strong, distinctive, and varied activities. The chemical structure of celastrol (CSL), a pentacyclic triterpene that was isolated from Tripterygium wilfordi \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among the many pharmacological effects of CSL are anti-inflammatory and antioxidant \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. CSL has been shown to inhibit several proinflammatory cytokines, such as nuclear factor-kappa B (NFκB), nitric oxide synthase, adhesion molecules, proinflammatory enzymes, interleukins (IL-2, IL-8), interferon-gamma (IFN-γ), and tumor necrosis alpha (TNF-\u003cem\u003eα\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, it shown encouraging effects in a variety of inflammatory conditions, including asthma, ulcerative colitis, Crohn's disease, and rheumatoid arthritis \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. CSL has also been shown to have anti-diabetic, neuroprotective, and anti-obesity properties\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, several investigations have proven CSL anticancer potential by encouraging apoptosis and preventing cell invasion, angiogenesis, and proliferation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Tripterygium wilfordis plant extract has been used in a number of clinical investigations because of its wide range of pharmacological activities. Furthermore, in an effort to assess CSL therapeutically for a more thorough examination of Castrol's effects on the kidney, randomised clinical trials were conducted on patients with a variety of conditions, including renal transplantation, rheumatoid arthritis, solid tumours, and diabetic nephropathy \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. CSL has been shown to be an antioxidant that can increase the expression and activity of heme oxygenase-1 (HO-1) and reduce the production of reactive oxygen species (ROS) in vascular smooth muscle cells in hypertensive rats \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Zhang et al. reported a CSL can improve the kidney injury induced by high-fat diet in obese mice by regulating kidney Keap1/Nrf2 pathway to increase antioxidant level \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Additionally, mechanistic investigations shown that CSL inhibited several processes in the production of oxidative stress and inflammation, such as the NF-κB signalling pathway \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. It has been proposed that NF-κB, a pleiotropic transcription factor, is crucial for gene regulation during oxidative stress and inflammation, which lead to atherosclerosis \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This study used SHR kidney injury as a model to explore whether CSL alleviates and improves the pathology of SHR kidney injury through the pathway, providing theoretical basis for the prevention and treatment of SHR kidney injury.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e The protocol was approved by the Committee on the Ethics of Animal Experiments of Jilin Normal University (Siping, Jilin, China) (Approval number: KJLL20250301).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal and Experimental design\u003c/h3\u003e\n\u003cp\u003eA total of 24 12-week-old male SHRs and 8 12 12-week-old male Wistar-Kyoto (WKY) rats with weights ranging from 180\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g [License Nomber SCXK (Beijing) 2016-0006] were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co.Ltd, were kept under controlled conditions for temperature, humidity and light, with unrestricted access to food and water available throughout all experimental stages. The SHRs were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;8). model group, L-CSL\u0026thinsp;+\u0026thinsp;SHR 0.02 mg/kg/d and H-CSL\u0026thinsp;+\u0026thinsp;SHR 0.04 mg/kg/d. The WKY rats were used for the control group. CSL drug injection solutions were prepared with physiological saline. The CON and SHR groups were given equal-volume physiological saline by intraperitoneal injection the L-CSL\u0026thinsp;+\u0026thinsp;SHR and H-CSL\u0026thinsp;+\u0026thinsp;SHR groups were given drug injections by intraperitoneal injection (drug concentrations were 0.02 mg/kg/d and 0.04 mg/kg/d, respectively). All rats were given intraperitoneal injections daily for 6 consecutive weeks.\u003c/p\u003e \u003cp\u003eTwenty-four hours after renal reperfusion, animals were sacrificed by decapitation under anesthesia, and blood samples were collected from different experimental groups. Serum samples were obtained via 15 min of blood centrifugation at 5000 rpm and were stored at \u0026minus;\u0026thinsp;20\u0026deg;C for subsequent measurements. The right kidney was rapidly removed and separated into two parts; the first part was homogenized and kept at \u0026minus;\u0026thinsp;80\u0026deg;C for biochemical analyses. The second part was kept in 10% formalin for histopathological examination.\u003c/p\u003e \u003cp\u003eIntraperitoneal injection of α-chloroaldose (15879-93-3, Sigma, MH, NE) (80 mg/kg) and ooligosaccharides (51-79-6, Sangon, SH, China) (800 mg/kg) to anesthetize rats. All experiments were carried out according to the guidelines.\u003c/p\u003e \u003cp\u003eAt the end of each experiment, the rats were euthanized by intravenous injection of 0.5 ml KCl solution (10%) under anesthesia.\u003c/p\u003e\n\u003ch3\u003eBlood analysis\u003c/h3\u003e\n\u003cp\u003eThe serum samples were defrosted at 4\u003csup\u003e◦\u003c/sup\u003eC and mixed evenly before determination. The levels REN, Angiotensin, ACE and ALD and levels of serum inflammatory factors TNF-\u003cem\u003eα\u003c/em\u003e, IL-1\u003cem\u003eβ\u003c/em\u003e and IL-6 were detected according to the kit instructions.\u003c/p\u003e\n\u003ch3\u003eAntioxidant Indexes\u003c/h3\u003e\n\u003cp\u003eThe contents of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) in the kidney were determined by spectrophotometry (PU 8720UV/VIS scanning spectrophotometer) and investigated. Commercial assay kits were provided by the Nanjing Jiancheng Institute of Bioengineering (Nanjing, China), and all procedures were performed following the kit\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch3\u003eHistology Examination and immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eKidney samples were collected and stored in 4% paraformaldehyde at 4\u0026deg;C overnight. The rest of the procedures followed the standard techniques published in previous papers \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor immunohistochemistry, the Kindey tissues were placed in Tissue-Tek OCT and snap-frozen in liquid nitrogen. Thereafter, the samples were cryosectioned (15 \u0026micro;m) and stained for target proteins. Antibodies raised against the following mouse antigens were used: Nrf2 antibody (1:300) and Keap1 antibody (1:200). Accordingly, secondary antibodies were used to amplify signals with AlexaFluor 488 anti-rat, Alexa Fluor 647 anti-guinea pig, and AlexaFluor 488 anti-rabbit IgG. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). Two slides of each rats (n\u0026thinsp;=\u0026thinsp;6 per group) were imaged using a Zeiss confocal Laser Scanning Microscope 780.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting Analysis\u003c/h2\u003e \u003cp\u003eThe mixture was used to homogenize kidney tissue after adding phosphatase and pro-tease inhibitors in cell lysis buffer (Biosharp, BL509A Hefei, China). Samples were separated on 10% SDS-PAGE gels after their protein content was equilibrated. After transfer to PVDF membranes (Millipore, IPVH00010, Burlington, CA, USA), samples were blocked for one hour using 5% skim milk. Membranes were first exposed for a 12 h incubation period at 4\u003csup\u003e◦\u003c/sup\u003eC with some primary antibodies before treatment with HRP-conjugated secondary antibodies. Finally, chemiluminescence was detected using a Dannon 5200 multi-imaging system and a high-sensitivity ECL kit (NCM Biotech, P2300, Suzhou, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReal-Time PCR\u003c/h3\u003e\n\u003cp\u003e The kidney tissues from rats were utilized to isolate using Trizol (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions and stored at -80\u0026deg;C. Then, the RNA was reverse-transcribed into cDNA according to the instructions (Vazyme, Nanjing, China). mRNA expression was determined according to the instructions (Vazyme, Nanjing, China), and its relative expression was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, and SPSS statistical software was used for single-factor variance analysis (ANOVA). Duncan's multiple-range test was used for comparison. GraphPad Prism software was used for plotting, The standard for statistical significance is p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CSL on serum biochemical indexes of SHR rats\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Compared with CON group, serum levels of REN, AngⅡ, ALD and ACE in SHR group were significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with SHR group, low and high dose CSL groups could significantly reduce the levels of these biochemical indexes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Among them, the content of AngⅡ (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and ACE (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in H-CSL SHR group was significantly different from that in L-CSL SHR group, indicating that CSL could inhibit the changes of plasma related indexes in SHR rats, and it was dose-dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CSL on the levels of inflammatory factors in SHR rats\u003c/h2\u003e \u003cp\u003eAs displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. for serum levels of TNF-\u003cem\u003eα\u003c/em\u003e and IL-6 in SHR group were significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to CON. CSL pretreatment could significantly inhibit the expression levels of TNF-\u003cem\u003eα\u003c/em\u003e, IL-1\u003cem\u003eβ\u003c/em\u003e and IL-6 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with low dose, high dose CSL could significantly inhibit IL-1\u003cem\u003eβ\u003c/em\u003e level (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It is suggested that CSL can improve the anti-inflammatory level of SHR rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CSL on oxidative stress-related indexes in SHR rats\u003c/h2\u003e \u003cp\u003eAs displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the activities of SOD, GSH-Px and CAT in kidney tissue of SHR group were significantly decreased, and the level of MDA was significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to CON. CSL pretreatment could significantly increase the activities of SOD, GSH-Px and CAT in kidney tissue of SHR rats, and decrease the level of MDA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the activities of SOD, GSH-Px and CAT in kidney of high-dose group were significantly increased compared with those of low-dose group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The results showed that CSL could improve the antioxidant capacity of kidney in SHR rats and had a protective effect on kidney.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CSL on renal pathology in SHR rats\u003c/h2\u003e \u003cp\u003eH\u0026amp;E staining was used to observe the pathological changes of renal tissue in each treatment group, and the results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In CON group, the glomeruli were normal in size and structure, and the basal membrane of renal tubules was normal, no diffuse infiltration and congestion of inflammatory cells were observed. In SHR group, the basal membrane of renal tubules was thickened, the epithelial cells of renal tubules were swollen, and the mesangial cells were hyperplasia. Compared with SHR group, CSL group showed a small amount of inflammatory cell infiltration, a slight thickening of the basal membrane of renal tubules, and a significant improvement in congestion, tubule epithelial cell swelling and mesangial cell proliferation. It is suggested that CSL can reduce the inflammatory cell infiltration and the swelling of renal tubular epithelial cells, and improve the renal inflammatory injury in SHR rats, and the therapeutic effect of high dose is more significant than that of low dose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of CSL on the expression of Nrf2 and Keap1 proteins in kidney of SHR rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of Nrf2 and Keap1 proteins in kidney tissues of rats in each group were detected by IHC and WB methods, and the results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Compared with CON group, Nrf2 protein expression in kidney tissue of rats in SHR group was extremely significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while Keap1 protein expression was extremely significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with SHR group, after CSL pretreatment, the expression of Nrf2 protein in kidney tissue was extremely significantly increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the expression of Keap1 protein was extremely significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The expression levels of Nrf2 and Keap1 protein in kidney tissues of CSL high-dose and low-dose groups were significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicated that Nrf2 and Keap1 proteins were involved in the process of renal tissue injury in SHR rats, and CSL could regulate Nrf2 and Keap1 proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of CSL on mRNA transcription of genes related to Nrf2/Ho-1 signaling pathway in kidney of SHR rats\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the mRNA relative expressions of Nrf2, Nqo1 and Ho-1 genes in kidney of SHR group were significantly down-regulated, and the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) Compared with CON group. his pathway is activated in the kidneys of hypertensive rats. The mRNA expressions of Nrf2 and Nqo1 in low-dose and high-dose CSL groups were significantly higher than those in SHR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the difference between the two groups was extremely significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting that CSL could exert therapeutic effect by regulating the expression of Nrf2/Ho-1 pathway gene in the kidney of SHR rats, and the therapeutic effect was positively correlated with dose.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThrough preventing NF-kB activation, CSL has been investigated for its ability to protect rats against renal IRI \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, in this work, we examined CSL antioxidant capacity by examining how it affects the Nrf2/HO-1 pathway. Oxidative stress and inflammation are the key factors leading to the sustained development of hypertensive kidney damage. This study is the first to investigate the molecular mechanism of CSL in improving kidney damage in SHR rats. Studies have shown that hypertensive renal damage mainly includes the damage of various endothelial cells and the infiltration of inflammatory cells. The kidney participates in the formation of blood pressure through the secretion of renin and the regulation of body fluids, and the imbalance of such regulation leads to hypertension\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Biological indicators related to renal function include REN, ALD, AngⅡ, and ACE, which are abnormal in SHR rats with kidney injury \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In this study, CSL can significantly inhibit the expression levels of renin, ALD, AngⅡ and ACE in SHR rats, and alleviate the pathological changes of kidney injury. Persistent hypertension can cause oxidative stress and inflammation in the kidney, and both play a combined role in hypertension-related kidney damage, exacerbating the pathological development of the kidney\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere is growing evidence that the fibrotic kidney is surrounded by a condition of inflammation due to the accumulation of inflammatory cytokines \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To slow the advancement of kidney damage in spontaneous hypertensive, it is thus essential to effectively decrease inflammation and ameliorate the inflammatory microenvironment. Anti-inflammatory medications are now extensively utilised in clinical settings to manage kidney inflammation, and in both experimental and clinical studies, tremendous progress has been achieved in the prevention and treatment of kidney fibrosis utilising natural ingredients \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. A Chinese medicinal herb called Tripterygium wilfordii Hook F has strong anti-inflammatory properties, particularly in rheumatoid arthritis. The active component of Tripterygium wilfordii Hook F, which is utilised in clinical settings to treat immunological disorders, is CSL \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. After kidney damage, CSL reduced the kidney's inflammatory response by improving the production of IL-10 cytokines and decreasing the release of TNF-\u003cem\u003eα\u003c/em\u003e, IL-1\u003cem\u003eβ\u003c/em\u003e, and IL-18 inflammatory factors. Wang et al. reported that significantly increase IL-10 and decreased level of TNF‐\u003cem\u003eα\u003c/em\u003e, IL‐1\u003cem\u003eβ\u003c/em\u003e and level of IL‐18 in kidney duo to supplementation of CSL \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In the current study, CSL treatment a decreased level of TNF-\u003cem\u003eα\u003c/em\u003e, IL-1\u003cem\u003eβ\u003c/em\u003e and IL-6 in kidney compared to SHR. Parallel to these previous studies, our findings also demonstrated that CSL had a potent anti-inflammatory effect on kidney damage in spontaneous hypertension, both in vitro and in vivo. This suggests that inflammation intervention was a useful tactic for controlling kidney damage in spontaneous hypertension.\u003c/p\u003e \u003cp\u003eIn the present study, increase the activities of SOD, GSH-Px and CAT and decrease the level of MDA in CSL groups compared with SHR. Younis et al. reported that CSL groups decrease level of MDA and increase content of GSH \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Increased ROS generation in SHR rats results in renal cell damage and apoptosis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Nrf2 is a crucial regulator that counteracts oxidative damage by activating genes that produce antioxidants, which helps protect against experimental acute kidney injury. Stress causes Nrf2 to evade its inhibitor (Keap1), enter the nucleus, and increase the production of defense-promoting enzymes such as superoxide dismutase (SOD) and heme oxygenase-1 (HO-1). Kidney damage from ischemia-reperfusion or toxins is lessened by this antioxidant response\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Heme oxygenase-1 (HO-1) is an essential cytoprotective mechanism that utilizes enzymatic breakdown to convert pro-oxidant heme into physiologically active chemicals. This catalytic reaction produces three beneficial components biliverdin and its metabolite bilirubin, and carbon monoxide (CO), a gaseous mediator that causes vasodilation and has strong antioxidant properties, with antiapoptotic effects. HO-1 is an essential defense mechanism against oxidative damage and cellular injury, as it simultaneously generates protective molecules and removes a harmful oxidant (heme) \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Our results showed an improve in gene expression Nrf2, HO-1 and Nqo1 and decreased Keap1 level in SHR as a defensive response to IR injury, and these results are consistent with a previous study \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Meanwhile, mice given CSL showed a significant increase in the gene expression of Nrf2, HO-1, and Nqo1, and a decrease in the level of Keap1 in the kidneys compared to mice treated with SHR. This indicates that CSL protects against SHR by activating Nrf2, which increases HO-1 and Nqo1 levels.\u003c/p\u003e \u003cp\u003eIn conclusion, CSL can alleviate the pathological changes of kidney injury in SHR rats, possibly by activating the expression of Nrf2/HO-1 pathway gene and protein to improve the level of kidney antioxidant, inhibit the expression of inflammatory factors and thus reduce the inflammatory response. This study provides a new pharmacological and mechanistic understanding of the effect of CSL on renal injury in SHR rats, and helps to better understand the application of CSL in the treatment of hypertensive nephropathy.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol was approved by the Committee on the Ethics of Animal Experiments of Jilin Normal University (Approval number: KJLL20250301). The studies were conducted in accordance with the local legislation and institutional requirements.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.W. and Y.Y. supervised the project and wrote the manuscript; Y.D. and M.Z. designed the experiments and\u0026nbsp;conducted\u0026nbsp;the ELISA and real-time RT-PCR experiments; J.W., Y.C. and M.W. performed a part of the immunoprecipitation and western blot analysis; X.L., X.W. and H.L. designed and conducted the animal studies. X.L., H.L. and B.G. conducted the western blot and immunofluorescent experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by research grants to Y.Y. from the Program for the Development of Science and Technology of Jilin Province (YDZJ202201ZYTS457);to R.W. from the National Nature\u0026nbsp;Science\u0026nbsp;Foundation of China (31871150) and the Science and Technology Research Project of Jilin Provincial Department of Education (JJKH20210446KJ); to H.L. from the Program for the Development of Science and Technology of Jilin Province (20220101322JC), to M.Z. from the Program for the Development of Science and Technology of Jilin Province (YDZJ202201ZYTS433).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo authors declare\u0026nbsp;that\u0026nbsp;the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang, Y. et al. 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Role of Nrf2 in protection against acute kidney injury. \u003cem\u003eKidney Int.\u003c/em\u003e \u003cb\u003e84\u003c/b\u003e, 1090\u0026ndash;1095 (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Celastrol, Leigong vine, spontaneous hypertension, kidney injury, Nrf2/Ho-1 pathway","lastPublishedDoi":"10.21203/rs.3.rs-6914458/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6914458/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe natural triterpenoid celastrol, which comes from Tripterygium wilfordii, has a variety of biological effects. We investigated Celastrol improves kidney damage in spontaneous hypertensive rats by regulating the Nrf2/Ho-1 pathway. A total of 24 12-week-old male spontaneous hypertensive rats (SHR) were randomly allotted to four groups [control group, SHR group, L-CSL\u0026thinsp;+\u0026thinsp;SHR group (0.02 mg/kg/d) and H-CSL\u0026thinsp;+\u0026thinsp;SHR group (0.04 mg/kg/d)]. The results showed that CSL group significantly decrease levels REN, Angiotensin, ACE and ALD and decrease expression levels of TNF-\u003cem\u003eα\u003c/em\u003e, IL-1\u003cem\u003eβ\u003c/em\u003e and increase expression levels of IL-6 in serum compared with SHR group. Kidney functions, CSL group significantly decrease level of MDA and increase SOD, GSH-Px and CAT compared with SHR group. CSL had a significant inhibitory effect on the increase in the relative expression abundance of Keap1. The Nrf2, Nqo1 and Ho-1 mRNAs were found to be significantly lower in the CSL compared with SHR group. The results show that CSL significantly reduces the pathology of kidney damage in spontaneous hypertensive rats by activating Nrf2/Ho-1, and provides treatment strategies for the kidney damage.\u003c/p\u003e","manuscriptTitle":"Celastrol improves kidney damage in spontaneous hypertensive rats by regulating the Nrf2/Ho-1 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-03 18:25:35","doi":"10.21203/rs.3.rs-6914458/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f9894c32-acbc-4bac-93bb-f2ae0be74049","owner":[],"postedDate":"July 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50922134,"name":"Biological sciences/Biochemistry"},{"id":50922135,"name":"Biological sciences/Biological techniques"}],"tags":[],"updatedAt":"2026-02-05T03:39:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-03 18:25:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6914458","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6914458","identity":"rs-6914458","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}