“Type A extracts” (Heinrich et al., 2022) are botanicals, the extracts of which are listed in national or regional pharmacopoeias as active metabolites in botanical medicines (licensed, marketed, or registered medicines) for controlled medical use. JT is a type A extract and the daily dosage of JT is as follows:Polygonatum cyrtonema[ Asparagaceae , Rhizoma siccum]:15 g;Rhodiola crenulata[ Crassulaceae , Radix et R. siccum]:10 g;Poria cocos[Polyporaceae;Sclerotium siccum];Polyporus umbellatus[Polyporus, S. siccum]:10 g;Alisma orientale[Alismataceae, Tuber siccum]:10 g;Cinnamomum cassia [ Lauraceae , Ramulus siccus]:5 g;Glycyrrhiza uralensis[ Fabaceae , Radix et R. siccum]:3 g;Rheum officinale[ Polygonaceae , Radix et R. siccum]:5 g;Atractylodes macrocephala [ Asteraceae , R. siccum]:10 g;Dioscorea nipponica [ Dioscoreaceae , R. siccum]:12 g. These samples were identified by Prof. Cheng Huanbo of Hubei University of Traditional Chinese Medicine (Table 1). The above drugs were used in accordance with the relevant provisions of the Chinese Pharmacopoeia, 2020 Edition (Part I). The voucher samples are stored at the Institute of Standardization, School of Pharmacy, Hubei University of Traditional Chinese Medicine. All of the plant names were checked at https://www.worldfloraonline.org. These botanical drugs met the relevant requirements of the Chinese Pharmacopoeia (Xu et al., 2021).

JT was prepared according to the classical method described in “Shang Han Lun”. After soaking in six volumes of water for 30 minutes, the herbs were boiled for 45 minutes. This process was repeated with another six volumes of water for 30 min. The combined filtrate was concentrated under reduced pressure at 60°C to a relative density of 1.08–1.10 and then spray-dried to obtain a dry extract powder. The extraction yield, measured by vacuum drying, was 17.61%. The dry extract was stored at −20°C. Valsartan (VAL,Cat. No. H20051350; Lunan Better Pharmaceutical Co., Jinan, China) was used as a positive control.

A Wayeal LC3600 series ultrahigh-performance liquid chromatograph (Anhui Wan Yi Technology Co., Ltd.) and a Luna^® Omega Polar C₁₈ column (150 mm × 2.1 mm, 1.6 μm, Phenomenex, United States) were used. The 5-hydroxymethylfurfural control (Batch No. 111626-202316, purity: 98.4%), R. rosea glucoside control (batch No. 110818-202210, Purity: 99.7%), chlorogenic acid control (batch No. 110753-202119, purity: 96.3%), cinnamic acid control (batch No. 110786-202305, purity: 98.8%), ammonium glycyrrhizinate control (batch No. 110731-202122, purity: 94.4%), rhubaric acid control (batch No. 110751-202122, purity: 94.4%), Purity: 98.8%, ammonium glycyrrhizinate control (batch No. 110731-202122, purity: 94.4%), rhubarbic acid control (batch No. 110757-201607, purity: 96.0%), rhubarbine control (batch No. 110756-201913, purity: 96.0%), blycyrrhizic hypophosphoric acid control (batch No. 110723-202316, purity: 99.6%), rhubarb phenol control (batch no. 110796-201922, purity: 99.4%), and aloe rhubarbin control (batch no. 110795-201710, purity: 98.3%) were purchased from the China Academy of Food and Drug Administration. Acetonitrile and phosphoric acid were chromatographically pure, and the water was ultrapure water. Samples S1 to S10 of Jingtian granules were prepared in the laboratory, samples S11 to S13 were prepared by Jin Brand Holding Zhengtang Pharmaceutical Company Limited, and the tablets used were purchased from Hubei Chenmei Traditional Chinese Medicine Company Limited.

Male SIRT3^−/− mice provided by Saiye Gene Co. (Suzhou, China) were bred in the laboratory. The experiment involved nine 6–8-week-old specific pathogen-free (SPF) male mice weighing 20 ± 2 g (from the Hubei Provincial Center for Disease Control and Prevention, Wuhan, China). The mice were acclimatized for 1 week under a 12-h light/dark cycle (55% ± 5% humidity, 23°C ± 2°C) with free access to food and water. The mice were split into the following groups at random (n = 9): (1) the control group, which was fed a regular diet; (2) the CKD group, which was fed a 0.2% adenine diet (w/w); and (3) the JT groups (high/medium/low), which were fed a 0.2% adenine diet and administered different doses of JT granules by gavage. In accordance with the “Pharmacological Methodology of Traditional Chinese Medicine (Zhou et al., 2011)”, JT granules were administered by gavage at low (0.24 g/kg), medium (0.48 g/kg), and high (0.96 g/kg) doses, with a gavage volume of 10 mL/kg body weight. The control and model groups were administered an equal volume of deionized water by gavage. Interventions lasted 2 weeks. (4) The VAL group was fed a 0.2% adenine diet and administered VAL by gavage for 2 weeks. In accordance with previous methods (Liu et al., 2023), the VAL group was gavaged the drug at 10 times the clinical dose (0.025 g/kg). The experiment lasted 6 weeks. At the end of the experiment, all the mice were euthanized, and their kidneys and plasma were collected. The kidneys were rinsed with isotonic saline and dissected to obtain renal tissue, which was stored in liquid nitrogen for the RNA-seq analysis. For additional analyses, the remaining samples were stored at −80°C. The requirements of the Animal Care and Use Committee of Hubei University of Traditional Chinese Medicine were met when animal research was conducted (HUCMS202302007).

Antigen retrieval was performed on kidney tissue sections from each group using the SP two-step method, and endogenous peroxidase activity was blocked according to the instructions of an immunohistochemistry kit. After the antibody incubation, the sections were observed and photographed using a microscope.

Kidney tissue was treated with RIPA buffer containing a protease inhibitor cocktail (Beyotime, Cat. No. P0013B) to extract total protein. The protein concentration was determined Using a BCA protein assay kit (Beyotime, Cat. No. P0011). Protein samples were separated on SDS‒PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies against SIRT3, GPX4, SLC7A11, P53, ac-P53, type I collagen and GAPDH. The membranes were incubated with an enhanced chemiluminescence (ECL) protein detection kit after being incubated with HRP-conjugated secondary antibodies for 1.5 h.

Human proximal tubular epithelial cells (HK-2) (Servicebio, Cat. No. STCC10303P-1) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified environment at 37°C with 5% CO₂. The main reagents used were as follows: CCK-8 kit (Servicebio, Cat. No. G4103-1 ML); DMEM/F12 (Thermo Fisher, Cat. No. A4192002); fetal bovine serum (Thermo Fisher, Cat. No. A5669701); erastin (Beyotime, Cat. No. SC0224-5 mg); ferrostatin-1 (MCE, Cat. No. HY-100579); and an ROS fluorometric assay kit (Beyotime, Cat. No. E-BC-F005). Eighteen healthy adult male SD rats weighing 180–220 g (from the Hubei Provincial Center for Disease Control and Prevention, Wuhan, China) were selected and adaptively fed under standard laboratory conditions for 1 week. The rats were randomly divided into three groups (n = 6) and administered different doses of JT (0.24 g/kg, 0.48 g/kg, or 0.96 g/kg) once a day for seven consecutive days. One hour after the last administration, the rats were anesthetized, and blood was collected from the abdominal aorta. Serum was obtained by centrifugation, complement was inactivated in a water bath at 56°C for 30 min, and the serum was prepared and stored at −80°C. The optimal concentration of JT-containing serum for intervention in the HK-2 cell model of erastin-induced injury was determined via the CCK-8 method. The corresponding drugs were administered, and the cells were incubated at 37°C with 5% CO₂ for 24 h and 48 h. The culture medium was then removed, and CCK-8 solution was added. The absorbance at 450 nm was detected after 2 h of incubation in an incubator, and HK-2 cell viability was calculated to obtain the growth inhibition rate. The groups included the blank control, erastin, Fer-1, and JT groups. The experimental setup for the cell seeding and CCK-8 assays was the same as that described above. HK-2 cell morphology was observed. HK-2 cells in each group were treated for 48 h. The cell morphology and growth status were observed under an inverted fluorescence microscope. ROS levels were detected using an assay kit. HK-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and incubated at 37°C with 5% CO₂. Each group was treated with working solution (1–10 μmol/L) and incubated at 37°C in the dark for 30–60 min. Fluorescence detection was performed using a microplate reader at an excitation wavelength of 300 nm or 488 nm and an emission wavelength of 610 nm.

Fresh kidney tissues (1 mm³) were fixed sequentially in 2.5% glutaraldehyde for 24 h, followed by 1% osmium tetroxide for 2 h. The samples were then dehydrated through a graded series of ethanol and acetone. After dehydration, the samples were infiltrated with a mixture of acetone and propylene oxide. Each sample was embedded in resin and then sliced using an ultramicrotome. The sections were stained with uranyl acetate and lead citrate. The ultrastructure of podocytes and the number of mitochondria in the CKD model mice were observed under a transmission electron microscope (TEM) (HITACHI, Cat. No. HT770).

The experimental findings were statistically analyzed with GraphPad 8.0. Differences between two groups were analyzed using an unpaired two-tailed Student’s t-test. The results are shown as x ¯ ± s. One-way analysis of variance (ANOVA) was used when the variance was homogenous and the data were normally distributed. Nonparametric tests were utilized when the variance was not homogenous or when the data did not follow a normal distribution. Statistics were deemed significant if P < 0.05.

One microliter each of the mixed control solution described in Section 2.3.2 and the test solution of JT described in Section 2.3.1 was added, and the mixture was injected into the ultrahigh performance liquid chromatograph according to the chromatographic conditions described in Section 2.1. The chromatographic peaks and separation of 5-hydroxymethylfurfural, R. rosea glycoside, chlorogenic acid, cinnamic acid, rhubarbic acid, ammonium glycyrrhizinate, rhodopsin, glycyrrhizic acid, rhubarb phenol, and Aloe barbadensis rhodopsin were good, as shown in Figure 1.

The UPLC chromatograms of the 13 batches of samples were obtained by preparing test solutions of the 13 batches of samples according to the method described in Section 2.3.1, injecting the samples according to the chromatographic conditions described in Section 2.3, and then importing them into the software “Fingerprint Evaluation System of Traditional Chinese Medicine Chromatograms (2012 version)” for analysis and evaluation. The similarity of the fingerprint profiles was above 0.9, and the similarity of the profiles of the Jingtian granules from the 13 batches was 0.985, 0.993, 0.992, 0.996, 0.983, 0.989, 0.955, 0.994, 0.978, 0.980, 0.994, 0.995, and 0.995, which indicated that the samples from each batch were highly similar to each other and that the process used to prepare the Jingtian granules from each batch was relatively stable. The granule preparation process was relatively stable, and the quality consistency was good.

The experimental flowchart is shown in Figure 1A. The data indicated that mice with adenine-induced CKD had significantly higher serum urea and creatinine concentrations than did the control mice. Following JT therapy, the concentrations of these biochemical markers were reversed in a dose-dependent manner, similar to the effects of valsartan (Figures 1B,C). In contrast to those in the group under supervision, iron (Figure 1D) and MDA (Figure 1F) levels in kidney tissue decreased in a dose-dependent manner after the JT intervention. Conversely, the GSH levels in the kidney tissue showed the opposite trend (Figure 1E). JT treatment was also associated with decreased typeI collagen protein levels (Figure 1G). In summary, these results demonstrate that JT can alleviate fibrosis levels in adenine-induced mice and reduce the expression of MDA and GSH in the kidneys.

After confirming the renoprotective effect of JT, RNA-seq analysis was performed to identify mRNAs that were differentially expressed in the kidneys of CKD mice before and after JT therapy to investigate the antifibrotic mechanism of JT. After the RNA-seq data were normalized and filtered, 6807 differentially expressed mRNAs were identified: 3,547 upregulated and 3,260 downregulated (Figure 2A). Principal component analysis (PCA) revealed three distinct clusters, highlighting differences in mRNA expression between the two experimental groups (Figure 2B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the mechanism of action of JT might involve the AGE-RAGE signaling pathway, apoptosis, the TNF-α signaling pathway, and the IL-17 and EGFR signaling pathways (Figure 2C). Notably, 20 mRNAs commonly associated with kidney diseases were significantly differentially expressed (Figure 2D). All analyses were based on an FPKM > 1 and absolute log fold change ≥ 1, defining log2 FC > 1 as upregulated and log2 FC < −1 as downregulated. Additionally, the expression of SIRT3 (a longevity gene of recent interest) and acyl-CoA synthetase long-chain family member 4 (ACSL4, a critical protein in ferroptosis) changed significantly. SIRT3 protein expression in kidney tissue was analyzed via immunohistochemistry to explore the “SIRT3-ferroptosis-fibrosis” pathway. The findings revealed a noteworthy increase in SIRT3-positive areas in JT-treated CKD mice (Figure 2E), suggesting that JT treatment affects the ferroptosis pathway.

H&E staining revealed that the glomeruli, renal tubules, and renal interstitial structure of the mice in the control group were normal. In the CKD group, the pathological changes included glomerular hypertrophy, renal tubular epithelial cell vacuolization, protein casts in the renal interstitium, and inflammatory cell infiltration. JT treatment reversed these histopathological changes, reducing inflammation and renal tubule atrophy. Prussian blue staining indicated that compared with the control, the JT intervention reduced Fe²⁺ deposition in renal tubules. Masson’s trichrome staining revealed a significant reduction in interstitial fibrosis in the CKD group after treatment. PAS staining revealed a significant disruption of the structural integrity and tubular dilation in CKD mice, effects that were partially reversed by the JT intervention. Western blot analysis revealed that SIRT3 protein expression was significantly decreased in the CKD group compared with the control group, whereas the JT intervention increased SIRT3 protein expression in a dose-dependent manner. GPX4 and SLC7A11 expression decreased in the CKD group but increased after treatment with different doses of JT. No discernible difference in P53 expression was detected across the groups, but the level of the acetylated form of P53 increased significantly in the CKD group and decreased significantly with JT and VAL treatment (Figure 3B). These results confirm the successful establishment of the adenine-induced CKD mouse model and show that JT has the potential to alleviate renal damage through a mechanism possibly related to ferroptosis.

The results of the CCK-8 assay revealed that 10 μmol/L erastin inhibited HK-2 cell activity after 24 h, following a dose‒response curve. Therefore, 10 μmol/L erastin was used to induce HK-2 cell injury in subsequent experiments. Similarly, based on the results of the cell viability assay, 5 μmol/L Fer-1 was used as the intervention dose. Medium and high doses of JT-containing serum promoted the activity of erastin-treated HK-2 cells; therefore, medium and high doses of JT were chosen for the cell-based experiments (Figure 4A).

The ROS staining results revealed low ROS levels in the control group. Compared with those in control cells, the ROS fluorescence signals were more intense in erastin-stimulated HK-2 cells. The ROS signal intensity in the erastin+Fer-1 group was weaker than that in the erastin group. Compared with those in HK-2 cells in the erastin group, the ROS fluorescence signals in the medium- and high-dose JT groups were less pronounced (Figure 4B).

The results of the Western blot analysis revealed a considerably higher level of the ac-P53 protein in the erastin group than in the control group, whereas it was lower in the Fer-1, JT-M, and JT-H groups. The erastin group presented decreases in Sirt3, GPX4, and SLC7A11 protein expression, whereas the Fer-1, JT-M, and JT-H groups presented substantial increases in the expression of these proteins. P53 protein expression did not change significantly in any of the groups (Figure 4C).

Experiments were conducted with control mice fed a normal diet, adenine diet, or adenine diet+JT-H and with SIRT3^−/− mice fed an adenine diet or adenine diet+JT-H to evaluate the role of SIRT3 in the protective effect of JT on CKD (Figure 5A). Significant renal fibrosis progression was observed after 6 weeks of adenine diet feeding. As shown in Figure 6, most of the deteriorating physicochemical parameters, including the serum urea, creatinine, GSH, MDA, and iron levels, in the CKD group did not improve in the SIRT3^−/− and SIRT3^−/−+JT-H groups (Figures 5B–F). Under JT treatment, the protein expression level of type I collagen was reduced. In contrast, after SIRT3 knockout, its expression level did not show significant changes compared with the CKD group (Figure 5G). We subsequently employed TEM to assess the ultrastructural changes in renal tubular cells following injury or necrosis. The TEM results revealed that in the CKD model group, the mitochondria in proximal renal tubular cells exhibited significant morphological alterations, characterized by decreased volume and reduced or absent mitochondrial cristae. Following JT treatment, these mitochondrial ultrastructural defects were reversed. However, this reversal was not observed in SIRT3 knockout mice (Figure 5H). In conclusion, these findings demonstrate that SIRT3 plays a crucial role in mediating the protective effects of JT on renal fibrosis and mitochondrial integrity in CKD. The absence of SIRT3 abrogates the beneficial effects of JT on renal function, oxidative stress markers, and mitochondrial ultrastructure, highlighting its essential contribution to the therapeutic potential of JT in CKD.

Compared with wild-type mice, SIRT3 knockout mice did not exhibit any protective effects on the renal pathological structure, fibrosis, or iron deposition (Figure 6A). Furthermore, a protein blotting analysis was performed to assess the expression of related signaling proteins in the kidneys to study the impact of JT on renal fibrosis signaling pathways. Under CKD conditions, GPX4 and SLC7A11 expression decreased; however, the expression of these proteins did not increase in the absence of SIRT3. Conversely, P53 levels remained unchanged in all groups, although ac-P53 levels were significantly increased in CKD mice (Figure 6B). These results suggest that JT does not significantly alleviate CKD progression in SIRT3^−/− mice.