Introduction

Hepatic ischemia-reperfusion injury (IRI) refers to the exacerbation of organ damage caused by hypoxia during blood circulation and oxygen delivery, involving multiple physiological processes. Ischemia restricts blood supply to organs, resulting in an imbalance of cellular metabolism. Subsequently, ischemia-reperfusion (IR) induces excessive damage and inflammatory reactions in the ischemic regions of organs and tissues. Hepatic IRI, as a common pathological phenomenon in clinical practice, occurs not only during hepatic transplantation and other hepatic surgeries but also triggers postoperative hepatic dysfunction and even multiple organ failure, which seriously endangers patient safety1,2,3. Consequently, an increasing number of researchers have focused on hepatic IRI. Recent studies indicate that the pathological process of hepatic IRI is highly complex, involving mitochondrial damage, metabolic acidosis, Kuppfer cell activation, calcium overload, oxidative stress, and the upregulation of pro-inflammatory factors4,5,6. Moreover, the liver, as the primary organ of metabolism, frequently develops metabolic diseases following transplantation. The incidence rate may reach 50–60%, steadily increasing as the postoperative period extends. CYPs and UGTs are highly expressed isoforms of drug-metabolizing enzymes in the mammalian liver7. These enzymes are critical to hepatic drug metabolism, influencing drug disposition and detoxification processes8,9. Furthermore, emerging evidence implicates these enzymes in the pathogenesis of IRI, particularly through their involvement in oxidative stress and inflammatory responses10,11. However, the alterations in CYPs and UGTs during hepatic transplantation, in conjunction with hepatic IRI, have yet to be elucidated.

Currently, protective treatments for hepatic ischemia-reperfusion injury (IRI) primarily consist of medications, non-pharmacological interventions12, surgical procedures, and gas inhalation or perfusion techniques13. However, most of these strategies require further research and refinement, as they are predominantly being studied in animal models at this stage. Pharmacological therapy for hepatic IRI has consistently been a focal point of research14,15,16,17. The principal therapeutic agents include antioxidants, energy metabolism regulators, adenosine receptor agonists, and calcium channel blockers. Although these medications exhibit beneficial therapeutic effects, their inevitable toxic side effects significantly limit their clinical applicability18.

Oridonin (ORI, Fig. 2 A), a bioactive ent-kaurane diterpenoid, is primarily extracted from the traditional Chinese medicinal herb Rabdosia rubescens19. Related studies have indicated that ORI possesses multiple pharmacological effects, including anti-tumor, anti-inflammatory, and immunomodulatory properties20. In clinical practice, ORI is predominantly utilized for the treatment of acute and chronic tonsillitis, pharyngitis, laryngitis, and stomatitis and exhibits protective effects on critical organs such as the heart, liver, and brain. For instance, ORI has been shown to protect mice from myocardial ischemia-reperfusion injury (IRI) by inhibiting oxidative stress and inflammatory responses21. Consequently, it is anticipated that ORI may also prevent hepatic ischemia-reperfusion injury (IRI).

In this study, we investigated the spatiotemporal alterations of CYPs and UGTs mediated by hepatic IRI, as well as the protective effects and mechanisms of ORI on hepatic IRI. The results indicated that after 6 h of hepatic ischemia-reperfusion, the mRNA levels of Cyp2c29, Cyp2c65, Cyp2c66, Cyp2c38, Cyp3a11, Cyp2d22, Ugt1a2, Ugt1A9, Ugt2a1, Ugt2a2, Ugt2b35, and Ugt2b36 significantly decreased, while the mRNA levels of Ugt1a1, Cyp2e1, and Cyp1a1 exhibited slight increases. Additionally, the protein level and activity of Cyp3a11 dramatically decreased. All levels gradually recovered after 24 and 72 h of hepatic IRI. Moreover, indicators of hepatic injury were correlated with the mRNA levels of Cyp2c29, Cyp2c66, Cyp2c38, Cyp3a11, and Cyp2d22. Meanwhile, ORI can prevent hepatic IRI by modulating the transcription levels of related CYPs and UGTs, activating the expression of the Nrf2/HO-1 pathway, and influencing the NLRP3 inflammatory signaling pathway.

Materials and methods

Materials

The origin of the ORI was Meilunbio (Dalian, China). The primary antibodies utilized for western blotting included: anti-NRF2 antibody (A1244, ABclonal, Wuhan, China), anti-HO-1 antibody (A1346, ABclonal, Wuhan, China), anti-IL-1β antibody (PK56396, Abmart, Shanghai, China), anti-IL-18 antibody (TD6252, Abmart, Shanghai, China), Total and Cleaved Caspase 1 antibody (P29466, Abmart, Shanghai, China), anti-GSDMD antibody (P57764, Abmart, Shanghai, China), anti-Bcl-2 antibody (P10415, Abmart, Shanghai, China), anti-CYP7A1 antibody (A10615, ABclonal, Wuhan, China), anti-UGT1A1 antibody (ab194697, Abcam, Cambridge, UK), anti-UGT1A9 antibody (ab180707, Abcam, Cambridge, UK), anti-NLRP3 antibody (ab263899, Abcam, Cambridge, UK), anti-ASC antibody (ab307560, Abcam, Cambridge, UK), anti-Gapdh antibody (ab8345, Abcam, Cambridge, UK), anti-LaminB1 antibody (ab16048, Abcam, Cambridge, UK), and HRP-conjugated Goat anti-Rabbit IgG (AS014, ABclonal, Wuhan, China).Phase I metabolism NADPH system Solution B (G6PDH) was obtained from Zhibang Biotechnology Co., Ltd. (Guangzhou, China). RAW264.7 cells were sourced from ATCC (Philadelphia, PA, USA). The mouse TNF-α (3511–1 A) and IL-1β (BE0246) ELISA kits were acquired from Xinbosheng Biotechnology Co., Ltd. (Shenzhen, China). Additionally, the following kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China): aspartate aminotransferase (AST) kit (C010-3-1), lactate dehydrogenase (LDH) kit (A020-1–2), alanine aminotransferase (ALT) kit (C009-3-1), malondialdehyde (MDA) kit (A003-1–2), glutathione peroxidase (GSH-PX) assay kit (A005-1–2), glutathione (GSH) assay kit (A006-1-1), myeloperoxidase (MPO) assay kit (A044-1-1), and superoxide dismutase (SOD) assay kit (A001-2-2). The Advantage RT for PCR Kit (639505) was procured from Takara Corporation (Japan), while the fluorescence quantitative PCR kit (LS2062) was obtained from Promega Corporation (USA). Finally, the RNA easy fast animal tissue/cell total RNA extraction kit (DP451) was sourced from Tiangen Biochemical Technology Co., Ltd. (Beijing, China).

Animal models

Six to eight-week-old SPF grade C57BL/6J male mice were procured from the Laboratory Animal Center of Southern Medical University in Guangzhou, China. The mice were maintained under pathogen-free conditions, with unrestricted access to water and chow, and were subjected to a 12-hour light/dark cycle. All animal experiments received approval from the Animal Ethics Committee of Southern Medical University and were conducted in compliance with the applicable guidelines and regulations established by the Committee. This study is reported in accordance with ARRIVE guidelines.

Drug solution preparation

A stock solution of Ori was prepared by dissolving 25.0 mg of Ori standard in 100 µL DMSO, yielding a final concentration of 250 mg/mL. For the preparation of working solutions, appropriate volumes of the stock solution were mixed with 200 µL PEG300 using a vortex mixer, followed by the addition of double-distilled water to adjust the total volume to 4 mL. Both the Sham and I/R groups received injections of an equivalent volume of the vehicle solution (DMSO/PEG300/water).

Hepatic IRI model and treatment with ORI

The mice were randomly divided into four groups, each consisting of eight animals: the Sham group, the I/R 6 h group, the I/R 24 h group, and the I/R 72 h group. A partial (70%) hepatic ischemia-reperfusion injury (IRI) model was established. Prior to the procedure, the mice were anesthetized with 1% pentobarbital at a dose of 10 mL/kg. The abdominal hair of the mice was wetted with 75% alcohol, followed by removal of the hair and disinfection with 75% alcohol. Surgical scissors were then employed to make an incision along the midline of the abdomen, allowing exposure of the left hepatic pedicle and middle hepatic lobes. Vascular clamps were applied to occlude the blood supply to the hepatic veins and arteries in these lobes. Under surgical light, the color of the left and middle hepatic lobes was observed for a few seconds; in comparison to the right lobe, which remained perfused, the occluded lobes turned significantly white, indicating successful blockage of blood supply. Following this, the liver was gently pressed to ensure contact with the abdominal skin using a moist cotton swab, and the abdomen was covered with a layer of moist gauze. Physiological saline was regularly supplemented to maintain moisture at the surgical site. After one hour of ischemia, the sham surgery group (Sham) did not experience occlusion of the hepatic portal vein or hepatic artery, and all other procedures were identical to those of the experimental group (I/R). In the experimental group, the vascular clamps were carefully removed to restore blood flow to the liver, after which the abdominal cavity was sutured. Subsequently, blood and liver samples were collected for analysis after 6 h, 24 h, and 72 h of hepatic I/R.

H/R cell culture model and treatments

To establish a hypoxia/reoxygenation (H/R) model23, RAW264.7 cells were cultured in serum- and glucose-free DMEM and incubated under hypoxic conditions (1% O2, 5% CO2, and 94% N2) for 6 h. Following this, the cells were reoxygenated by returning them to normoxic conditions (21% O2, 5% CO2, and 74% N2) in complete DMEM for an additional 6 h. The control group was maintained under normoxic conditions. Ori was dissolved in DMSO to prepare a 10 mM stock solution. For Ori treatment, the stock solution was added to the complete DMEM medium at concentrations of 2.5 µM, 5 µM, and 10 µM prior to the hypoxic treatment.

Biochemical analysis

In accordance with the manufacturer’s instructions, various reagent kits from Jiancheng Bioengineering Institute (Nanjing, China) were employed to measure the levels of ALT, AST, and LDH in mouse serum, as well as MDA, GSH, GSH-PX, MPO, and SOD in hepatic tissue. The absorbance of the samples was quantified using a spectrophotometer (Infinite M1000 Pro, Switzerland), and the concentrations of the various indicators were determined based on standard curves.

Enzyme-linked immunosorbent assay

According to the manufacturer’s instructions, the mouse ELISA kit from Xinbosheng Biotechnology Co., Ltd. (China) was used to detect the levels of TNF-α and IL-1β in mouse serum. Finally, the absorbance was measured at 450 nm by a spectrophotometer (Infinite M1000 Pro, Switzerland), and the sample concentration was calculated based on the standard curve.

Real-time PCR

Total RNA was isolated using the RNAprep Kit (TianGen, China) following the manufacturer’s guidelines. The RNA samples underwent reverse transcription to cDNA utilizing the Takara PrimeScript RT reagent kit. The real-time fluorescence quantitative PCR was conducted with the LightCycler480II instrument (Roche, Switzerland), employing a two-step PCR amplification protocol. Results were quantitatively analyzed using the ΔΔCt method, and Table 1 presents the primer sequences for all detected genes.

Table 1 Sequences of RT-qPCR primers.
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Western blot analysis

Protein samples (20–30 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using a Bio-Rad Trans-Blot wet transfer apparatus. The membrane was then sealed with 5% skim milk powder. Following this, the PVDF membrane was incubated overnight at 4 °C with the primary antibody specific to the target protein (1:1000 dilution) and internal reference primary antibodies, Gapdh or LaminB1 (1:4000 dilution). Afterward, the HRP-conjugated sheep anti-rabbit secondary antibody (1:4000 dilution) was applied and incubated at room temperature. Finally, the protein bands were visualized using the FluorChem R system (ProteinSimple, San Francisco, USA), and the grayscale values of the proteins were quantified using ImageJ software.

MTT assay

For the MTT assay, 10 µL of MTT solution (5 mg/mL) was added to 100 µL of the culture medium in each well of a 96-well plate and incubated at 37 °C in the dark for 4 h. Following incubation, the working solution was removed, and each well was treated with 200 µL of DMSO to dissolve the formed formazan crystals. Absorbance was subsequently measured at 560 nm to evaluate cell viability.

Pathological examination and evaluation

Hepatic tissues from modeling mice were excised and fixed in a 10% formalin solution for 24 h. The tissues were then subjected to a dehydration process using low and high concentrations of ethanol for 20 min each. Following dehydration, the hepatic tissues were embedded in wax, cooled, and solidified before being sliced and dried at 45 °C. The slices were immersed in a transparent agent twice, with each immersion lasting 20 min. Subsequently, the slices were dewaxed and washed with water. The dewaxing procedure involved treating the slices with anhydrous ethanol twice for 5 min each, followed by a 5-minute treatment with 75% alcohol. The slices were stained with hematoxylin for five minutes, after which they underwent a gentle rinse with water for 20 min. Following a brief differentiation step using hydrochloric acid solution, the slices were washed with distilled water for 20 min and stained with eosin solution for five minutes to restore the blue hue. The slices were then immersed three times for five minutes each in anhydrous ethanol, followed by a single immersion in N-butanol for five minutes, and two additional immersions in a transparent agent for five minutes each. Finally, the slices were sealed with neutral gum. Image acquisition was conducted under a microscope, and the results were scored according to Suzuki’s score criteria (refer to Table 2).

Table 2 Suzuki’s score criteria.
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Immunohistochemical staining

After dewaxing the above embedded hepatic tissue, it was hydrated with alcohol and washed three times with distilled water for 5 min. The slices were soaked in a slice box containing 1 x sodium citrate antigen repair solution, heated in a microwave oven at high heat for 10 min, and then removed and allowed to cool naturally. Subsequently, the slices were washed three times with distilled water on a shaker, each time for 5 min. Afterwards, the 100 µL of 3% hydrogen peroxide blocking solution was added in the slices dropwise and blocked for 10 min. The slices were washed with PBS three times, each time for 5 min. The surrounding water of the slices was absorbed by filter paper. The slices were incubated with 5% BSA for 60 min and Nrf2 primary antibody overnight at 4 ℃. After incubating, the slices were washed three times with PBS, absorbed the surrounding and incubated with secondary antibodies at room temperature for 60 min. Then, the slices were washed three times with PBS and performed color development by DAB color solution in each group with the same color development time. After color development is completed, the slices were washed with PBS three times for 5 min each time, stained with hematoxylin for 20 s, washed with PBS for 1 min, differentiated with alcohol, washed with PBS for 30 min, and then dehydrated and sealed. Finally, the histological images were observed and collected by a microscope.

Establishment of the CYP metabolism incubation system for testosterone in vitro

The 5 µL liquid of testosterone was mixed with 250 µL KPI buffer solution and 5 µL hepatic microsomal enzyme solution into a 10 mL glass tube. The mixed solution was incubated at 37 °C for 5 min. Subsequently, the solution was mixed with 15 µL Solution A (NADP+, Glc-6-PO4), 3 µL Solution B (G6PDH), and 5 mL solution of ORI (30 mM) and incubated at 37 °C for 1.5 h. After incubation, 500 µL of acetonitrile containing an internal standard was added to the sample, and the reaction was terminated after shaking and mixing. The supernatant of sample was taken and dried in a vacuum oven. After drying, the sample was added to 100 µL of methanol aqueous solution (1:1) and redissolved via vortex for 1 min. Finally, the supernatant of sample was analyzed by UPLC-MS/MS.

Statistical analysis

All experimental results were plotted and statistically analyzed using GraphPad Prism 8.0 and SPSS 22.0. Independent sample t-test was used for differences between two groups, one-way ANOVA was used for comparisons between multiple groups, and LSD-t test was used for pairwise comparisons between multiple groups. P < 0.05 was considered to be significant (*P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance).

Results

Alterations and correlations in hepatic injury indicators and CYPs/UGTs with different durations of hepatic ischemia-reperfusion

To investigate the alterations in hepatic injury indicators and CYPs/UGTs across various ischemia-reperfusion (IR) time intervals, we established a mouse model of hepatic ischemia-reperfusion injury (IRI). As illustrated in Fig. 1, the duration of hepatic IR significantly influenced various injury indicators, which exhibited differing degrees of change. Compared to the control group (0 h), serum injury indicators such as ALT, AST, and LDH levels increased significantly after 6 h of hepatic IR. Conversely, after 24 h and 72 h of hepatic IR, the levels of ALT, AST, and LDH decreased significantly relative to the 6-hour group, demonstrating a gradual decline over time (Fig. 1A). This suggests that damage was most pronounced at 6 h of hepatic IR, with subsequent recovery observed at 24 h and 72 h. Additionally, at 6 h of hepatic IR, the levels of MDA and MPO, which serve as indicators of oxidative stress in hepatic tissue, increased significantly compared to those at 0 h, while GSH content and SOD activity decreased markedly, gradually returning to near-normal levels at 24 h and 72 h (Fig. 1B). These findings indicated that the organism’s capacity to manage oxidative stress was significantly diminished at 6 h of hepatic IR, with a gradual restoration and reduction of damage observed at 24 h and 72 h. Furthermore, hepatic IR triggered an inflammatory response, evidenced by a significant increase in serum inflammatory factors TNF-α and IL-1β at 6 h. The levels of these inflammatory factors began to normalize at 24 h and 72 h (Fig. 1C). Collectively, these results demonstrate that the liver experienced the most severe damage at 6 h of IR, with significant impacts on the body’s oxidative stress and inflammatory response, while the extent of damage gradually decreased with prolonged IR duration.

Hepatic ischemia-reperfusion (IR) not only caused varying degrees of damage to the organism but also affected the levels and activities of CYPs and UGTs. Real-time quantitative polymerase chain reaction (RT-qPCR) results demonstrated a significant decrease in the mRNA levels of Cyp2c29, Cyp2c65, Cyp2c66, Cyp2c38, Cyp3a11, Cyp2d22, Ugt1a2, Ugt1a9, Ugt2a1, Ugt2a2, Ugt2b35, and Ugt2b36 at 6 h following hepatic IR. In contrast, the mRNA levels of Ugt1a1 and Cyp2e1 were found to be increased. Subsequently, these mRNA levels gradually returned to baseline at 24 and 72 h (Fig. 1D). The temporal discrepancy between mRNA and protein expression may account for this phenomenon. Given that hepatic IR can influence CYP3A11 protein levels, we proceeded to investigate CYP3A11 activity. The activity of CYP3A11 can be indirectly assessed by measuring 6β-hydroxytestosterone, a phase I-specific metabolite of testosterone. The level of 6β-hydroxytestosterone was significantly reduced at 6 h after hepatic IR, indicating a marked decrease in CYP3A11 activity at that time (Fig. 1E). These findings suggest that hepatic IR for 6 h significantly affects the expression levels and activities of CYPs and UGTs. Furthermore, the observed alterations in CYP and UGT activities highlight the need for careful consideration of drug matching following hepatic IR.

We investigated the relationship between indicators of hepatic injury and the levels of CYPs and UGTs, as hepatic ischemia-reperfusion (IR) may influence both hepatic injury indicators and enzyme levels. Our results indicated that the mRNA expression levels of Cyp2c29, Cyp2c38, Cyp2c66, Cyp3a11, Ugt1a2, and Ugt2b35 were negatively correlated with the hepatic serum indicators of injury, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), while showing positive correlations with the hepatic indicators of oxidative stress, glutathione (GSH) and superoxide dismutase (SOD). In contrast, Cyp1a1 and Cyp1b1, which are associated with cancer-related drug metabolism, exhibited positive correlations with ALT and AST levels, respectively, and negative correlations with GSH and SOD levels in relation to their mRNA expression levels (Fig. 1F). These findings suggest important clinical implications for the use of medications during hepatic IR, highlighting a correlation between the expression of CYPs/UGTs and indicators of hepatic injury in this context.

Fig. 1
figure 1

Alterations and correlations in hepatic injury indicators and CYPs/UGTs with different durations of hepatic ischemia-reperfusion. (A) Serum levels of ALT, AST, and LDH during different time periods of hepatic IR (n = 6). (B) The levels of GSH, MDA, SOD, and MPO in hepatic tissue during different time periods of hepatic IR (n = 6). (C) The levels of serum inflammatory factors TNF-α and IL-1β during different time periods of hepatic IR (n = 6). (D) The mRNA levels of CYPs/UGTs during different time periods of hepatic IR (n = 6). (E) The concentration of metabolite 6β-hydroxytestosterone in the enzymatic experiment of testosterone (n = 6). (F) The correlation between CYPs/UGTs and hepatic injury indicators. (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance, vs. 0 h; #p < 0.05, ##p < 0.01, vs. 6 h).

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Effects of ORI on cyps/ugts and normal mouse liver

Our laboratory previously discovered that the mRNA levels of Ugt1a1, Ugt1a6, Ugt1a9, Ugt2b7, Cyp3a5, and Cyp4f11 in HepG2 cells could be upregulated by 10 µM ORI. Consequently, we employed gavage in C57 mice to evaluate the toxicity of various doses (25 mg/kg, 50 mg/kg, 100 mg/kg, and 200 mg/kg) of ORI, as well as its effects on CYPs and UGTs in vivo. Mice were continuously administered ORI via gavage for 14 days, with liver tissue and blood samples collected on Day 15. The results demonstrated that, across all dose levels, there was no discernible difference in the serum damage indicators of normal mice in the ORI-treated groups (Fig. 2B). Additionally, there was no significant distinction between the liver HE-stained sections of the treated and control groups, both of which exhibited normal hepatic cord and sinusoidal morphology, as well as the absence of hepatocellular necrosis and vacuolar degeneration (Fig. 2 C). Based on these findings, mice administered the specified dosage range of ORI did not exhibit any overtly harmful side effects. Furthermore, in normal mice, ORI was able to dose-dependently upregulate the mRNA levels of Cyp3a11, Cyp2c37, Cyp2c39, Cyp2d22, Cyp2e1, Cyp1a2, Ugt1a1, Ugt2b37, Ugt1a2, and Ugt1a9 (Fig. 2D).

Fig. 2
figure 2

Effect of oridonin on liver and CYPs/UGTs of normal mice. (A) Chemical structure of oridonin. (B) The plasma ALT and AST levels of normal mice treated with oridonin (n = 6). (C) HE staining results of hepatic tissue in normal mice treated with oridonin (n = 3). (D) Induction of CYPs/UGTs in normal mice by oridonin (n = 6). (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance)

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ORI alleviates hepatic IRI and reduces oxidative stress in mice

To further investigate the protective effect of ORI on IRI, we selected a 6-hour time point, which is associated with the most severe I/R injury, to construct our models. ORI was administered intraperitoneally to the treatment group seven days prior to the experiment at doses of 2.5 mg/kg and 5.0 mg/kg (Fig. 3A). The results indicated that the mRNA levels of Cyp3a11, Cyp2c38, Cyp2c40, Ugt2b36, Ugt1a2, and Ugt1a9 in the I/R group mice were significantly downregulated. Following the administration of ORI at both doses, the mRNA levels of primary hepatic enzymes in the I/R group were elevated, although they did not return to normal levels (Fig. 3B). This suggests that the downregulation of mRNA levels caused by hepatic IRI can be dose-dependently reversed by oridonin. Compared to the I/R group, serum levels of AST and ALT in mice treated with 2.5 mg/kg and 5.0 mg/kg ORI significantly decreased (Fig. 3C). Concurrently, the levels of hepatic oxidative stress indicators, including GSH, GSG-PX, and SOD, significantly increased, while the level of MDA significantly decreased (Fig. 3F). Furthermore, the pathological states of the livers in each group were assessed using HE staining, and the extent of hepatic tissue destruction was evaluated by Suzuki’s score. We found that the hepatic lobule structure, hepatic sinusoids, and hepatocytes in the Sham group of mice were well-preserved, exhibiting no visible pathological abnormalities. In contrast, the hepatocytes in the I/R group exhibited significant balloon-like degeneration, congestion, and necrosis of hepatic lobules, along with infiltration of inflammatory cells. Compared to the I/R group, the livers of mice in the I/R + ORI2.5 group showed partial necrosis and tissue congestion, demonstrating significant improvement, while the livers of the mice in the I/R + ORI5 group exhibited considerably less tissue necrosis and congestion, revealing distinct hepatic cords (Fig. 3D). Based on the results of Suzuki’s score, the I/R group’s score was significantly higher than that of the Sham group, while the treatment group’s score was notably lower than that of the I/R group. Furthermore, the degenerative state of the hepatic tissue in the I/R + ORI5 group was superior to that in the I/R + ORI2.5 group (Fig. 3E). These findings suggest that ORI can effectively mitigate hepatic injury and oxidative stress induced by hepatic ischemia-reperfusion.

Fig. 3
figure 3

ORI alleviates hepatic IRI and reduces oxidative stress in mice. (A) Schematic diagram of oridonin treatment in mice with hepatic IRI. (B) Oridonin intervened in the mRNA levels of CYPs/UGTs in I/R mice (n = 6). (C) The levels of serum ALT and AST in each group (n = 6). (D) HE staining section images of hepatic in each group (①-④ are magnified 100 times, ⑤-⑧ are magnified 400 times). (E) Suzuki’s score semiquantitative evaluation of the degree of hepatic injury in each group (n = 3). (F) The levels of oxidative stress indicators in hepatic tissues of each group (n = 6). (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance).

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ORI inhibits the NLRP3 pathway to reduce the inflammatory response caused by hepatic IR

Hepatic IR can induce inflammatory responses alongside oxidative stress and hepatic damage. Therefore, we investigated the capacity and mechanism of oridonin to suppress the inflammatory response elicited by hepatic IR. ELISA results indicated that mice in the I/R group exhibited significantly elevated serum levels of the inflammatory cytokines TNF-α and IL-1β compared to those in the Sham group. In contrast, mice in the I/R + ORI2.5 and I/R + ORI5 groups demonstrated a dose-dependent reduction in the serum levels of these cytokines relative to the I/R group (Fig. 4A). Additionally, qPCR results revealed that in the I/R + ORI2.5 and I/R + ORI5 groups, there was a dose-dependent decrease in the mRNA levels of TNF-α and IL-1β compared to the I/R group (Fig. 4B). NLRP3, a well-established inflammatory pathway, plays a crucial role in the inflammatory process induced by hepatic IR. Western blot analysis showed that the protein levels of IL-1β, IL-18, GSDMD and ASC were upregulated in the I/R group, indicating significant activation of the NLRP3 pathway. In comparison to the I/R group, the levels of these downstream proteins were markedly downregulated in the I/R + ORI2.5 and I/R + ORI5 groups, demonstrating that oridonin can partially inhibit the activation of the NLRP3 pathway (Fig. 4C, D). These findings suggest that oridonin may mitigate the inflammatory response triggered by hepatic ischemia-reperfusion injury by inhibiting the NLRP3 inflammatory signaling pathway.

Fig. 4
figure 4

Oridonin alleviated the inflammatory response induced by hepatic ischemia-reperfusion injury via the NLRP3 pathway. (A) The levels of serum inflammatory factors in each group (n = 6). (B) The mRNA levels of inflammatory factors in hepatic tissues of each group (n = 6). (C) The protein levels of the NLRP3 pathway in hepatic tissues of each group (n = 3). D. Grayscale analysis of the protein levels related to the NLRP3 pathway. (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance).

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ORI reduces hepatic IRI via activating the NRF2/HO-1 pathway

Subsequent mechanistic research demonstrated that ORI can dose-dependently upregulate the mRNA levels of Nrf2, HO-1, and NQO1, highlighting its protective effects against hepatic ischemia-reperfusion injury (IRI) (Fig. 5A). Western blot analysis revealed that the stress response elevated the protein levels of Nrf2 and HO-1 in the hepatic tissue of the I/R group. In the hepatic tissue of the I/R + ORI2.5 group, there was a modest increase in the protein levels of Nrf2 and HO-1 compared to the I/R group. However, a substantial increase in the hepatic tissue was observed in the I/R + ORI5 group for all three proteins (Fig. 5B, C). We further assessed the expression levels of Nrf2 protein in the nucleus, as this protein must translocate to the nucleus to activate its function. The conclusion that nuclear Nrf2 protein levels in the I/R + ORI5 group were markedly elevated compared to the I/R group was corroborated by the immunohistochemistry results (Fig. 5D). In summary, ORI exerts its antioxidant capacity by activating the NRF2/HO-1 pathway, thereby mitigating hepatic IRI.

ORI inhibits hepatocyte apoptosis resulting from hepatic IRI

Given the close relationship between hepatocyte apoptosis and hepatic ischemia-reperfusion injury (IRI)22, the ability of ORI to prevent hepatocyte apoptosis is crucial. The quantitative PCR results revealed that the pro-apoptotic protein Bax exhibited significantly elevated mRNA levels in the I/R group, while the mRNA levels of the anti-apoptotic protein Bcl-2 were reduced. Conversely, in the I/R + ORI2.5 and I/R + ORI5 groups, the mRNA levels of Bax and Bcl-2 demonstrated dose-dependent significant decreases and increases, respectively (Fig. 5E). Furthermore, western blot analyses indicated that Bcl-2 protein levels were markedly elevated in the I/R + ORI2.5 and I/R + ORI5 groups (Figs. 5F). These findings suggest that ORI inhibits hepatocyte apoptosis induced by hepatic IRI.

Fig. 5
figure 5

The antioxidant and anti-apoptotic mechanisms of oridonin on hepatic ischemia-reperfusion injury. (A) The mRNA levels of the Nrf2-related pathway genes in each group (n = 6). (B) The expression levels of the Nrf2 pathway related proteins in hepatic tissues of each group (n = 3). (C) Grayscale analysis of the expression levels of the Nrf2 pathway related proteins in hepatic tissues. (D) The level of Nrf2 protein entering the nucleus was detected via immunohistochemistry in each group (n = 3). (E) The mRNA levels of the apoptosis related pathway genes in each group (n = 6). (F) The expression levels of proteins related to the apoptotic pathway in hepatic tissues of each group and grayscale analysis (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance).

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ORI inhibited the inflammation of macrophages induced by H/R

To further validate the results of animal experiments, we utilized the mouse monocyte macrophage leukemia cell line (RAW264.7 cells) to examine the effects of ORI on a cell hypoxia/reoxygenation (H/R) model, which is a well-established in vitro model of hepatic ischemia-reperfusion injury (IRI)23. The MTT assay results indicated that treatment with ORI at concentrations ranging from 0 to 10 µM did not significantly affect cell survival after 24 and 48 h. However, concentrations of 20 µM and 50 µM began to demonstrate notable cytotoxicity (Fig. 6 A). Consequently, we selected ORI doses of 2.5, 5.0, and 10.0 µM for the H/R model. The findings revealed a dose-dependent reduction in the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in each ORI treatment group, while the H/R group exhibited significantly elevated levels of these factors (Fig. 6B). Furthermore, the H/R group showed significantly increased mRNA levels of inflammatory signaling factors, including IL-1β, TNF-α and IL-6. In contrast, the ORI treatment groups effectively downregulated the mRNA levels of these inflammatory signaling factors (Fig. 6 C). Compared to the control group, the protein expression levels of NLRP3 and Caspase 1 were significantly higher in the H/R group; however, the ORI treatment groups successfully downregulated these protein levels induced by H/R (Figs. 6D). In summary, ORI exhibited an anti-inflammatory effect in the cell H/R model, thereby further corroborating the results of hepatic IRI observed in vivo.

Fig. 6
figure 6

The anti-inflammatory effect of oridonin on macrophage inflammation induced by hypoxia/reoxygenation (H/R). (A) The effect of oridonin on the activity of RAW264.7 cells (n = 6). (B) The levels of inflammatory factors in the supernatant of RAW264.7 cells treated with H/R and oridonin (n = 6). (C) The mRNA levels of inflammatory factors in RAW264.7 cells treated with H/R and oridonin (n = 6). (D) The expression levels of the NLRP3 pathway related proteins in RAW264.7 cells treated with H/R and oridonin and grayscale analysis (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance)

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Discussion

Hepatic IRI is a frequent complication in surgeries such as hepatic transplantation and hepatic lobectomy. It leads to the production of excessive reactive oxygen species (ROS), resulting in oxidative stress, biomolecule oxidation, and mitochondrial dysfunction24,25. This oxidative stress can initiate apoptosis and necrosis of hepatic cells, triggering inflammasome assembly and subsequent activation and secretion of pro-inflammatory cytokines, which drives an inflammatory response. Research indicates that alcoholic liver injury is associated with a notable increase in the expression and activity of CYP2E1, which contributes to lipid peroxidation and ROS formation, ultimately leading to cell apoptosis26,27. In CYP2E1 knockout mice with non-alcoholic steatohepatitis, upregulation of CYP4A10 and CYP4A14 protein expression correlates closely with in vivo lipid peroxidation28. These findings suggest that changes in the expression and activity of CYPs/UGTs are involved in hepatic disease damage. However, there is limited research on CYPs/UGTs alterations during hepatic IRI and their relationship with injury. Our study showed that, while levels of hepatic oxidative stress indicators MDA and MPO significantly increased, serum damage markers ALT, AST, and LDH sharply rose 6 h after hepatic IRI. Antioxidant indicators GSH and SOD dropped significantly, while inflammatory markers TNF-α and IL-1β increased. As hepatic IRI progressed, these indicators gradually returned to normal levels, suggesting severe hepatic IRI at 6 h with subsequent recovery. Post-6 h of hepatic IRI, mRNA levels of Cyp2e1 and Ugt1a1 increased, while those of Cyp3a11, Cyp2c66, Cyp2c38, Cyp2d22, Cyp3a16, Ugt2a1, Ugt1a1, Ugt1a9, and Ugt2b36 decreased. The significant elevation of serum ALT/AST/LDH levels and hepatic MDA/MPO content at post-6 h of hepatic IRI indicates the peak occurrence of mitochondrial ROS burst and neutrophil infiltration during this phase. Notably, metabolic enzyme families exhibit marked isoform-specific regulatory patterns. While most CYPs demonstrate suppressed expression during the acute phase of IRI, the early upregulation of Cyp2e1 manifests dual pathophysiological implications. Initially, it may exert compensatory detoxification effects through elimination of lipid peroxidation products (e.g., MDA) generated during oxidative stress. However, persistent overexpression of Cyp2e1 potentially exacerbates mitochondrial dysfunction and promotes hepatocyte apoptosis via catalyzing ROS cascade amplification through futile cycle mechanisms. Of particular interest is the specific upregulation of Ugt1a1 within the UGT family. Contrasting with the general suppression of other UGT isoforms at post-6 h of IRI, Ugt1a1 upregulation likely facilitates adaptive clearance of toxic bilirubin or lipid peroxides that accumulate during IRI. At the same time, the selective induction of Ugt1a1 could be driven by Nrf2, which is known to transcriptionally regulate Ugt1a1 via antioxidant response elements (AREs). CYP3A11 protein levels and activity also significantly decreased before gradually normalizing. This suggests that varying degrees of hepatic IRI affect the mRNA levels, protein levels, and metabolic activity of crucial CYPs/UGTs. Bioinformatics correlation analysis revealed a relationship between the mRNA levels of key CYPs/UGTs and several hepatic IRI indicators. Hepatic oxidative stress indicators GSH and SOD positively correlated with mRNA levels of Cyp2c29, Cyp2c38, Cyp2c66, Cyp3a11, Ugt1a2, and Ugt2b35, while serum damage indicators ALT and AST showed a negative correlation. The mRNA levels of Cyp1a1 and Cyp1b1 negatively correlated with GSH and SOD but positively correlated with ALT and AST. These findings are valuable for the rational use of medications following hepatic IRI.

The primary therapeutic approaches for hepatic IRI include drug intervention and surgical methods. Drug intervention mainly involves the use of anesthetic agents. Research indicates that sevoflurane and propofol can alleviate hepatic IRI in animal models, mainly due to their anti-apoptotic and antioxidant properties29. Additionally, numerous studies have highlighted the preventive effects of Chinese herbal components against hepatic IRI, operating through mechanisms such as endoplasmic reticulum stress inhibition, antioxidant stress mitigation, anti-inflammatory responses, and anti-apoptotic actions30. Our previous research demonstrated that ORI can alleviate acute liver injury by inhibiting inflammatory pathways31. Furthermore, ORI has been shown to reduce cardiac and cerebral IRI in animals by blocking inflammatory pathways21,32. Therefore, we investigated whether ORI could also prevent hepatic IRI.

In normal mice, a 200 mg/kg dose of ORI did not cause significant liver damage, indicating its low toxicity. ORI also dose-dependently enhanced the mRNA expression of various CYPs/UGTs, including Cyp3a11, Cyp2c37, Cyp2c39, Cyp2d22, Ugt1a1, Ugt1a2, Ugt2b36, and Ugt1A9. In the I/R group, ORI intervention (at doses of 2.5 mg/kg and 5.0 mg/kg) not only reversed changes in the mRNA expression of these CYPs/UGTs but also decreased levels of serum injury markers (ALT, AST, LDH), hepatic oxidative stress indicators (MDA, MPO), and inflammatory factors (TNF-α, IL-1β), while increasing antioxidant indicators (GSH, SOD). In addition, ORI facilitated the overexpression of the anti-apoptotic protein Bcl-2, thereby preventing hepatic cell apoptosis associated with hepatic IRI. However, it should be noted that the current study primarily relied on Bax/Bcl-2 ratio as an indirect indicator of apoptosis. While this ratio provides valuable insights into mitochondrial apoptotic pathways, it does not fully distinguish apoptosis from alternative cell death mechanisms such as necrosis. To address this limitation, future studies should integrate techniques to validate the occurrence of cell apoptosis from multiple perspectives. These efforts will provide a more thorough understanding of the protective effect of ORI on cell apoptosis in hepatic I/R injury.

The NLRP3 pathway is a key player in inflammatory responses, and research has shown that ORI can inhibit this pathway, reducing inflammation associated with hind limb IRI33. In this study, ORI effectively suppressed the expression of NLRP3 and its downstream proteins, including GSDMD, ASC, IL-1β, and IL-18. These findings suggest that ORI mitigates inflammatory responses during hepatic IRI by blocking activation of the NLRP3 pathway.

In addition to the inflammatory signaling pathway, the oxidative stress signaling pathway also plays a key role in hepatic IRI. The Nrf2 pathway, an endogenous antioxidant mechanism, helps reduce oxidative stress in vivo. Recent research has shown that ORI inhibits the progression of atherosclerosis in apolipoprotein E-deficient mice by activating Nrf234. Similarly, our findings suggested that ORI increased the expression of downstream proteins HO-1, thereby activating the Nrf2 pathway and mitigating the oxidative stress response associated with hepatic IRI.

Conclusion

In conclusion, our study revealed that hepatic IRI triggers inflammatory and oxidative stress responses while also mediating spatiotemporal changes in CYPs/UGTs. We clarified the relationship between hepatic injury markers and the mRNA levels of CYPs/UGTs. Additionally, ORI was found to reverse changes in CYPs/UGTs, activate the Nrf2 pathway, inhibit the NLRP3 pathway, and prevent hepatic cell apoptosis, jointly providing protection against hepatic IRI. These findings suggest that ORI holds promise as a potential therapeutic agent for hepatic IRI. Furthermore, the oxidative stress signaling pathway, particularly the Nrf2 pathway, plays a significant role in hepatic IRI. As an endogenous antioxidant mechanism, Nrf2 reduces oxidative stress in vivo. Recent studies have shown that ORI can inhibit atherosclerosis progression in apolipoprotein E-deficient mice by activating Nrf2. Consistent with this, our findings demonstrated that ORI increased the expression of downstream proteins HO-1, thereby activating the Nrf2 pathway and alleviating oxidative stress associated with hepatic IRI.