DHM was purchased from Mansite Bio-Technology (China) and Sigma-Aldrich (United States). LPS and 3-TYP were purchased from MedChemExpress (United States). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl (polyethylene glycol)-2K] (PEG2K-DSPE) was obtained from Xi’an Ruixi Biological Technology (China). ELISA kits were obtained from Shanghai FANKEL Industry (China). The murine macrophage cell line RAW264.7 and exclusive complete medium (icell-m047-001b) were obtained from ICell Bioscience Inc., China. IR-808 was provided by the Institute of Combined Injury of Army Medical University.

Briefly, 93 mg soy phosphatidylcholine, 20 mg cholesterol, and 27 mg PEG2K-DSPE were dissolved in 5.6 ml dichloromethane to obtain the aqueous phase. Subsequently, 7 mg DHM was added and solubilized in 5.6 ml methanol under magnetic stirring for 30 min to produce the lipid phase. The aqueous and liquid solutions were added dropwise to 28 ml methanol/dichloromethane (4:1) mixture solvent at the same time under magnetic stirring for 5 min. The harvested solution was placed on the rotary steamer under reduced pressure rotary steamer (40°C, 80 r/min). Then, 20 ml PBS was added and processed with ultrasound. The solution was filtered by the 0.45 um film and 0.22 um film sequentially. Unencapsulated DHM was cleared by dialysis at 4°C against PBS using Slide-A-Lyzer dialysis cassettes (JingKeHongDa Biotechnology Co., China) (Bartneck et al., 2015). Ultimately, a homogeneous and translucent DHM liposome solution was obtained. The blank liposomes were also prepared by a similar method.

To verify the distribution of DHM-Lipo in vivo, its surface was further labeled with a near-infrared (NIR) heptamethine cyanine dye (IR-808). IR-808 belongs to a class of Cy7 NIR probes that have been reported with strong fluorescent emission near 800 nm (Tan et al., 2012). IR-808 was synthesized according to previously established methods (Luo et al., 2016). Briefly, DHM-Lipo was mixed with IR-808 (10 mM) in 1.0 ml ultrapure water and shaken for 30 min. With hydrophobic heptamethine core and negative carboxyl terminus, IR-808 was efficiently labeled on the surface of PEG-DSPE modified liposomes through hydrophobic and electrostatic interaction. IR-808 labeled DHM-Lipo was purified by centrifuge filtration (3,000 rpm for 5 min) through centrifugal filters (10 kDa), washed three times with ultrapure water to remove free IR-808. The final product was concentrated at 1 ml and stored at 4 °C for further use.

The morphology of the DHM-Lipo was examined by the transmission electron microscope (TEM, TECNAI 10, Philips, United States). Briefly, the DHM-Lipo solution was added to the copper mesh with the film and stained by phosphotungstic acid negative stain solution for 2 min. TEM was used for examining the morphology of the liposomes after air drying. A laser particle size analyzer (Zetasizer Nano, MALVERN, UK) was used to analyze the particle size of DHM-Lipo.

The high-performance liquid chromatography (HPLC, Waters, India) was used to acquire the encapsulated efficiency by analyzing the supernatant after centrifugation of liposomes. The experiment demonstrated out on C18 column at 30°C. The encapsulation efficiency% was calculated using the following equation (Moghimipour et al., 2018). encapsulation   efficiency % = 100 TD − FD TD Whereas TD is the amount of DHM originally added to the formulation and FD is the amount of the free DHM in the supernatant after centrifugation.

2 ml DHM-Lipo was supplemented into a dialysis bag (MWCO 5000 Da) and then dialyzed against 500 ml PBS at 25°C for 100 h. At designed intervals, 10 ml of sample from the reservoir was harvested, and 10 ml of fresh PBS was supplemented again. The DMH concentration of the harvested samples was analyzed by HPLC. The’ release kinetic curve was calculated to reflect the accumulated release percentage.

Cell viability was investigated by Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Briefly, RAW264.7 cells were replated at 8000 cells/well in 96-well plates. Then, the 2 mg/ml DHM-Lipo was added. At the time point of 24 and 48 h, 10 μL of CCK-8 solution (Dojindo, Japan) were added and incubated for 2 h. The optical density (OD) value was measured at 450 nm with a microplate reader (Bio-Rad Laboratories, United States).

Male C57BL/6J mice were fed in a condition as previously reported (Zhou et al., 2021). The experimental procedure of each group was illustrated in Figure 1A. After 1 week of adaptive feeding, DHM was administered intraperitoneally for 1 week. Then, mice executed adaptive swimming exercises for 1 week and exhaustive swimming exercises for another week, following the protocol of previous studies with slight modification (Kim et al., 2014; Yuan et al., 2018). Briefly, swimming was carried out with ten mice per plastic box (90 × 50 × 40 cm) filled to a depth of 30 cm with water maintained at a temperature of 34 ± 1°C. All mice were adapted to swimming for 5 days initially: Day 1, two times of 30-s swimming with a 2 min rest interval between the swimming periods; Day 2, two times of 2 min of swimming with a 2 min rest interval; Day 3, three times of 10 min of swimming with a 5 min rest interval; Day 4, two times of 15 min of swimming with a 5 min rest interval; Day 5, a period of 30 min with no pause. After that, except for the mice in the control group, all mice started exhaustive exercise training in the fourth week. During this period, mice should swim until exhaustion once a day for seven consecutive days. Exhaustion was defined by two criteria: greater than 10 s spent below the water surface and lack of a “righting reflex” when placed on a flat surface (Dawson and Horvath, 1970; Thomas and Marshall, 1988). All efforts were made to minimize animal suffering after the experiments. Serum and liver tissues were collected and stored at 80°C. Part of the mouse was used for Flow Cytometry analysis. All the animal experiments were approved by the Animal Care and Use Committee of Third Military Medical University (Chongqing, China; Approval SYXC-2017–0002).

Serum was prepared by solidification and centrifugation (4 °C, 3000 g, 10min) and stored at -80 °C (Zeng et al., 2019). Biochemical measurement of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), and total bilirubin (TBIL) was performed on an Olympus AV5400 auto analyzer.

TNF-α, IL-1β, and IL-6, from the cultured macrophage supernatant, the mice serum and liver samples were quantified by TNF-α (#F2132), IL-1β (#F2040) and IL-6 (#F2163) ELISA kits (Fankew, China) according to the manufacturer’s instructions. The SpectraMax^® M2 spectrophotometer (Molecular Devices Corp., United States) was used to obtain the OD value at 450 nm.

RNA was extracted with TRIzol reagent (Invitrogen, United States). We used qTower 2.2 real-time PCR system (Analytik Jena, Germany) to run qRT-PCR with SYBR Premix Ex Taq II (Takara Bio, Japan). All primers are listed in Table 1. Relative mRNA expression levels were normalized to those of β-actin and calculated by the 2^−ΔΔCt method.

Immediately after animals were sacrificed, liver samples were fixed with 4% paraformaldehyde and embedded in paraffin. The hematoxylin and eosin (H&E) were performed on the liver sections.

For visualization of inflammatory cytokines distribution in the liver, the samples were then stained with TNF-α (1:20 dilution, Abcam, # Ab183218) and IL-1β (1:200 dilution, Abcam, # Ab9722). Following a standard staining protocol. The section results were scanned by a high-resolution digital slide scanner (VS-200, Olympus, Japan).

For immunofluorescence, liver frozen sections were processed using anti-F4/80 antibody (1:50 dilution, Abcam, #Ab60343). And anti-CD206 antibody (1:100 dilution, Abcam, #Ab64693), anti-iNOS antibody (1:50 dilution, Abcam, #Ab3523), anti-SIRT3 antibody (1:100 dilution, Abcam, #Ab189860), and anti-HIF-1a antibody (1:100 dilution, Abcam, #Ab228649). Then, the liver sections were treated with proteinase K (1:200, Solarbio, China) for 30 min, next by Triton X-100 (0.1%, Beyotime, China) for 30 min, and then with normal goat serum (ZSGB-BIO, China) for 30 min. Subsequently, the sections were incubated by the primary antibodies at 4 °C overnight, and then the secondary antibodies (1:1000, #Ab150165, #Ab150062, Invitrogen, United States) for 1 h at room temperature. Finally, DAPI solution (0.1%, Beyotime, China) was applied to visualize the cell nucleus for 10 min at room temperature. The immunostaining image was captured by the confocal microscopy system (LSM780, ZEISS, German) and analyzed by the matching software (Zen 2.3, ZEISS, German).

The cell line RAW264.7 of murine macrophage and exclusive complete medium (icell-m047-001b) were obtained from ICell Bioscience Inc., China. Cells were cultured at 37°C in a 5% CO₂ atmosphere. To establish the model of inflammation, RAW264.7 cells were treated with LPS in sequential concentrations (0, 25, 50,100, and 200 ng/ml) for 24 h. To explore the effect of DHM, cells were preincubated with DHM at various concentrations (0, 10, 20, and 30 μM) for 2 h and thereafter treated by LPS (100 ng/ml) for 24 h. Moreover, SIRT3 inhibitor 3-TYP (50 μM) was used 1 h before DHM treatment to investigate the molecular mechanism of RAW264.7 cells in response to DHM. The small interfering RNA (siRNA) targeting Sirt3 were purchased from RiboBio (China). The sequence of the mouse siRNA was as follows: Sirt3, 5-ACU​CCC​AUU​CUU​CUU​UCA​C-3. Twenty-four hours after seeding, cells were transiently transfected with 100 nM siRNA per dish at 80% confluence using the lipofectamine 2000 (Invitrogen Life Technology, Carlsbad, CA, United States). The knockdown efficiency of the target proteins was measured with western blot assay. All experiments were repeated at least three times.

Mice were sacrificed and the liver was harvested for analysis ex vivo as previously described (Park et al., 2017). Cells were isolated and stained with anti-mouse CD45 (1:100 dilution, Biolegend, #103116), anti-mouse F4/80 (1:100 dilution, Biolegend, #123108), anti-mouse CD206 (1:100 dilution, Biolegend, #141720), and anti-mouse CD11c (1:100 dilution, Biolegend, #117310), and then tested using flow cytometry (LSRFortessaTM cell analyzer, BD, United States). Data were analyzed with FlowJo V10.6.

Protein expression was investigated by western blot analysis as in previous methods (Zhou et al., 2021). The primary antibodies were used to study the protein expression of sirtuins-3 (SIRT3, 1:1000 dilution; Cell Signaling Technology, #2627S), hypoxia-inducible factor-1α (HIF-1α, 1:300 dilution; Proteintech, #20960-1-AP), and β-actin (1:1000 dilution, Santa Cruz, #47778), respectively.

Statistical analysis of in vitro and in vivo studies was performed using GraphPad Prism 9 (GraphPad Software Inc., CA). A one-way analysis of variance (ANOVA) was performed to determine significance by the Turkey-Kramer post hoc test, which was set at p < 0.05. Quantitative data are presented as the mean ± standard error of mean (X ± SEM) values. All experiments were repeated independently at least three times.

Exhaustive exercise leads to liver injury caused by inflammation (Huang C. C. et al., 2013; Huang K. C. et al., 2013). Mice were randomly assigned to five groups (n = 15/group): control, exhaustive exercise (EE), and EE + DHM (frequent administration, FA) at 2, 4, and 8 mg/kg body weight (Figure 1A). There were no significant differences in body weight between each group (Figure 1B). The liver index represents the ratio of liver weight to body weight. The liver index of the EE group was increased compared with that of control mice, and this trend was reversed after DHM administration (p < 0.05, Figure 1C). Furthermore, the serum levels of ALT, AST, GGT, and TBIL in mice in the EE group were significantly elevated compared with those in mice in the control group (p < 0.01, Figure 1D–G, n = 5/group). Surprisingly, the EE-induced effect was ameliorated by DHM administration (p < 0.05, Figure 1D–G). Moreover, the EE-induced increase in inflammatory cytokines in the serum and liver, including tumor necrosis factor-a (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6), was strongly inhibited by DHM (2 mg/kg) administration (p < 0.05, Figure 1H-M). Moreover, the increased hepatic mRNA levels of Tnfa and Il1b induced by EE were also significantly reversed by DHM in vivo (p < 0.01, Figure 1N–O). Overall, 2 mg/kg DHM exhibited the most hepatoprotective effect on EE-induced liver injury. Previous studies reported that poor bioavailability limits the clinical application of DHM (Liu et al., 2019). To explore the influence of bioavailability on DHM efficacy, we adjusted the dosing interval of DHM administration into two groups: EE + DHM (FA) (1 administration every day) and EE + DHM (infrequent administration, IFA) (1 administration every 3 days) (Figure 1A). As expected, EE-induced notable inflammatory infiltration was ameliorated by DHM (FA) administration, as evidenced by histology with H&E staining and immunohistochemistry (IHC) for TNF-α and IL-1β (Figure 1P). Moreover, EE-induced expression of the inflammatory cytokines TNF-α, IL-1β, and IL-6 was decreased by DHM (FA) administration, as determined by ELISA analysis (Figure 1Q–S). Regrettably, the DHM (IFA) group showed a certain difference in the expression of inflammation compare with DHM (FA) group (Figure 1P–S), suggesting that DHM (IFA) may lead to a significant compromise in the anti-inflammatory effect.

Our results in Section 3.1 and previous studies indicate that the poor aqueous solubility and low bioavailability of DHM diminished its hepatoprotective effect (Liu et al., 2017a). Therefore, searching for an efficient drug delivery strategy to improve DHM bioavailability and prevent premature release before reaching the desired site is necessary. Liposomes have shown promising potential for drug delivery. Its outstanding performance in resisting degradation, penetrating pathogen barriers, and adjusting the release behavior makes it a valuable prospect (Liu et al., 2020). As described in the Methods section, we synthesized long-circulating liposomes for favorable DHM delivery. The micromorphologies of the DHM-Lipo were characterized by transmission electron microscopy, as depicted in Figure 2A. The liposomes exist as nanosized spheres with smooth surfaces and good dispersibility. The particle size of the tested sample presented a normal distribution, and the average particle diameter of the DHM-Lipo was 116.1 nm (Figure 2B). The release kinetics of DHM is shown in Figure 2C. Within 1 h, the DHM was released in a burst with a percentage of 17.9%. Subsequently, the sustained release percentage of DHM decreased and stabilized gradually during the middle stage of release from 2 to 24 h. At this stage, additional 30% of DHM was released from the nanoparticles. After 3 days, the cumulative release percentage reached 65.5 ± 3.1%. The encapsulation efficiency of DHM in the liposomes was 45%. This result indicates that DHM loaded in the liposomes could be released stably for an extended time. The CCK-8 assay was conducted to quantify the cytotoxicity of DHM-Lipo on RAW264.7 cells. As shown in Figure 2D, the cell proliferation activity of the DHM-Lipo group showed no obvious difference compared to that of the control group at 24 h or 48 h (p > 0.05). In brief, the results demonstrated that DHM-Lipo exhibited desirable characteristics of nanosized microspheres, good dispersion, stable release, and favorable cytocompatibility.

As stated above, C57BL/6J mice were used to establish the EE-induced liver inflammation model. DHM (IFA), DHM-Lipo, and liposomes were administered to the EE-induced mouse model were administered every 3 days, as illustrated in Figure 1A (n = 5). The single intraperitoneal dose of the DHM (IFA) group was set to 2 mg/kg DHM based on Section 3.1, and the DHM-Lipo group was set at 40 mg/kg liposomal DHM. With a loading efficiency of 5% for the liposomes, the DHM content is identical. As expected, EE-induced inflammatory injury in the liver was ameliorated by infrequent administration of DHM-Lipo. Histologically, the anti-inflammatory effect of the DHM-Lipo was more powerful than that of free DHM, as evidenced by a greater reduction in the secretion of TNF-α and IL-1β. There were no significant differences between the EE group and EE + Lipo group, indicating that blank liposomes could not exert an anti-inflammatory effect on liver injury (Figure 3A). Furthermore, the serum levels of ALT and AST in mice in the EE + DHM-Lipo group were significantly decreased compared with those in the EE group (p < 0.05) but ALT was not decreased in the EE + DHM (IFA) group or EE + lipo group (Figure 3B,C). Moreover, EE-induced inflammatory cytokine levels in the serum (Figure 3D–F) and liver (Figure 3G–I) were significantly inhibited by DHM-Lipo administration, but not by the EE + DHM (IFA) group except for Liver TNF-α. In brief, these data demonstrate that DHM-Lipo administration notably attenuated liver injury and inflammation in EE-treated mice. The hepatoprotective effect of DHM-Lipo was more effective than that of free DHM (IFA), indicating that DHM-Lipo overcomes the limitation of poor bioavailability of DHM.

In recent years, lipid nanoparticles have been developed to passively and actively target drugs in the liver (Böttger et al., 2020). Thus, we hypothesized that the hepatoprotective effect of the DHM-Lipo was possibly related to its liver-targeting bioactivity. To verify this hypothesis, we prepared DHM-Lipo@IR-808 and subsequently performed in vivo NIR fluorescence imaging to assess the liver targeting capability after intraperitoneal injection of DHM-Lipo@IR-808. The fluorescent signals associated with the liver region sites can be visualized with low background interfering fluorescence 1–48 h after injection (Figure 4A). Next, we performed fluorescence reflectance imaging (FRI) ex vivo scans of the heart, liver, spleen, lung, kidney, intestine, and blood. The fluorescence intensity of the dissected organs further confirmed the preferential accumulation of DHM-Lipo@IR-808 in the liver (Figure 4B). Quantification of fluorescence revealed that the liver is the predominant organ for DHM-Lipo uptake. 48 h after injection, fluorescently tagged liposomes predominantly accumulated in the liver, where they were widely distributed (Figure 4C). We also detected the distribution of DHM-Lipo@IR-808 in liver macrophages (Figure 4D). Visualization of DHM-Lipo@IR-808 in vivo showed that the macrophages in the liver exhibited a favorable uptake of DHM-Lipo@IR-808 at 48 h, as evidenced by DHM-Lipo@IR-808 mainly accumulating in F4/80⁺ macrophages in different samples. In contrast, no colocalization of free IR-808 and F4/80 was observed in the liver. Taken together, these results indicate that DHM-Lipo@IR-808 accumulated in hepatic macrophages.

To further investigate the effects of DHM-Lipo on liver macrophages, we performed immunofluorescence staining for M1 and M2 macrophage markers in liver tissue. Staining of hepatic M1 macrophages with the specific marker of colocalization of iNOS with F4/80 revealed that DHM-Lipo reduced the amount of M1 macrophages in the liver (Figure 5A). DHM-Lipo treatment was almost exclusively related to reduced numbers of M1 but not M2 macrophages, as evidenced by an increase in the colocalization of CD206 with F4/80 that was induced by DHM-Lipo (Figure 5B). This phenomenon was confirmed by flow cytometric determination (Figure 4C–F). We analysed M1 (CD45⁺ F4/80⁺ CD11c⁺) and M2 (CD45⁺ F4/80⁺ CD206⁺) macrophages from the liver tissues of mice by flow cytometry according to previous study (Zhang X. et al., 2019). DHM-Lipo significantly decreased the percentage of M1-like macrophages within the total macrophage population that was activated by EE (p < 0.01, Figure 5C,D). Simultaneously, DHM-Lipo increased the percentage of M2-like macrophages (p < 0.01, Figure 5E,F). These results demonstrate that M1 macrophages (inflammatory) in the liver are polarized towards the alternatively activated (anti-inflammatory) M2 subtype.

To further verify the effect of DHM on macrophage polarization in vitro, we established an inflammatory cell model using RAW264.7 cells treated with a series of concentrations (0, 25, 50, 100, and 200 ng/ml) of lipopolysaccharides (LPS) for 24 h. Compared to the control group, the mRNA expression of Il1b and Il6 was significantly increased, especially in the 100 ng/ml group (Figure 5G,H). To further explore the anti-inflammatory effect of DHM, RAW264.7 cells were preincubated with a series of concentrations (0, 10, 20, and 30 μM) of DHM for 2 h before treatment with LPS (100 ng/ml) for 24 h. Compared with treatment with LPS alone, DHM administration, especially at 10 μM, significantly suppressed the LPS-induced increase in Il1b and Il6 mRNA expression (p < 0.01, Figure 5I,J). In addition, the mRNA expression of iNos, which represents M1-like macrophages, was significantly increased in the LPS group and was significantly reversed by DHM, as expected (p < 0.05, Figure 5K). Moreover, the mRNA expression of CD206, Fizz1, Ym1, and Il10, which represent M2-like macrophages, was significantly increased in the LPS + DHM groups compared with the LPS group (p < 0.05, Figure 5L, Supplementary Figure S1). Collectively, these results demonstrate that DHM administration can regulate macrophage polarization. DHM effectively inhibited M1-like macrophages and enhanced M2-like macrophages in vivo and in vitro.

Many studies have found that the polarization phenotype of immune cells is related to their energy metabolism (Orihuela et al., 2016). SIRT3 is a classical regulatory molecule of energy metabolism that is involved in the regulatory mechanism of a variety of immune cell phenotypes in previous studies (Nogueiras et al., 2012). Furthermore, HIF-1α, as an important target of SIRT3, has also been shown to be involved in the regulation of the immune cell phenotype (Nogueiras et al., 2012). Thus, we investigated whether SIRT3 and HIF-1α are involved in the regulatory effect of DHM on macrophage polarization. In vivo, the mRNA and protein expression levels of SIRT3 were notably increased in the EE + DHM-Lipo group compared with the EE group (p < 0.01, Figure 6A,C,D). However, the mRNA and protein expression of HIF-1α was increased in the EE group and decreased in the EE + DHM-Lipo group (p < 0.01, Figure 6B,C,E). As expected, comparable results were shown in the mRNA and protein expression levels of SIRT3 and HIF-1α in vitro. However, these benefits of DHM were abolished by treatment with 3-TYP, a SIRT3 inhibitor (Figure 5F–J), and Sirt3 siRNA (Supplementary Figure S2). Moreover, the DHM-induced elimination of the expression of the M1-like macrophage marker (iNos) and the upregulation of the expression of the M2-like macrophage marker (CD206, Fizz1, Ym1, and Il10) in LPS-treated RAW264.7 cells was abolished by treatment with 3-TYP (Figure 6K, L, Supplementary Figure S3). Collectively, these results demonstrate that DHM suppresses M1-like macrophages and upregulates M2-like macrophages by activating the SIRT3/HIF-1α signaling pathway.