Phospholipid complex-based microemulsion for treating concurrence of primary sclerosing cholangitis and inflammatory bowel disease via gut-liver crosstalk
Yihao He , Ru Guo , Lu Yang , Ling Li , Tong Zhang , Bing Wang , Yongzhuo Huang
Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver and biliary system illness that can significantly shorten life expectancy and often require liver transplantation. It was declared one of the biggest unmet needs in hepatology at the International Liver Congress 2016 [1]. Approximately 50% of individuals with inflammatory bowel disease (IBD) will experience extraintestinal manifestations at some point in their lives, which can occasionally include PSC [2]. PSC is an infrequent, chronic liver condition marked by the inflammatory degeneration of bile ducts within and outside the liver, leading to progressive hepatic disease [3]. PSC prevalence is roughly 10 cases per 100, 000 individuals [4]. Notably, between 60% and 80% of those diagnosed with PSC also suffer from IBD (referred to as PSC with concurrent IBD). On the other hand, the incidence of PSC among patients with IBD is estimated to range from 0.5% to 8.1% [5, 6]. In patients with PSC associated with IBD, over 80% have PSC-ulcerative colitis (UC), approximately 10% have PSC—Crohn's disease (CD), and another 10% have indeterminate colitis [7]. In the liver, genetic susceptibility and immune-mediated pathways are primarily involved in the pathogenesis of PSC, and bile acid (BA) homeostasis is impaired [8]. Changes in BAs directly affect the gut microbiota, leading to ecological imbalance and increasing the passage of microorganisms and toxins through the damaged intestinal barrier. Conversely, intestinal leakage can cause inflammation of the bile ducts and liver, and microbial imbalance further exacerbates liver inflammation due to BA disorders [9]. PSC-IBD is characterized by increased intestinal permeability and pro-inflammatory responses in the liver, including the polarization of naïve T cells to Th17 [10]. Additionally, the abnormal migration of "gut-tropic" lymphocytes contributes to inflammation and fibrosis in the liver and bile ducts [10,11]. There is currently no effective treatment for PSC-IBD; existing medications for individual PSC or IBD, including ursodeoxycholic acid (UDCA), obeticholic acid (OCA), and 5-aminosalicylic acid (5-ASA), have limited therapeutic effects and significant side effects [12-14], highlighting the urgent need for more effective therapies for PSC-IBD.
Paeoniflorin (PAE) is the main active compound derived from Paeonia lactiflora Pall., which is a Chinese traditional medicine clinically used for over 1000 years. PAE exhibits therapeutic effects on IBD by repairing the intestinal barrier and reducing inflammation [15,16], and has shown antioxidant effects and benefits in reducing hepatocyte damage and bile duct inflammation [17]. Macrophages, integral to various tissues, play an important role in inflammatory responses, immune regulation, and antigen presentation [18]. In PSC, they are recruited to the biliary microenvironment and promote the occurrence of injury and cholestasis [19]. Initial inflammatory responses in PSC promote macrophage infiltration around the bile duct [19]. During PSC-IBD, the intestinal barrier damage, including alterations in the mucus layer and disruptions in intercellular connections, allows intestinal pathogen-associated molecular patterns (PAMPs) to enter the liver through the lymphatic vessels or biliary tract, thereby stimulating Kupffer cells and macrophages to release chemokines/cytokines and induce cholangitis [20,21]. Therefore, given the mutual actions of macrophages on PSC-IBD, they thus can be a therapeutic target.
PAE can suppress pro-inflammatory M1 macrophages while enhancing M2 macrophage activity [22-24]. Therefore, we propose that PAE is a potential therapeutic candidate for PSC-IBD. However, PAE is a highly water-soluble compound with low membrane permeability (biopharmaceutics classification system (BCS) class Ⅲ) and poor bioavailability (reportedly only 3.6%) [25]. To address this issue, phospholipid (PL) complex (PLC)-based microemulsions were applied to improve PAE's membrane permeability. PLs, amphiphilic molecules, and a crucial component of cell membranes can bind with various phytomolecules via complex, and PLC is a useful technique for enhanced delivery of phytomedicine [26]. The drug/PLC rely on hydrogen bonds and/or hydrophobic interactions [27]. In the form of a PLC, drugs exhibit different physical and chemical properties, including alterations in oil-water partition coefficient, solubility, water dispersion properties, and phase transition characteristics [28]. As a result, the pharmacokinetic profiles (e.g., biodistribution and cell permeability) of drugs change accordingly.
Monocytes recruited to the liver differentiate into macrophages at inflammation sites [29, 30]. Suppression of this recruitment and differentiation can alleviate PSC-like liver injury [19]. Notably, the liver macrophages can actively engulf nanoparticles at the site of inflammation [31]. It provides a basis for drug-targeting delivery to macrophages. In this work, we prepared the PAE-PLC. Then we constructed an oil-in-water microemulsion system (PAE-ME), with PAE-PLC encapsulated into the oil inner phase. It aimed to address the low permeability of PAE and achieve macrophage-targeting delivery. It was expected that PAE-ME could regulate the imbalance of chronic intrahepatic BA accumulation, maintain intestinal epithelial barrier function, and carry out therapeutic effects on the liver-gut axis.
The microemulsion system of PAE-PLC primarily comprises PAE, PL, surfactants, and co-surfactants (Fig. 1a). Soybean PLs exhibited no absorption peak in the 200–400 nm wavelength range. The ultraviolet (UV) spectra of PAE, PAE/PL physical mixture (PAE/PM), and PAE-PLC were similar, with absorption peaks at 230 nm (Fig. 1b). This similarity suggests that the chromophore groups of PAE did not change after preparation into the PLC. Moreover, in the Fourier transform infrared spectroscopy (FTIR) spectra, the spectrum of PAE displays characteristic peaks at 1470 cm−1 (C═C, aromatic ring stretching vibration) and 1713 cm−1 (C═O, carbonyl stretching vibration). The spectrum of PL shows peaks at 1736 cm−1 (C═O stretching vibration), 1217 cm−1 (P═O stretching vibration), and 1086 cm−1 (P-O-C stretching vibration). When comparing the absorption peaks of PAE/PM with those of PAE-PLC, the peak for the carbonyl group of PAE at 1713 cm−1 shifts to a higher wavenumber in PAE-PLC, moving to 1725 cm−1. Similarly, the PL peak at 1736 cm−1, corresponding to the C═O stretching vibration, shifts to a lower wavenumber at 1725 cm−1 (Fig. 1c). Additionally, it is observed that the shape and width of the absorption peaks at 1281 and 1077 cm−1 in PAE-PLC undergo certain changes, indicating that some weak physical interactions occur between PAE and PL during the composite formation. X-ray diffraction (XRD) experiments revealed that the diffraction pattern of PAE displays strong diffraction peaks at 12.5°, 14.9°, 17.1°, 18.7°, and 20.4°, indicating a highly crystalline structure of PAE. In contrast, no obvious crystalline peaks were observed in the PL, indicating that the PL structure is amorphous. The XRD pattern of PAE/PM shows mainly the crystalline peaks of PAE, but the peak intensity is significantly reduced. Compared to PAE-PLC, the crystalline peaks of PAE disappear in the composite, indicating a significant change in the crystal structure of PAE (Fig. S1b in Supporting information). It indicated the formation of PAE-PLC. The differential scanning calorimetry (DSC) analysis indicates that the DSC curve of PAE (Fig. S1c in Supporting information) exhibits a sharp exothermic peak at 118 ℃, suggesting that the carbon chains in PAE may have undergone melting, isomeric changes, or crystallization. In contrast, the DSC curve of PAE-PLC shows no discernible peaks, indicating no PAE crystallization within the PAE-PLC; instead, PAE is likely dispersed molecularly or amorphously within the PL molecules.
The PAE-ME was prepared, with a clear and transparent appearance with blue opalescence. The mean size of the PAE-ME was 51.69 nm (Figs. 1d and e) with a polymer dispersity index (PDI) of around 0.13, and the zeta potential was −1.67 mV (Figs. 1f and g). The transmission electron microscopy results showed spherical or sphere-like particles (Fig. 1h). The drug-loading capacity (DL%) and encapsulation efficiency (EE%) of PAE-ME were measured to be 5.8% and 89%, respectively (Table S1 in Supporting information). The microemulsion exhibited good stability in phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) and a sustained-release pattern (Figs. 1i and j).
PAE-ME showed high biocompatibility with macrophages, maintaining cell viability above 80% even at a high concentration of 25 µmol/L (Fig. 2a). At 12 h, the intracellular drug concentration in the PAE-PLC group is 2.9 times that of the PAE group (Fig. S2a in Supporting information). M1$\mathit{Φ}$ cells treated with coumarin 6 (Cou-6)-labeled PAE-ME exhibited a significantly higher mean fluorescence intensity (MFI) than those treated with free Cou-6 solution (Figs. S2b and c in Supporting information), demonstrating that PAE-ME significantly facilitated cellular uptake of the encapsulated agent. The PAE-PLC significantly increases the concentration and uptake efficiency of PAE in macrophages, thereby effectively improving the permeability of PAE and achieving targeted delivery to macrophages. PAE showed the ability to suppress pro-inflammatory M1-type macrophages, as evidenced by the down-regulation of M1-associated genes (e.g., CD86 and C—C chemokine receptor 7 (CCR7)) (Figs. 2b and c). Furthermore, PAE-ME facilitated M1 → M2 repolarization of macrophages (Fig. 2d), indicated by the up-regulation of an M2-related marker CD206 (Fig. 2e) and down-regulation of M1-related markers CD86 and inducible nitric oxide synthase (iNOS) (Figs. 2f and g). Western blot results further confirmed this repolarization effect (Fig. 2h). The flow cytometric analysis showed that PAE-ME treatment increased the percentage of F4/80+CD206+ macrophages by 16% (Figs. S3a and b in Supporting information).
Following a 24-h treatment with PAE-ME, mRNA levels of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) in M1 macrophages were significantly reduced (Figs. 2i–k). By contrast, the levels of anti-inflammatory transforming growth factor beta (TGF-β) were up-regulated (Fig. 2l). M1 macrophages exhibited elevated reactive oxygen species (ROS) levels, which were reduced after PAE-ME treatment (Fig. S4 in Supporting information). These findings indicate that PAE-ME exerts anti-inflammatory effects through cytokine modulation and ROS scavenging.
Transcriptome data of autoimmune cholestatic liver disease and IBD patients were analyzed using bioinformatics approaches on the gene expression omnibus (GEO) database. The gene set enrichment analysis (GSEA) analysis and immune cell infiltration analysis were performed on these datasets. Gene set enrichment analysis revealed that both PSC and IBD patients showed enrichment in two identical inflammatory pathways, namely IL-6_JAK_STAT3_SIGNALING and TNFα_SIGNALING_VIA_ NF-κB, similar to healthy controls (Figs. S5 and S6 in Supporting information). Single-sample gene set enrichment analysis (ssGSEA) showed the immune cell infiltration differences in 28 immune cell-related pathways between the PSC and IBD groups compared to healthy controls. Statistical analysis using the Wilcoxon test with false discovery rate (FDR) correction revealed the differences in immune pathway enrichment scores between patient and healthy control groups; specifically, PSC patients have higher levels of eosinophils, macrophages, memory B cells, and Th17 cells in their blood samples than healthy individuals. By contrast, natural killer cells, central memory CD4+ T cells, and effector memory CD8+ T cells are relatively low in PSC patients (Fig. S5c). Similarly, IBD patients have a higher proportion of neutrophils, dendritic cells, memory B cells, and Th17 cells in their blood samples than healthy individuals, while γδT cells, follicular helper T cells, Th2 cells, MDSCs, natural killer cells, natural killer T cells, and effector memory CD8+ T cells are relatively low (Fig. S5b). The results indicate that PSC and IBD share similar immune profiles, which may play a role in inflammation regulation and provide a clue for investigating how PAE treats PSC-IBD.
An in vivo study was conducted to further investigate the effects of PAE-ME on PSC and IBD (Fig. 3a). The animal feed formulation is shown in Table S2 (Supporting information). All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Shanghai Institute of Materia Medica Chinese Academy of Sciences, and experiments were approved by the Animal Ethics Committee of the Shanghai Institute of Materia Medica. The results indicated that after 2-week treatment with PAE-ME, there was a significant amelioration of PSC-IBD symptoms, with a reduction in both the body weight loss (Fig. 3b) and disease activity index (DAI) (Fig. 3c). Figs. 3d and e show the appearances of livers and gallbladders after treatment in different experimental groups. Colon length shortening, indicative of colitis severity, was notably less in the mice treated with PAE-ME compared to the untreated colitis mice (Figs. 3f and g).
Histological examination showed mucosal epithelium injury and ulcer formation in the colon segments of the untreated colitis mice, along with increased neutrophil infiltration in the ulcer and loss of crypts in the lamina propria. PAE-ME treatment effectively protected against colon epithelium damage and reduced inflammatory cell infiltration (Fig. 4a). Furthermore, immunofluorescence results indicated decreased expression of tight junction protein zonula occludens-1 (ZO-1) in the colon tissue of untreated mice compared to the normal group (Fig. 4b). However, ZO-1 expression was upregulated after PAE-ME treatment, signifying the reparative effect on the intestinal mucosal barrier.
Serum alkaline phosphatase (ALP) is an indicator of the severity of PSC [32]. In this study, serum ALP levels were significantly elevated in the PSC model group but decreased after PAE-ME treatment (Fig. 4c). Following intravenous administration in mice, the drug concentration in liver tissue for the PAE-ME group was significantly higher compared to the PAE group. Notably, at 12-h post-administration, the concentration in the PAE-ME group was 4.5 times greater than that observed in the PAE group (Fig. S7 in Supporting information).
TNF-α acts as a signaling molecule in cholangitis inflammation [33]. Western blot results revealed decreased IL-6 and TNF-α levels in the liver tissue of PSC-IBD mice after PAE-ME treatment (Fig. 4e). Enzyme-linked immunosorbent assay (ELISA) results demonstrated significant reductions in serum TNF-α levels in the PAE-ME treatment group (Fig. 4d), suggesting superior anti-inflammatory effect compared to PAE. Furthermore, PAE-ME displayed an evident inhibitory effect on the NF-κB p65 expression in the liver (Fig. 4e). These findings suggest that PAE-ME can suppress the TNF-α/NF-κB pathway in the liver of PSC-IBD mice and regulate inflammatory responses.
The liver tissues of the PSC model mice exhibited luminal porphyrin plugs, slight periportal ductular reaction, and inflammatory cell infiltration (Fig. 4f). PAE-ME treatment showed significant improvement in liver damage, with reduced inflammatory cell infiltration in the portal tract, decreased liver cell necrosis, and suppressed bile duct reaction. Sirius red staining highlighted fibrosis in the liver tissues of the PSC model group but showed reduced collagen deposition after treatment. Immunohistochemical results demonstrated that PAE-ME decreased the expression of CK19 induced by the DDC modeling agent, indicating its suppressive effect on the proliferation of biliary epithelial cells.
The biological safety of PAE-ME was evaluated preliminarily. The organ coefficients showed no significant difference among all groups (Fig. S8 in Supporting information). HE-staining results displayed that the hearts of mice treated with PAE and PAE-ME were normal without any apparent pathological changes (Fig. S8). The white and red pulp structure of the spleen was intact, and there was no damage to the capsule, trabeculae, and lymphoid tissue. The morphology of the lungs and kidneys appeared normal, with no signs of tissue inflammatory reaction, abnormal proliferation, or pathological changes. These results indicated good biosafety.
BAs constitute the key elements of the bile and are essentially a set of cholic acids synthesized from cholesterol in the liver. These acids can trigger bile secretion, while deoxycholic acid and ursodeoxycholic acid can unclog bile ducts and promote liver cells to secrete bile, consequently eliminating stasis. The metabolism of BAs is moderately associated with biliary damage [34]. We explored the role of BAs in PSC-IBD treatment with PAE-ME by incorporating targeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) techniques. By using principal component analysis (PCA), we developed the models for samples from each group and observed a clustering phenomenon among sample points within each group (Fig. S9a in Supporting information). This finding suggests that there are certain discrepancies in the metabolic profiles of sample groups, including the normal group, model group, PAE treatment group, and PAE-ME treatment group. The BA contents in each sample were computed, and a stacked column chart displaying the top 20 BAs in order of content was constructed (Fig. S9b in Supporting information). The analysis indicated that there were variances in the metabolic composition of liver tissues among the diverse groups. A box plot to visualize the BAs showed significant differences, as identified through univariate statistical analysis and meeting the established criteria. BAs that significantly increased in the liver tissues of the model group, as compared to the normal group, include tauro-alpha-muricholic acid (T-α-MCA), tauro-beta-muricholic acid (T-β-MCA), thyronine conjugated cholic acid (THCA), taurocholic acid (TCA), thyronine conjugated deoxycholic acid (THDCA), tauroursodeoxycholic acid (TUDCA), beta-muricholic acid (β-MCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), beta-ursodeoxycholic acid (β-UDCA), and nor-cholic acid (NorCA). Compared to the PSC-IBD model group, PAE and PAE-ME notably reduced the levels of BAs, such as T-α-MCA, THCA, CDCA, TCDCA, β-UDCA, and NorCA (Fig. S9a). These findings suggest that PAE and PAE-ME can potentially regulate abnormal BA metabolism under inflammatory conditions.
Traditional Chinese herbal medicine contains numerous highly water-soluble drugs, most of which encounter challenges related to poor permeability, short half-life, and low bioavailability [35,36]. To address these issues, those active compounds can be subjected to structural modifications, PLC, or nanocarrier technology to alter their structural characteristics and physicochemical properties [25,37]. Among them, phospholipid complexation technology is beneficial for its easy preparation and scalability. Phospholipids can interact with functional groups like hydroxyl, carbonyl, and amino groups of many natural product compounds via hydrogen bonds, thereby forming phospholipid complexes and enhancing their biopharmaceutical properties. The complexes not only act as prodrugs but can also be assembled into vesicles.
In order to overcome the low lipophilicity of PAE, the PAE-PLC is prepared. Optimally increasing the proportion of PLs enhances the permeability of PAE and prevents drug leakage [38]. The amphiphilic nature of PAE-PLC facilitates the transportation and permeation of drug molecules through biological membranes [39]. The PLC tends to dissolve in the oil phase and be encapsulated by oil droplets, forming an oil-in-water microemulsion, in which the PLC engages with the aqueous phase via its phosphoric head group, while the hydrophobic fatty acyl group interacts with oil phase, thereby stabilizing the phase interface and facilitating emulsification. Microemulsions in the bloodstream can be captured by the reticuloendothelial system, potentially achieving a liver-targeted effect [40]. Moreover, due to the phagocytic activity of inflammatory macrophages in the liver, PAE-ME may accumulate at the site of biliary lesions to exert an effective therapeutic outcome.
In PSC-IBD, there is a significant accumulation of macrophages around the bile ducts, which triggers inflammatory responses [41]. OCA and 5-ASA are anti-inflammatory medications employed in the treatment of PSC and IBD, respectively [13,42]. OCA is a farnesoid X receptor agonist commonly used to treat various liver diseases. However, there is no approved drug for PSC, but in a Phase Ⅱ study of OCA in PSC, it was found to reduce disease severity during the initial 24 weeks of treatment [13]. 5-ASA is a commonly used drug for the treatment of IBD. PAE-ME showed an improved therapeutic outcome compared to the combo of OCA and 5-ASA in the PSC-IBD mice. It implied the therapeutic potential of PAE-ME through immunomodulation.
The increased ratio of M1/M2 macrophage phenotypes is positively correlated with the severity of intestinal inflammation [43]. Targeting macrophages has become a leading strategy for immune imbalance-related diseases [44]. ROS serves as a critical nexus of cellular homeostasis and ROS regulation is a useful approach for modulating the immune microenvironment [45,46]. BAs increase intracellular ROS levels, thereby triggering bile duct cell proliferation [47]. We hypothesize that PAE may reduce the production of ROS in the bile duct epithelial cells with cholestasis, and inhibit the proliferation and inflammation of bile duct cells.
IBD is a chronic, recurrent condition characterized by inflammation of the gastrointestinal tract and encompasses two primary subtypes: CD and UC [48]. The predominant features of UC include diarrhea, mucosal ulceration, and rectal bleeding [49]. The loss of barrier integrity is distinctive in IBD [50-52]. Altered expression of tight junction proteins has been observed in patients with IBD [53]. Notably, PAE-ME can upregulate the expression of intestinal epithelial tight junction protein ZO-1. In conjunction with intracellular signaling proteins, tight junction proteins activate numerous cellular processes to preserve the integrity of the barrier [54]. However, damage to the intestinal barrier from inflammation has been found to have a protective effect on the liver [55]. This novel perspective sheds light on the longstanding association between PSC-IBD and provides robust empirical evidence for clinical research on multi-organ treatment strategies for PSC.
The synthesis and regulation of BAs offer promising approaches for treating cholestatic liver diseases. A variety of BAs, such as TCA, deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), chenodeoxycholic acid (CCDC), and taurolithocholic acid (TLCA), can stimulate liver cells to release cytokines, thus initiating an inflammatory response [56]. Liver inflammation triggered by chronic colitis is mitigated by inhibiting BA synthesis, thereby reducing bile stasis-related liver damage and bile duct fibrosis [55]. The abnormal metabolism of BAs can directly trigger necrosis or apoptosis of bile duct cells and hepatocytes, with toxic BAs playing a particularly prominent role in this process. In the PSC-IBD model, there is a notable increase in the levels of TCDCA in liver tissue. TCDCA is a conjugated BA formed by combining CDCA and taurine, and high levels can induce cholestasis-related hepatocyte apoptosis [57]. The transport of these BAs across the PL bilayer of cell membranes into hepatocytes and intestinal epithelial cells primarily relies on transporter proteins, including organic solute transporter alpha/beta (OSTα/β), sodium taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2) [58]. PAE-ME can effectively ameliorate the accumulation of various BAs in the livers of PSC-IBD mice, potentially due to their regulatory effects on BA transport proteins.
We have developed a microemulsion drug delivery system for the water-soluble drug PAE modified with soybean-derived PLs. The microemulsion we prepared exhibits a consistent particle size, excellent dispersion, and stability, with the additional benefit of sustained-release properties. In vitro, experiments have demonstrated that PAE-ME effectively inhibits the production of reactive oxygen species and inflammatory cytokines in macrophages. Furthermore, in vivo, pharmacological studies using a mouse model of PSC-IBD have shown that both PAE and PAE-ME alleviate liver-gut injury, effectively inhibit colon shortening, and improve the pathological condition of the liver and intestine. Moreover, PAE-ME can regulate disrupted levels of BAs caused by biliary inflammation, thereby optimizing the therapeutic efficacy within this specific mouse model. These significant findings pave the way for new strategies and disease models in drug delivery for the treatment of PSC-IBD.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Yihao He: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Ru Guo: Methodology, Investigation. Lu Yang: Methodology, Investigation. Ling Li: Methodology, Investigation. Tong Zhang: Writing – review & editing, Supervision, Resources, Conceptualization. Bing Wang: Writing – review & editing, Supervision, Project administration, Conceptualization. Yongzhuo Huang: Writing – review & editing, Writing – original draft, Supervision, Formal analysis, Conceptualization.
We are thankful to National Key Research and Development Program of China (No. 2022YFE0203600, China), National Natural Science Foundation of China (Nos. 82341232, 81925035), Department of Science and Technology of Guangdong Province (No. 2021B0909050003), and Chinese Academy of Sciences President's International Fellowship Initiative (No. 2024VBB0004), and the Scientific Innovation Group Project in Zhongshan (No. CXTD2022011). This work was also supported by grants from the Program of Shanghai Committee of Science and Technology, China (No. 22S21902900).
Supplementary material associated with this article can be found, in the online version, at doi:
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Figure 1 Characterization of PAE-ME. (a) Schematic illustration of PAE-ME. (b) Ultraviolet absorption spectrum. (c) FTIR spectra of PAE, PL, PAE/PM, and PAE-PLC. (d, e) Size distribution of PAE-ME. (f, g) PDI and ζ potential measurement. (h) Cryo-electron microscopy (Cryo-EM) of PAE-ME. Scale bar: 200 nm. (i) Stability of PAE-ME in a serum-containing PBS. (j) In vitro release of PAE from PAE-ME. All data are expressed as mean ± standard deviation (SD) (n = 3).
Figure 2 PAE-ME promoted macrophage repolarization from M1- to M2-like phenotype exhibited anti-inflammatory effects. (a) MTT assay of RAW264.7 cells incubated with PAE and PAE-ME. The mRNA levels of CCR7 (b), and CD86 (c). (d) Illustration of macrophage repolarization after PAE-ME treatment. (e–g) The mRNA level of M1 macrophage markers (CD86 and iNOS) in the activated macrophages and M2-related markers (CD206). (h) PAE-ME upregulates CD206 and downregulates CD86 protein. (i–k) The down-regulation of IL-6, IL-1β, and TNF-α in M1 macrophage after treatment. (l) The mRNA levels of anti-inflammatory cytokines in the drug-treated M1 macrophage. Data are presented as mean ± SD (n = 3). ns, no significance. P < 0.05, **P < 0.01, ***P < 0.001. PAE (15 µmol/L); PAE-ME (equal to 15 µmol/L PAE).
Figure 3 In vivo treatment study. (a) Schematic illustration of DDC/DSS-induced PSC-IBD in C57BL/6 mice and the treatment regimen (a part of this illustration drawn by Figdraw). (b) Changes in body weight during the treatment. (c) DAI record. (d) Images of livers. (e) Images of gallbladders. (f) Representative colon tissues of each group. (g) Statistical analysis of colon length. Data are presented as mean ± SD (n = 5). P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4 Anti-inflammatory mechanisms. (a) HE-stained histological sections of colonic tissues. The panels (scale bar: 250 µm) show the loss of surface epithelium marking ulceration (blank arrowhead), accompanied by inflammatory cell infiltration (red arrowhead). (b) Immunofluorescence showing the colon tissue expression of ZO-1. Scale bar: 100 µm. (c) Levels of ALP in serum. (d) Levels of TNF-α in serum. (e) The liver protein expression of IL-6, TNF-α, P-p65, and p65 after one week of PAE and PAE-ME. (f) Sirius red staining and HE staining for light microscopy analysis of liver morphology, and immunohistochemical staining showing the liver tissue expression of CK19. Scale bar: 250 µm. Data are presented as mean ± SD (n = 3). P < 0.05, **P < 0.01. DAPI, 4′, 6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IκB, inhibitor of kappa B; TNFR, tumor necrosis factor receptor.
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