PA stock solution was a gift from Good Doctor Pharmaceutical Group (Sichuan, China). P407 was purchased from H and C Fine Chemical (Shanghai, China) was purchased from BASF SE (Ludwigshafen, German). e-Poly-L-lysine (ε-PL) was purchased from Aladdin (Shanghai, China) and CMCS was procured from Yuanye Bio-Technology (Shanghai, China). Chondroitin sulfate (CS), Rhodamine B (RB) and Fluorescein isothiocyanate (FITC) fluorochrome were purchased from Solarbio (Beijing, China). DSS (MW 36–50 kDa) was purchased from MP Biomedicals (Santa Ana, CA, United States). LPS (E. coli serotype O55:B5) was purchased from Sigma-Aldrich (Missouri, United States). Mouse IL-6 (Interleukin-6), IL-1β, IL-18, and TNFα (Tumor necrosis factor α) ELISA (enzyme-linked immunosorbent assay) kits were purchased from Elabscience Biotechnology (Wuhan, China).

Primary antibodies for Zonula occludens-1 (ZO-1, ab96587), Claudin-3 (ab15102) and Occludin (ab216327) were procured from Abcam (Cambridge, UK). Primary antibodies for E-Cadherin (#3195), RIP1 (#3493), RIP3 (#13526), Phospho-RIP3 (#91702), MLKL (#14993), Phospho-MLKL (#37333), NF-κB p65 (#4764) and F4/80 (#30325) were purchased from cell signaling technology (Danvers, MA, United States). Primary antibody against NLRP3 (NBP2-12446) was purchased from Novus Biologicals (Littleton, Colorado, United States), Caspase-1 (p20) (AG-20B-0042-C100) was purchased from Adipogen (San Diego, United States) and beta-actin (AF7018) was purchased from Affinity (Melbourne, Australia). Anti-mouse (abs20001) and anti-rabbit (abs20002) IgG conjugated with horseradish peroxidase (HRP) were purchased from Absin (Shanghai, China) and CoraLite594 conjugated goat anti-rabbit IgG (SA00013-4) was purchased from Proteintech (Wuhan, China).

CCK8 assay kit was procured from Med Chem Express (New Jersey, United States). Immunohistochemical (IHC) staining kit was purchased from ZSJQ-BIO (Beijing, China). Hematoxylin and eosin (H&E) staining kit, DAPI and bicinchoninic acid (BCA) protein kit were purchased from Beyotime Biotech (Shanghai, China).

CCMTS: 53 mg CMCS was weighed and added into a 50 mL round-bottom flask, 10 mL purified water was added and dissolved. 25 mg N-hydroxysuccinimide and 21 mg 1-ethyl- (3-dimethylaminopropyl) carbonyl diimide hydrochloride were added and reacted for 2 h in dark. After adjusting the pH to 7.5 with 0.1 mol/L sodium hydroxide solution, 30 mg CS was added and the reaction was allowed to continue for 24 h. After pure water dialysis for 24 h (MW3500), the reaction was freeze-dried to obtain the polymer, chondroitin sulfate modified carboxymethyl chitosan (CCMTS). Certain amount of CCMTS, CMCS, and CS were dissolved in heavy water (D₂O) for ¹H NMR analysis (AVANCE Ⅲ, German).

P407-CHO: 100 mg P407, 366 mg p-aldehyde benzoic acid, 629 mg dicyclohexyl carbimide and 29.50 mg 4-dimethylaminopyridine were weighed, and then successively added to 50 mL double-necked round-bottom flask. Then 9.5 mL tetrahydrofuran was added and the reaction was stirred under nitrogen protection for 12 h. The filtrate of the reaction solution was dispersed in diethyl ether, and the precipitate was obtained by centrifugation. After repeating the above steps for three times, the precipitate was dried at 40°C to obtain aldehyde P407 (P407-CHO). The product was pressed with potassium bromide and analyzed by FT-IR (SHIMADZU, Japan). Certain amount of P407-CHO and P407 were dissolved in dimethyl sulfoxide for analysis by ¹HNMR.

Preparation: CCMTS, P407-CHO, and P407 were slowly added to purified water under stirring. They were allowed to stand until full swelling in the refrigerator at 4°C for 12 h. Then P188 solution, ε-PL solution and PA concentrate were added and PA/CCMTS-P was made by stirring. Among them, the mass concentration of P407 was 21%, P188 was 0.2%, and polylysine was 0.4%. The concentration of PA in PA/CCMTS-P was consistent with its concentration in the patented Chinese medicine Kangfuxin liquid.

Phase-transition temperature determination: 10 g PA/CCMTS-P TISG and a stirrer were put into a vial, and a precision thermometer with an accuracy of 0.1°C was inserted into it. The mercury sphere of the thermometer was completely immersed in the gel solution. The gel was placed at a low temperature (<10°C) water bath with a rotating speed of 300 rpm/min, and the water temperature raised continuously and slowly about 1°C–2°C/min. The temperature at which the stirrer stopped rotating completely was set as the gelation temperature, and the measured time was averaged after repeating the above procedure thrice.

Rheological characterization: At 37°C, the gelled PA/CCMTS-P was placed on the measuring mold of the rotary rheometer (Anton Paar, Austria). Firstly, the amplitude of the PA/CCMTS-P was scanned to determine the linear viscoelastic region, that is, the storage modulus and loss modulus of the sample were independent of the applied strain. After that, the storage modulus (G′) and loss modulus (G″) at different angular frequencies were tested at 37°C and in the measured linear viscoelastic region with a frequency range of 0.1–100 rad/s.

Raw264.7 cells were obtained from Cell Center of Chinese Academy of Sciences (Shanghai, China) and cultured in dulbecco’s modified eagle medium (DMEM, KeyGEN, Jiangsu, China) containing 20% fetal calf serum (Gibco, California, United States), 1% penicillin/streptomycin sulfate (Beyotime, Beijing, China) at 37°C in a humidified 5% CO₂ atmosphere.

Cell viability was assessed by the CCK8 assay. Raw264.7 cells were plated into 96-well plates (1 × 10⁴ cells/well) and stimulated with the PA stock solution at different concentrations (0.05, 0.1, 0.2, 0.4 mg/mL), with different dilutions of CCMTS-P (1:225, 1:450, 1:900, 1:1800) and at different concentrations of PA/CCMTS-P (containing PA stock solution 0.05, 0.1, 0.2, 0.4 mg/mL) for 24 h. Meanwhile, different concentrations of LPS (0, 25, 50, 100, 200, 400 ng/mL) were added to Raw264.7 cells for 24 h to identify the optimal stimulus concentration. 10μL CCK-8 solution was then added to each well. After incubating at 37°C for 1–2 h, the optical density (OD) values were measured at 450 nm using a scanning multi-well spectrophotometer (Biotek, United States).

Quantitative and qualitative cellular uptake was measured by flow cytometry (Agilent, United States) and fluorescent microscope (Olympus, Japan). Raw264.7 mouse macrophage cells were seeded into 12-well plates (1 × 10⁶ cells/well). After stimulation with LPS (100 ng/mL) for 24 h, fresh DMEM containing 450 times diluted CCMTS and 5 μg/mL RB were added. For the blank group, DMEM containing 5 μg/mL RB alone was added for 1 h. After incubation for 1, 2, and 4 h, the cells were washed twice with PBS, gently dissociated with plastic pipette, centrifuged at 1,500 g for 5 min, resuspended in 0.2 mL of PBS, and analyzed by flow cytometry. Furthermore, fluorescent microscope was used to observe the distribution of RB labeled CCMTS in Raw264.7 cells after incubation with RB@CCMTS for 1, 2, and 4 h. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 10 min and treated with DAPI for staining the nuclei. The images were collected by fluorescent microscope at ×200 magnification.

Female C57BL/6 mice (weight 18–21 g) were obtained and housed in the Medical Experimental Animal Centre of Xuzhou Medical University. All experiments were approved by the Ethics Committee of Xuzhou Medical University. The mice were randomly divided into six groups (6 mice per group), including NS, Model, PA (ig), CCMTS-P, PA/CCMTS-P (low dose) and PA/CCMTS-P (high dose) group. The NS group received no treatment, while the remaining five groups had free access to drink 3% DSS for 7 days before reverting to normal drinking water. On day 8, mice in the PA (i.g.) group were gavaged with 1:10 times diluted PA stock solution (5 μL/g per day). For the CCMTS-P group, 5 μL/g CCMTS-P was administered by enema per day. PA/CCMTS-P (low dose) and PA/CCMTS-P (high dose) groups were given 5 μL/g CCMTS-P containing PA stock solution 0.09 g/mL and 0.18 g/mL, respectively, by enema every day. The NS and Model groups were given normal saline (5 μL/g) every day. The body weights were monitored daily on fixed time. On day 12, all mice were sacrificed under anesthesia, and the colon tissue was measured and collected.

To further study the targeted permeability of CS-functionalized CCMTS-P into macrophages in vivo, the fluorescent probe FITC and F4/80 were used to label CMTS-P, CCMTS-P and macrophages, respectively. Then, 3% DSS-induced UC mice were injected with FITC@CMTS-P (no CS) or FITC@CCMTS-P (linking CS) through rectal enema administration. 2 h after enema administration, mice were sacrificed and their colon tissues were extracted, rinsed thoroughly with PBS, and embedded in optimal cutting temperature (OCT) compound. Colon sections (20 μm) were stained with F4/80 and DAPI. Images of the tissue sections were acquired by fluorescent microscope (Olympus, Japan).

The harvested colon tissues were fixed in 4% paraformaldehyde, embedded in paraffin and then cut into 3 μm sections. The tissues were stained with hematoxylin and eosin (H&E). Degree of inflammation and mucosal damage were assessed by Olympus microscope (Olympus, Japan) at ×40 and ×200 magnification in a blinded fashion.

Raw264.7 cells were seeded into 24-well plates (5 × 10⁵ cells/well), LPS (100 ng/mL) was added with PA (0.2 mg/mL) or PA/CCMTS-P (containing PA 0.2 mg/mL) for 24 h. Following stimulations, the supernatants were collected and stored at −80°C until further analysis. The expression of inflammatory factors including TNF-α, IL-6, IL-1β and IL-18, were measured in the supernatants using ELISA kits according to the manufacturer’s instructions. Similarly, the levels of IL-1β, and IL-18 in the colon tissue homogenate were measured based on the manufacturer’s instructions for the ELISA kits. Concentration of the cytokines were calculated using standard curves.

Total protein was extracted from the colon tissues after homogenizing and lysing the samples in matrix tubes (MP Biomedicals). The protein concentration was determined using the BCA protein kit. Equal amounts of protein extracts were separated on 10% SDS-polyacrylamide gels and trans-blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with skimmed milk at room temperature for 1 h and incubated overnight at 4°C with the respective primary antibodies (1:1,000 dilution). The next day, the membranes were incubated with the secondary antibody (1:5,000 dilution) for 2 h at room temperature. Finally, after washing in the TBS-T washing solution three times, the protein bands were visualized with an enhanced chemiluminescence detection system (Bio-rad, United States). The protein expression in each sample was normalized with respect to the loading control beta-actin, with the software ImageJ 1.47V (National Institutes of Health, United States).

Serial sections of embedded colon tissue from each group were used for IHC analysis by using IHC staining kit. After being deparaffinized, hydrated, and heated in citric acid buffer at 95°C for 15 min, the slides were blocked with 5% bovine serum albumin (BSA) for 30 min at 37°C and incubated overnight at 4°C with primary antibodies against ZO-1 (1:300), Claudin-3 (1:100), Occludin (1:200) and E-cadherin (1:400), respectively. Then the sections were incubated with the respective secondary antibodies at room temperature for 1 h before being visualized with diaminobenzidine. Images were assessed by Olympus microscopes (Olympus, Japan) at ×40 and ×200 magnification in a blinded fashion.

All data were analyzed using SPSS 23.0 software (IBM SPSS Statistics, NY, United States). The data are presented as the mean ± standard deviation. One-way analysis of variance was performed among the groups and p < 0.05 was considered to be statistically significantly different.

FT-IR profiles of P407 and P407-CHO are shown in Figure 1A. The C-H stretching vibration peaks of aldehyde group were located at 2,900, 2,838, and 1700 cm⁻¹ corresponded to the stretching vibration peak of the aldehyde carbonyl group, which suggested that the aldehyde group was successfully connected to P407 through reaction. As shown in Figure 1B, the proton peak of the benzene ring was linked to the aldehyde group at δ = 7.9 ppm, and δ = 10.0 ppm corresponded to the proton peak of the aldehyde group, which demonstrated the successful synthesis of P407-CHO. As shown in Figure 1C, δ = 1.88 ppm was the methyl proton peak linked to the double bond, which indicated the successful grafting of CS to CMCS and the successful synthesis of CCMTS.

The phase change temperature of PA/CCMTS-P was determined as (32.9 ± 0.4) °C (n = 3), higher than room temperature and slightly lower than the rectal temperature, which was suitable for enema administration.

Rheological property analysis: The hydrogel is subjected to cyclic shear stress from body fluids in the patients’ body. We tested the stability of the gel under cyclic stress by exposing to angular frequency scanning of the rheometer. The storage modulus (G′) and loss modulus (G″) of PA/CCMTS-P were tested, where G′ represented the elastic property and G″ represented the viscous property of the hydrogel. As shown in Figure 2A, in the whole frequency scanning range, G′ curve was always higher than the G″ curve, which showed that the hydrogel was obviously dominated by elastic properties. Besides, G′ was relatively stable without significant frequency dependence, indicating that the gel always had a stable three-dimensional network structure.

As shown in Figure 2E, LPS was used to stimulate Raw264.7 cells to establish an inflammation model in vitro (He and Frost, 2016). It was found that 25 and 50 ng/mL LPS did not affect the viability of Raw264.7 cells after 24 h treatment, and the cell viability decreased to nearly 90% at the concentration of 100 ng/mL. However, 200 and 400 ng/mL of LPS caused obvious cytotoxicity to Raw264.7 cells. Therefore, 100 ng/mL LPS was chosen to stimulate Raw264.7 cells for 24 h and to establish an in vitro inflammation model.

The cytotoxicity of PA stock solution, CCMTS-P and PA/CCMTS-P were tested in Raw264.7 cells after 24 h treatment. The results showed that there was change in the viability of RAW264.7 cells when the concentration of the PA stock solution was below 0.4 mg/mL. Similarly, PA/CCMTS-P containing below 0.4 mg/mL of PA stock solution did not induce cytotoxicity to Raw264.7 cells (Figures 2B, C). As shown in Figure 2D, 1:225 diluted solution of CCMTS-P had obvious cytotoxic effects on Raw264.7 cells. Hence, 0.2 mg/mL PA, PA/CCMTS-P, and 1:450 dilution of CCMTS-P were selected for cellular uptake and inflammatory cytokines tests.

To investigate whether the higher anti-inflammatory effect of CCMTS was related to its ability to target of macrophages, RB was used to label CCMTS. As shown in Figure 3A, the fluorescence of free RB in Raw264.7 cells was weak and almost invisible, while the fluorescence signal in the CCMTS group was bright and intense. Cellular uptake tests in vitro showed that in comparison with the blank group, CCMTS was internalized more effectively by Raw264.7 cells after LPS stimulation. Meanwhile, the fluorescence intensity in Raw264.7 cells increased with longer incubation time, indicating that the uptake ability of CCMTS was time-dependent. After 4 h, the accumulation of RB labeled CCMTS in nucleus was significantly higher than that at 2, 1 h, which suggested the time-dependent uptake of CCMTS by Raw 264.7 cells through endocytosis. Similarly, the results of the flow cytometry histogram showed that the uptake of RB labeled CCMTS by Raw 264.7 cells was also time-dependent. As shown in Figure 3B, the flow intensity of CCMTS was significantly higher than that of the blank group, and the flow intensity was remarkably increased after 4 h (vs. 2 h, 1 h and blank group, p < 0.01).

To verify whether FITC labeled CS-functionalized CCMTS-P could specifically penetrate into macrophages in the colitis tissues, the frozen sections were immunostained with the F4/80 antibody. As shown in Figure 3C, the red fluorescence of the F4/80 antibody showed the location of macrophages and the green fluorescence of FITC showed CMTS-P and CCMTS-P. F4/80 was only rarely distributed in the colon tissue of control group, while in the other three groups, the fluorescence intensity was brighter and stronger. In the FITC@CCMTS-P group, FITC was highly overlapped with the red fluorescence signal of F4/80, and the fluorescence intensity was brighter than that of the FITC@CMTS-P group. These results indicated that FITC@CCMTS-P had better selectivity for colon macrophages than FITC@CMTS-P, mainly because of the ability of CS to specifically conjugate to the CD44 receptor on macrophages. The above results confirmed that CCMTS-P had good macrophage and colon targeting ability.

Based on the role of DSS in UC induction, we explored the protective efficacy of PA/CCMTS-P in DSS-induced mouse model of UC. The body weight of the mice was recorded daily. As shown in Figure 4A, the body weight in the model group revealed a dramatic decrease after 7 days of 3% DSS drinking, while treatment with PA/CCMTS-P significantly improved the weight loss caused by DSS. Meanwhile, on day 12, compared with the PA (i.g.) and CCMTS-P groups, high dose of PA/CCMTS-P showed better results on weight loss. Colon length reflects the severity of inflammatory infiltration in the colon and is also an important indicator for evaluating the efficacy of UC treatment strategies. The images of the collected colon samples showed that the colon length of the model group was significantly shortened. While in the PA/CCMTS-P high dose group, after 5 days of therapy, colonic shortening was effectively alleviated and there was no obvious difference in the colon length between the NS and PA/CCMTS-P high dose group (Figures 4B, C).

H and E staining reflects the inflammatory injury and epithelial integrity of colon tissues. As shown in Figure 4D, the model group mice showed severe gland disappearance, inflammatory cell infiltration, obvious edema and extensive necrosis of the intestinal crypt. However, the administration of PA/CCMTS-P effectively protected the structure of the crypts with better epithelial integrity, fewer ulcers and milder inflammatory cell infiltration. Moreover, the PA/CCMTS-P high dose group had the most obvious protective effects on colon epithelial cells relative to the PA (i.g.), CCMTS-P and PA/CCMTS-P low dose group. These results showed that PA/CCMTS-P alleviated inflammation and accelerated wound healing after DSS injury in mice.

The tight connectivity of intestinal tight junctions (TJs) and adherent junctions (AJs) proteins are important for the formation of an intact intestinal immune barrier. An intact intestinal mucosal barrier prevents harmful substances such as bacteria or toxins from entering other tissues or blood circulation through the intestinal mucosa. Destruction of the intestinal barrier function is the typical clinical manifestation of UC (Mehandru and Colombel, 2021). ZO-1 is one of the representative tight junction proteins connected with the cytoskeleton protein, and Occludin plays a vital role in the formation and regulation of TJs and AJs. Claudin-3 plays a major role in TJ-specific obliteration of the intercellular space. In addition, E-Cadherin belongs to a superfamily of transmembrane glycoproteins, which mediates calcium-dependent cell-cell adhesion and plays a critical role in normal tissue development (Yin Y et al., 2022). These four indexes reflect the integrity and permeability of the intestinal barrier. The immunohistochemistry results are shown in Figures 5A, B. Compared with the NS group, the expression levels of ZO-1, Occludin, Claudin-3 and E-Cadherin were significantly decreased in the model group, indicating that the intestinal barrier was seriously damaged. While PA/CCMTS-P (low and high dose) dramatically increased the expression levels of these four proteins in the DSS-induced UC colon, and the effect of high dose PA/CCMTS-P group was higher than that in the PA (i.g.) and CCMTS-P treatment groups, indicating that PA/CCMTS-P treatment was effective at protecting the intestinal mucosal barrier.

Inflammation is a typical manifestation of UC and the imbalanced secretion of inflammatory factors is closely associated with the occurrence and development of UC. To demonstrate the anti-inflammatory effects of PA and PA/CCMTS-P, LPS stimulated Raw264.7 cells were used as inflammation model in vitro. As shown in Figure 6A, after stimulation with LPS for 24 h, the levels of proinflammatory cytokines such as TNFα, IL-6, IL-18 and IL-1β were obviously increased, while PA and PA/CCMTS-P had good regulatory effects on these proinflammatory factors. Both PA and PA/CCMTS-P significantly reduced the levels of TNFα, IL-6, IL-18, and IL-1β. Additionally, PA/CCMTS-P was more effective than PA at decreasing the level of IL-1β (p < 0.01). The above results showed that PA/CCMTS-P had excellent anti-inflammatory effects in vitro.

Since inflammatory cytokine expression plays an essential role in the pathogenesis of UC and the severity of UC is directly related to the number of inflammatory cytokines, we studied the anti-inflammatory effects of PA/CCMTS-P in the DSS-induced UC model. As shown in Figure 6B, in the Model group, IL-1β and IL-18 were significantly increased compared to the NS group (p < 0.01), while the levels of IL-1β and IL-18 in the DSS-induced UC model were significantly reduced after PA/CCMTS-P treatment. Furthermore, PA/CCMTS-P showed higher anti-inflammatory effects as compared with the PA (ig) and CCMTS-P groups (p < 0.01) and this was dose-dependent. These results were consistent with the anti-inflammatory findings in vitro.

In order to determine whether PA/CCMTS-P was involved in the modulation of necroptosis-mediated inflammasome signaling, we measured the expression of several key proteins in colon tissues through Western blotting. As shown in Figures 7A, B, significant differences were observed in expression of NF-κB p65, NLRP3, Caspase-1 (p20), RIP1, RIP3, and MLKL between the NS and Model group. (p < 0.01). However, PA/CCMTS-P treatment remarkably decreased the expression of NF-κB p65, NLRP3, Caspase-1 (p20), RIP1, RIP3 and MLKL (p < 0.01). Compared with the PA (i.g.) and CCMTS-P groups, PA/CCMTS-P treatment reduced the expression of NF-κB p65, NLRP3, Caspase-1 (p20), RIP1, RIP3, and MLKL more effectively, and this effect was dose-dependent. MLKL is a key player in necroptotic signaling, and its phosphorylation is a specific marker of necroptosis activation (Wang H et al., 2014). As shown in Figures 7A, B, the level of RIP3 and MLKL phosphorylation in the model group were significantly increased in comparison to the control group. Additionally, compared with the Model, PA (ig) and CCMTS-P groups, the expression level of phosphorylated RIP3 and MLKL were significantly reduced in the PA/CCMTS-P group in a dose-dependent manner. All these results revealed that PA/CCMTS-P strongly inhibited RIP1/RIP3/MLKL mediated necroptosis as well as NF-κB and NLRP3 inflammasome activation in UC mice model, which further alleviated inflammation in the DSS-induced UC model.