Antibodies and Reagents

The molecular weight of the DSS, which was between 36,000 and 50,000, was given by MP Biomedicals LLC. The main antibodies that were used were: Sources from which the following reagents were procured: Atg12 antibody (#4180T, Cell Signaling Technology), p62 antibody (#5114T, Cell Signaling Technology), Beclin-1 antibody (#3495T, Cell Signaling Technology), LC3A/B antibody (#12741T, Cell Signaling Technology), β-actin antibody (#4970T, Cell Signaling Technology), β-Tubulin antibody (#2128, Cell Signaling Technology), JAK2 antibody (ab108596, Abcam), Phosphorylated JAK2 antibody (P-JAK2, ab32101, Abcam), STAT3 antibody (ab68153, Abcam), Phosphorylated STAT3 antibody (P-STAT3, ab267373, Abcam), MUC2 antibody (ab272692, Abcam), Claudin-1 antibody (ab211737, Abcam), Occludin antibody (ab222691, Abcam), ZO-1 antibody (ab216880, Abcam), Elabscience (Wuhan) was also the source for the following ELISA kits: The following ELISA kits were purchased from Elabscience in Wuhan: Mouse TNF-α (E-EL-M3063), Mouse Interleukin-1β (IL-1β) (E-EL-M0037c), Mouse IL-6 (E-EL-M0044c), Mouse Interleukin-10 (IL-10) (E-EL-M0046c), Human TNF-α (E-EL-H0109c), Human Interleukin-1β (E-EL-H0149c), Human IL-6 (E-EL-H6156), and Human Interleukin-10 (IL-10) ELISA kits (E-EL-H6154). Yeasen Biotech Co., Ltd. supplied the PAGE gel fast preparation kit (Catalog number: 20325ES), while Abbkine supplied the ultra-sensitive ECL luminous liquid (Catalog number: BMU102-CN). We purchased the SYBR® Green Premix Pro Taq HS qPCR Kit II (Catalog number: AG11702) and the Evo M-MLV Mix Kit with gDNA Clean for qPCR reagents (Catalog number: AG11728) from Hunan Accurate Biotechnology. Sigma supplied the lipopolysaccharide (LPS) (L2880, St. Louis, USA), and MedChemExpress the agar (AG490, HY-12,000, Monmouth Junction, NJ, USA). From the Jiangsu Provincial Traditional Chinese Medicine Hospital, we acquired the traditional Chinese medicines Xianhecao and Huanglian. These were soaked, decocted, filtered, and concentrated to 1 g/mL in our hospital’s preparation room for use in future research, and then put in a freezer set to -80 °C.

Experimental Animals

A total of seventy SPF male C57BL/6 N mice, aged 9–12 weeks, with a body weight of (22 ± 2) g, originated from the Vital River Laboratory Animal Technology Co., Ltd. in Beijing, with the following license number: SCXK (Beijing) 2021-0006. The mice were given free reign over their nourishment plus water sources.

Modeling, Grouping, and Drug Administration

The mice were then divided into five separate groups: one set of controls (n = 6), a DSS group (n = 16), a group receiving a 3% DSS + Xianhecao-Huanglian drug pair in a 10:1 ratio (referred to as A10:C1; with Xianhecao administered at 5.45 g/kg/d and Huanglian at 0.55 g/kg/d, n = 16), a group receiving a 3% DSS + Xianhecao-Huanglian drug pair in a 5:1 ratio (referred to as A5:C1; with Xianhecao administered at 5 g/kg/d and Huanglian at 1 g/kg/d, n = 16), and a group receiving a 3% DSS + Xianhecao-Huanglian drug pair in a 2:1 ratio (referred to as A2:C1; with Xianhecao administered at 4 g/kg/d and Huanglian at 2 g/kg/d, n = 16). After a week of adaptive feeding, control, and DSS group mice had free access to water and a normal diet. Treatment groups received preventive intervention for 5 days, starting on the third day of adaptive feeding. From day 7, DSS and treatment groups had free access to a 3% DSS solution, while the treatment group continued traditional Chinese medicine intervention for 7 days. Mice were euthanized 8 days post-decapitation, as illustrated in Fig. 1. A daily check was made on the mice’s weight throughout this timeframe. Following the experiment, in C57BL/6 N mice Whole blood was acquired through orbital blood collection using a heparin sodium anticoagulant tube. After centrifugation at 4 ℃, 3000 rpm/min for 10 min, the obtained serum was stored at -80 ℃ for testing. Mice were euthanized by decapitation, and prompt dissection provided colon samples. The colon length was measured, and after washing feces, it was opened longitudinally, rinsed with pre-cooled saline, and measured without external force. LCM of the obvious lesion was taken, frozen in liquid nitrogen, and then transferred to a -80 ℃ refrigerator for storage, Retain the obvious lesion LCM and fix it in 4% paraformaldehyde for 24 h, followed by making pathological sections for the purpose of conducting staining with hematoxylin and eosin (HE), periodic acid-Schiff (PAS) analysis, immunohistochemistry, and immunofluorescence. Extract the spleen of the mouse, dry the residual blood with filter paper, and weigh. In the case of rats, fasted for 12 h before the last administration and couldn’t help but water. Following the last gavage, mice were anesthetized with pentobarbital sodium (SIGMA company, USA) for 1 h. Blood was collected from the abdominal aorta and allowed to stand for 1 h. Centrifugation at 3000 rpm for 10 min yielded upper clear liquid, combined with the same serum group. This mixture was subjected to complement inactivation at 56 ℃ in a water bath for 30 min. Subsequently, the serum underwent filtration and sterilization using a 0.22 µ m microporous filter membrane within a sterile ultra-clean workbench. After packaging, it was sealed and stored in a -80 ℃ refrigerator for future use.

Drug-Contained Serum Formulation

A total of twenty SPF male SD rats weighing 200 ± 20 g, were segregated to split into two categories: the serum groups that did not include any drugs and those that did both, each consisting of ten rats. These rats were obtained under license number SCXK (Beijing) 2021–0011 via the Beijing-based Vital River Laboratory Animal Technology Co., Ltd. The optimal drug pair ratio of 5:1 was selected based on prior experimental outcomes to prepare the decoction of traditional Chinese medicine. The medicated group was orally administered with a Xianhecao-Huanglian drug pair medicinal decoction at a dose of 3.24 g/kg per day, in contrast to the blank serum group, which was given the same amount of saline twice da, for four consecutive days. One hour following the final administration, blood samples were collected from the rats’ abdominal aorta, centrifuged, complement-inactivated, sterilized via filtration, and stored for subsequent cellular experiments.

Colon Histological Analysis

Colonic tissue samples were extracted, rinsed with PBS, and preserved in a 4% paraformaldehyde solution for at least 24 h. After dehydration in an ethanol gradient, the slices of 5 μm were cut from tissues that had been fixed in paraffin. The samples were stained using the standard method of H&E., and after sealing, the structural integrity of the colon was examined under a microscope. Periodic Acid-Schiff (PAS) staining, following established protocols, was carried out to evaluate goblet cell growth and mucus secretion.

Disease Activity Index (DAI) and Histological Evaluation

During the experiment, body weight, stool character, and fecal occult blood were recorded. The disease activity index (DAI) was calculated based on the scoring system [17] and histological evaluation [18] in accordance with previously published methods (Table 1).

Table 1 The Disease Activity Index (DAI) scoreImmunohistochemistry (IHC) Staining of Colonic Tissue

Sections of paraffin-embedded tissue were processed for routine deparaffinization, followed by antigen retrieval, endogenous peroxidase inhibition, and sealing. Subsequently, the pieces were incubated with primary antibodies, Muc2 (1: 2000), Occludin (1:250), ZO-1 (1:1000), and Claudin-1 (1:250), at 4 °C for the night., followed by secondary antibodies and DAB staining. The nuclei of the cells were counterstained with hematoxylin. The slices were dehydrated, mounted using neutral glue, dried out, and then taken and viewed under a microscope.

Imaging Using Fluorescence Microscopy

Immunofluorescence staining was conducted on paraffin-embedded sections of colonic tissue. After deparaffinization, sections of paraffin were soaked and then the extraction of the antigen was performed in a citrate buffer (pH 6.0). To minimize endogenous peroxidase operation, 3% H2O2 was added. After applying serum blocking, the samples were incubated overnight at 4 °C using primary antibodies that were properly diluted. The cell nuclei were stained with DAPI, and then fluorescent secondary antibodies were added. After 5 min of incubation under light-protected conditions, the slices were carefully sealed, examined, and photographed under a microscope.

Cell Culture and Treatment

NCM460 intestinal epithelial cells and LOVO colon cancer cells were procured from Wuhan Procell and cultured in RPMI-1640 and Ham’s F-12 K media, respectively, both incubated at 37 °C with 5% CO2 and contained 10% FBS as well as 1% penicillin/streptomycin. The cells were depleted of serum for 24 h before every test. By incubating LoVo as well as NCM460 cells with 10 µg/mL of LPS, an inflammatory cell model for IBD was created.Cells were treated with varying concentrations of Xianhecao-Huanglian drug pair medicated serum (0%, 10%, 20%, 30%, 40%, and 50%) for 24 h to investigate the effect of the drug-loaded serum on cell survival rates. First, the cells were treated with 20% medicated serum and compound AG490, either individually or in combination. The medicated serum was added 2 h prior to AG490, followed by 24 h of LPS stimulation at 37 °C. The influence of Xianhecao-Huanglian drug pair medicated serum on LOVO and NCM460 cells under LPS stimulation was assessed.

Monitoring the Survival of Cells

The amount of cells planted in a 96-well plate was 1 × 105 cells/mL, with 100 µL of medium per well. They were allowed to adhere overnight. Different concentrations of Xianhecao-Huanglian drug pair medicated serum were introduced into their corresponding wells, after which LPS was added to the cells, either individually or in combination, for 24 h. When the procedure was finished, 10 µL of CCK-8 solution was transferred to every well and allowed to incubate for another 2 h in the incubator. A plate reader was used to detect absorbance around 450 nm in order to ascertain the vitality of the cells.

ELISA

Mouse colonic tissue was collected, weighed, homogenized in PBS, and then centrifuged to obtain the supernatant. A 6-well plate was used to seed the cells, which were cultivated overnight at a density of 1 × 105 cells/mL in 1 mL of media per well. The ELISA kit’s instructions were followed to quantify the amounts of TNF-α, IL-10, IL-1β, as well as IL-6 in the obtained tissues, blood, and cell supernatants from mice.

RT-qPCR

After deciding on the RNA content, the entirety of RNA was isolated through colonic tissues. Then, utilizing the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728, ACCURATEBIOTECHNOLOGY, HUNAN, Co., Ltd.), cDNA was produced employing reverse transcription. Afterward, the cDNA was produced for RT-qPCR amplification by the SYBR® Green Premix Pro Taq HS qPCR Kit II (AG11702, ACCURATE BIOTECHNOLOGY, HUNAN, Co., Ltd.). The 2-ΔΔCt technique was used for data analysis, with β-actin acting as the internal reference. In Table 2 you can find the primer sequences that were needed for this study.

Table 2 The primers information of genes in this studyWestern Blot

Protein extraction was performed on cells as well as the mouse colonic tissue using a lysis buffer containing RIPA (Beyotime, China), along with proteinase as well as phosphatase inhibiting agents. The amount of protein was determined using the BCA kit. 20 µg of each protein was poured into each well, and SDS-PAGE gel electrophoresis was performed on eighty volts to distinguish the intended proteins. The concentration of the gel used for SDS-PAGE was 8%, 12.5%, and 15%. Afterward, protein fragments were moved to a PVDF membrane from Millipore. The cell membrane was blocked using 5% skim milk at ambient temperature over 1 h, then subjected to an overnight soak at 4 °C with particular primary antibodies. The antibodies used in this study comprised ATG12, p62, LC3A/B, Beclin-1, STAT3, p-STAT3, ZO-1, MUC2, β-actin, and β-Tubulin, each at a 1:1000 dilution, while JAK2 and p-JAK2 were utilized at a 1:500 dilution ratio. On the next day, the membrane was carefully washed multiple times via PBST. Then, HRP-conjugated goat anti-rabbit or anti-mouse IgG (1:5000, 111-035-003/115-035-003; Jackson) was put on and incubated at ambient temperature for 1 h. After rinsing with TBST, we proceeded with ECL development as well as exposure. The grayscale measurements for every single protein band were subsequently evaluated using Image J software, just like a biologist would do.

Data Analysis

For every circumstance, a minimum number of three separate studies were conducted, as well as the outcomes are displayed as the mean ± standard deviation. The statistical significance was evaluated using an analysis of variance (ANOVA) for comparing each group, and then Tukey’s t-test was used for post hoc evaluation. The statistical evaluation was performed employing GraphPad Prism version 9.5.0 (GraphPad Software Inc 9.5.0). Statistical significance was determined by a p-value lower than 0.05.