Morinda officinalis (no. 20150603) was obtained from Shenzhen Lush Pharmaceutical Co., Ltd. Positive drug mesalazin enteric-coated tablets (no. 150308) was bought from Losan Pharma GmbH (Germany). DSS was purchased from MP Biomedicals (MW; 36,000–50,000; MP Biomedicals, Solon, OH, USA). The enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-6, and IL-17 was bought from Huamei (Cusabio Biotech Co. Ltd., China). All plastic materials were purchased from Falcon Labware (Becton-Dickinson, Franklin Lakes, NJ, USA). RPMI Medium 1640, fetal bovine serum (FBS), and phosphate buffer saline (PBS) were obtained from GIBCO Laboratories (Grand Island, NY, USA). Penicillin G/streptomycin, MTT, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Apoptosis Assay Kit was purchased from Lianke Biology Inc. (Hangzhou, China). APC antimouse CD3 antibody, PE/Cy7 antimouse CD4 antibody, and APC antimouse CD8 antibody were purchased from BioLegend (USA). All other chemicals used were analytical grade and were supplied by the Beijing Chemical Agents Company (China).

The stem of M. officinalis was used as explants for the induction of hairy roots. The excised root segments were preincubated on half-strength MS (MS/2) solid medium for 3 days before infection. A. rhizogenes strain MSU440 was cultivated overnight and collected the bacterial suspension of A. rhizogenes by centrifugated at 6,000 rpm for 5 min. The explants were soaked in the overnight grown bacterial suspension of A. rhizogenes strain MSU440 (OD₆₀₀ = 0.6–0.8) for 15 min and dry blotted on sterile filter paper. The explants were cultured on 1/2 MS-based solid medium in the dark at 28 ± 1°C. After 3 days of cocultivation, the explants were transferred to 1/2 MS-based solid medium of hormone free which containing sodium cefotaxim (300 mg/L) to eliminate the residual bacteria and incubated in the dark at 25 ± 1°C. The healthy hairy roots were obtained and used after once subculture every 2 weeks in this study.

The powder of root M. officinalis or hairy roots of M. officinalis was heated to reflux for 2 h with 8 times volume 85% alcohol. The extraction was repeated twice. The extracting solution was filtered to remove the residue. And then it was concentrated by rotary evaporator. The concentrate was freeze dried under vacuum. Dry MORE and MOHRE were used to treat mice with colitis.

The HPLC analyses of MORE and MOHRE were performed by an Agilent 1260 High Performance Liquid Chromatograph System.

The iridoids analysis was done by using a Phenomenex Luna C18 column (250.0 × 4.6 mm, 5.0 µm). The column temperature was 25.00°C. The mobile phases were 0.4% H₃PO₄ solution (A) and methanol (B) with the flow rate at 1.000 mL/min. The gradient elution was 0–13 min, A 95.0%; 13–14 min, A 95.0–91.6%; 14–65 min, A 91.6–91.3%; 65–70 min, A 91.3–86.0%; 70–175 min, A 86.0–84.0%; 75–100 min, A 84.0–80.5%; 100–105 min, A 80.5–95.0%; and postrun 5.00 min. The UV detection wave length was 233 nm, and the injection volume was 10.00 µL. The quantitative analysis of iridoids was carried out by the external standard method.

The anthraquinones analysis was done with an Agilent Eclipse XDB-C18 column (150.0 mm × 4.6 mm, 5.0 µm). The column temperature was 30.00°C. The mobile phase was 0.2% H₃PO₄ solution (A) and acetonitrile (B) with the flow rate at 1.000 mL/min. The gradient elution was 0–15 min, A 90.0–80.0%; 15–50 min, A 80.0–70.0%; 50–70 min, A 70.0–60.0%; 70–100 min, A 60.0–50.0%; 100–120 min, A 50.0–20.0%; 120–125 min, A 20.0%; 125–130 min, A 20.0–90.0%; and postrun 5.00 min. The UV detection wave length was 277 nm, and the injection volume was 15.00 µL. The quantitative analysis of anthraquinones was carried out by the external standard method.

Adult male and female Kunming (KM) mice (18–22 g) were purchased from the Laboratory Animal Services Center, Guangzhou University of Chinese Medicine (Guangzhou, China). All animals were raised in accordance with the National Institutes of Health Guide for Laboratory animals’ use. The study was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine. Animals were housed under standard environment condition of temperature at 20–25°C under a 12 h dark/light cycle and allowed free access to sterilized water and standard food.

Acute oral toxicity in mice was performed by using the limit dose test of up-and-down procedure according to OECD guideline (OECD 425) (19). Healthy KM mice were randomly assigned to three groups, each group with six mice (three males and three females). A single dose of 5,000 mg/kg of extracts (MORE or MOHRE) were administered orally to each group. Each mouse was continuously observed for 4 h after the treatment. The mice were further observed once daily for 14 days and the number of deaths, body weight and toxic parameters were recorded.

A total of 108 male mice were included in this experiment. Chronic colitis was induced by oral administration of 3% DSS for three cycles (Figure 1), as described by Farkas et al. (20) and Okayasu et al. (21), with slight modification. Randomly selected 12 mice as the normal group, only received tap water every day. The others were induced by three cycles that a 7-day treatment with DSS dissolved in drinking water (3%, w/v) followed by 14 days of recovery with water. After the first cycle, according to their body weight, 96 modeled mice were randomly divided into eight groups (n = 12/group): DSS group, mesalazin (50 mg/kg/day) in positive control group, three groups received treatment with MORE (20, 40, and 80 mg/kg/day), the other received treatment with MOHRE (20, 40, and 80 mg/kg/day). The dose selection of extracts was based on clinical practice (usually 6,000 mg/day, 70 kg body weight). Except for the control group, the others were still administered with 3% DSS as described previously, while all drug groups were given the corresponding does of drugs. The treatment lasted for two other cycles and body weight, behavior, stool consistency, fecal occult blood (both visible and occult bleeding), disease activity index (DAI), mortality, food, and water intake of all animals were monitored daily. At the end of the experiment, animals were fasted for 12 h and then anesthetized with pentobarbital sodium (70 mg/kg, i.p.). Blood samples were collected directly from the orbit, which were transferred to 2 mL EP tubes followed by centrifuged at 3,000 rpm for 10 min at 4°C and finally stored at −80°C for biochemical assays. The spleens were weighted and the colons were quickly removed and gently rinsed with sterile and cold saline solution, then moved to cold plate for measuring the length of the colon, colon macroscopic evaluation. Subsequently the colons were divided into two parts, one was fixed in 4% paraformaldehyde for histological examination and the other was stored at −80°C for biochemical assays. Spleen was also fixed in 4% paraformaldehyde for histological examination.

Segments of colon and spleen were immediately fixed in 4% paraformaldehyde for 24 h, then washed with water, dehydrated with alcohol, and finally embedded in paraffin. Tissues were cut into standard sections and stained with hematoxylin–eosin (H&E) for histological examination. Segments of colon were evaluated according to the method previously reported by Boirivant et al. (25) and described as follows: 0, normal and no inflammatory cell infiltration; 1, slight inflammatory cell infiltration and no injure in submucosal tissues; 2, moderate inflammatory cell infiltration and submucosal tissues are destroyed (damage range between 10 and 25%); 3, obvious inflammatory cell infiltration, submucosal tissues are destroyed and colonic wall thickening (damage range between 25 and 50%); and 4, serious inflammatory cell infiltration, large scale colon tissue damage (damage range > 50%) and colonic wall thickening.

The systemic levels of TNF-α, IL-6, and IL-17 in the serum were measured by using a commercially available ELISA according to the manufacturer’s instructions.

At the end of the experiment, the splenocytes and peripheral blood (PB) lymphocytes were isolated from colitis mice. The method was described previously by Su et al. (26). The cells (2 × 10⁶ cells/mL) were stained for surface markers, antimouse CD3-APC, antimouse CD4-PE/Cy7, or antimouse CD8-APC in PBS for 20 min at RT in the dark. After washing, cells apoptosis were detected by the AnnexinV/PI detection kit, according to manufacturer’s instruction, briefly, cells were washed twice with precold PBS, then suspended in binding buffer and stained with 5 µL annexin V-FITC and 10 µL PI for 15 min at 25°C in the dark. Annexin V⁺/PI⁻ cells were considered early apoptotic cells. Annexin V⁺/PI⁺ cells were considered late apoptotic cells. Apoptosis analysis was detected by FACS canto™ flow cytometer.

Splenocytes were isolated as described previously by Su et al. (26), with slight modification. Mice were sacrificed and the spleens were aseptically separated, washed with precold PBS. The spleens were ground and through a 70 µm strainer. After centrifuge at 300 × g for 5 min at 4°C, ACK Lysis Buffer was used to remove erythrocytes. Cells were collected by centrifuged at 300 × g for 5 min at 4°C and washed twice with PBS. Splenocytes were resuspended and cultured in RPMI1640 medium supplemented with 10% (v/v) FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) under a humidified 5% (v/v) CO₂ atmosphere at 37°C. Cells viability analysis was assessed by using trypan blue dye exclusion staining and all cases cell viability was higher than 95%.

Cytotoxicity assay was evaluated by MTT assay, 100 µL of splenocytes were seeded in 96-well plates at 3 × 10⁶ cells/mL, treated with 100 µL MORE and MOHRE (50–400 µg/mL) for 24 h, respectively. After that, 20 µL of MTT (5 mg/mL) was added to each well and further incubated for 4 h at 37°C with 5% CO₂. 150 µL of DMSO was added into 96-well plates, and finally the plates were read at 490 nm with Multiskan Go. Without cytotoxic and optimal doses of MORE and MOHRE were selected for further apoptosis assessments. The formula was applied to calculate the cell viability (%): Cell viability(%)=(AS−Ab)(Ac−Ab)×100 where A_s, A_c, and A_b represent the absorbance of the different concentrations wells of drugs, control wells, and blank wells, respectively.

Splenocytes were separated and cultured by the same methods in the section cell preparation. 100 µL of splenocytes were seeded in 96-well plates at 3 × 10⁶ cells/mL. The cells were stimulated with 5 µg/mL of Concanavalin A (ConA) and treated with 100 µL MORE and MOHRE (50, 100, and 200 µg/mL) for 24 h, the range of concentrations was optimal values according to the cytotoxicity. The formula was applied to calculate the cell proliferation by the same methods in the section Cytotoxicity Assay.

Splenocytes were seeded in 48-well plates at 3 × 10⁶ cells/mL, treated with different concentrations of MORE and MOHRE (50, 100, and 200 µg/mL) for 4 h following by 5 µg/mL ConA stimulation or no stimulation with ConA for another 24 h. Apoptosis analysis was tested according to the method in section apoptosis evaluation by Annexin-V/PI in vivo. Apoptosis analysis was detected by FACS canto™ flow cytometer and fluorescence staining.

Statistical analysis was performed using SPSS software (version 18.0; SPSS, Chicago, IL, USA). The analysis of survival was conducted using Kaplan–Meier method (log-rank test). All results are expressed as mean ± SEM. Results are using one-way ANOVA or Student’s t-test when appropriate. Values of p < 0.05 were considered statistically significant.

Iridoid and anthraquinones are the main active ingredients of M. officinalis. Here, the chromatographic peaks of hairy roots extract of M. officinalis (MOHRE) were more abundant than roots extract of M. officinalis (MORE) (Figure 2), suggesting that there were more secondary metabolites in the MOHRE extracts, especially anthraquinones. The content of monotropein is 2.12 and 1.50% in MORE and MOHRE, respectively, while the content of Desacetyl asperulosidic acid is 0.98% in MORE and 1.51% in MOHRE. In addition, the contents of rubiadin-1-methyl ether, 2-hydroxy-1-methoxy-anthracene and 2-hydroxy-3 (hydroxymethyl) anthraquinone are 0.04, 0.07, and 0.03% in MORE, as well as the contents of rubiadin-1-methyl ether, 2-hydroxy-1-methoxy-anthracene, and 2-hydroxy-3(hydroxymethyl)anthraquinone are 0.11, 0.19, and 0.31% in MOHRE. Suggesting that the content of active iridoid is similar to MORE and MOHRE, the anthraquinones are derived from the MOHRE is higher than MORE.

Roots extract of Morinda officinalis and MOHRE did not caused obvious toxic reactions and mortality with the dose of 5,000 mg/kg by oral administration. In addition, during the 14 days observation time, all animals looked bright and the body weight did not have significant change (Table 1).

In this study, the body weight of colitis mice induced by DSS decreased significantly from day 7, and the animal condition continued to deteriorate until the end of the experiment in DSS group. All drug-administration groups significantly restored body weight (Figure 3A). The enteritis induced by DSS significantly increased the mortality of animals. Treatment with mesalazin as a positive drug, MORE, and MOHRE reduced the death of animals (Figure 3B). In addition, food intake and water intake also were reduced in DSS group. Treatment with mesalazin, MORE, and MOHRE could improve food intake and water intake to a certain extent (Figures 3C,D). DAI is a most important indicator of the effect evaluation in IBD model. In this study, the DAI score increased significantly in DSS group, indicating a severe condition of colitis. By oral administration of mesalazin, MORE, and MOHRE, the DAI scores were decreased significantly (Figure 3E). The roles of MORE and MOHRE revealed a dose-dependent manner. And there were not obvious differences on reversing these pathological symptoms of UC between MORE and MOHRE under the same concentration. These results showed that both MORE and MOHRE could attenuate DSS-induced experimental chronic UC with similar effect.

The colon length is inversely associated with the severity of DSS-induced colitis. Our results showed that the colon from mice in mesalazin, MORE, and MOHRE groups is longer than in the DSS group (Figures 4A,B). Colon thickness and morphology score are also the important indicators to evaluate the therapeutic effect of drug. We found that mesalazin, MORE, and MOHRE reduced colon thickness and morphology score (Figures 4C–E). In addition, the H&E staining of colonic tissues revealed the anti-inflammatory effects of mesalazin, MORE, and MOHRE in colon. Compared with the normal structure of the colon, DSS group showed obvious inflammatory cell infiltration, absence of epithelial cells and goblet cells. However, significant reductions in the inflammatory cell infiltration and mucous membrane ulcer were observed after treatment with mesalazin, MORE, and MOHRE (Figures 4F,G). Furthermore, these positive effects of MORE and MOHRE displayed a dose-dependent manner. An insignificant difference between MORE and MOHRE was observed at the same concentration. These results indicated that both MORE and MOHRE could effectively protect DSS-induced colon injury in mice.

It is well known that inflammation plays an important role in the pathogenesis of UC. The abnormal activation of immune system can cause excessive secretions of proinflammatory cytokines. To understand the anti-inflammatory effect of MORE and MOHRE in DSS-induced chronic UC in mice, we measured the serum levels of proinflammatory cytokines TNF-α, IL-6, and IL-17 were significantly increased in DSS group, which were all decreased significantly in mesalazin, MORE, and MOHRE groups (Figure 5). There are still no significant differences between MORE and MOHRE.

Chronic colitis often causes the disorder of immune function. In this study, we found that oral administration of DSS significantly increases spleen weight, and MORE and MOHRE significantly inhibited spleen swelling (Figures 6A,B). In addition, we analyzed the degree of spleen damage by H&E staining. We also found that DSS led to the change of spleen structure, swelling, and the accumulation of inflammatory cells. However, MORE and MOHRE could significantly effectively alleviate the injury of the spleen (Figure 6C). This protective effect between MORE and MOHRE was not significantly different.

The apoptosis of T cells has been confirmed to be critically involved in the pathogenesis of UC. In the present study, splenocytes and PB lymphocytes from chronic colitis mice were analyzed by flow cytometry after two cycle’s treatment of MORE and MOHRE. To analyze the subsets of splenic and PB T cells that suffer apoptosis following MORE and MOHRE treatment in chronic colitis mice, we applied annexin V⁺/PI⁺ among total T cell (CD3⁺), helper T cells (CD3⁺CD4⁺) and cytotoxic T cell (CD3⁺CD8⁺) in spleen and PB. We found that the early and late apoptosis of splenic CD3⁺ T were significantly decreased in DSS treated mice when compared with the control group, and early apoptosis of splenic CD4⁺ and CD8⁺ T cells were also significantly decreased in DSS group. Meanwhile, except PB CD3⁺ T cells were significantly reduced in late apoptosis, the apoptosis of PB CD4⁺ and CD8⁺ T cells had no difference in DSS group when compared with the control group. Interestingly, MORE and MOHRE significantly induced the early and late apoptosis of splenic CD3⁺ T cells in a dose-dependent manner in vivo. In addition, the early apoptosis of splenic CD4⁺ and CD8⁺ T cells in colitis mice were also significantly increased. However, there is no significantly difference in the induction of PB T cells apoptosis after treatment with MORE and MOHRE (Figures 7 and 8). The data showed that MORE and MOHRE could obviously induce the apoptosis of splenocytes, but not PB lymphocytes in chronic colitis mice. Meanwhile, the apoptosis effect of MOHRE on splenocytes or PB lymphocytes in vivo is similar to MORE.

According to the results from in vivo study, we found that splenic lymphocytes were much more sensitive to MORE- and MOHRE-induced apoptosis. Here, we further investigated the effect of MORE and MOHRE on splenic lymphocytes in vitro. The different concentrations of MORE and MOHRE were added into splenic lymphocytes in different 96-well plates for 24 h. The data showed that MORE and MOHRE had no toxicity to splenic lymphocytes (Figure 9A). However, after ConA stimulation, MORE and MOHRE inhibited the proliferation of splenic lymphocytes in a dose-dependent manner (Figure 9B), possessing unsignificant difference between them at the same concentration. The results revealed that MORE and MOHRE exert immunoregulatory properties for the proliferation of ConA-activated T cells in vitro.

To explore the relationship between the inhibition of splenic T cells proliferation and apoptosis, we incubated splenic lymphocytes with MORE and MOHRE in the presence or absence of ConA. Without ConA stimulation, MORE and MOHRE were unable to significantly induce lymphocytes apoptosis (Figure 10). However, after stimulation with ConA for 24 h, both MORE and MOHRE can dose-dependently induce the apoptosis of ConA-activated T lymphocytes (Figure 11). Meanwhile, MORE and MOHRE also could induced the early apoptosis of activated T lymphocytes at 48 in the presence of ConA stimulation (Presentation S1 in Supplementary Material). In addition, we further to explore whether CD3⁺, CD4⁺, and CD8⁺ T cells population is susceptible to cause apoptosis by MORE and MOHRE in both unstimulated and Con A-stimulated lymphocyte. Our data indicated that the early and late apoptosis ratios of CD3⁺, CD4⁺, and CD8⁺ T cells were no statistically differences in the unstimulated lymphocyte (Figure 12). However, the lymphocyte activated with ConA and exposed to MORE and MOHRE could dose-dependently increased the early and late apoptosis ratios of CD3⁺, CD4⁺, and CD8⁺ T cells (Figure 13). The difference of these effects between MORE and MOHRE was not significant at the same concentration.