Fermentation products of Danshen relieved dextran sulfate sodium-induced experimental ulcerative colitis in mice

With the increased incidence and recognition, ulcerative colitis (UC) has become a global public health problem in the world. Although many immunosuppressant and biological drugs have been used for UC treatment, the cure rate is still very low. It is necessary to find some safe and long-term used medicine for UC cure. Recently, the Chinese traditional herb Danshen has been investigated in the treatment of UC. However, it is a limitation of Danshen that many of the active components in Danshen are not easily absorbed by the human body. Probiotics could convert macromolecules into smaller molecules to facilitate absorption. Thus, Lactobacillus rhamnosus (F-B4-1) and Bacillus subtillis Natto (F-A7-1) were screened to ferment Danshen in this study. The fermented Danshen products were gavaged in the dextran sulfate sodium (DSS)-induced UC model mice. Danshen had better results to attenuate symptoms of DSS-induced UC after fermented with F-B4-1 and F-A7-1. Loss of body weight and disease activity index (DAI) were reduced. The abnormally short colon lengths and colonic damage were recovered. And fermented Danshen had the better inhibitory effect than Danshen itself on pro-inflammatory cytokine expression during DSS-induced UC. The results indicated that compared with Danshen, fermented Danshen relieved DSS-induced UC in mice more effectively. Danshen fermented by probiotics might be an effective treatment to UC in clinic stage in the future.

Gastric and intestinal fluid tolerance. The bacterial strain was inoculated in 5 mL MRS liquid medium for 24 h at 37 °C. After centrifuged at 3000 rpm for 10 min, the supernatant was discarded and rinsed with PBS buffer (pH 7.0) for 3 times. After suspended the bacterial in 5 mL sterile saline, 1 mL bacterial suspension was mixed with 9 mL artificial gastric juice (NaCl 0.2 g, pepsin 0.35 g, ddH 2 O 100 mL, pH 2.5) or artificial intestinal juice (trypsin 0.1 g, NaHCO 3 1.1 g, NaCl 0.2 g, bile salt 1.8 g, ddH 2 O 100 mL, pH 8.0) and cultured at 37 °C for 3 h or 4 h. Then, the culture medium was diluted with normal saline. 1 mL diluent was mixed with 30 mL MRS solid medium and cultured at 37 °C for 48 h. To determine the bacterial survival, viable cell counts were determined at 0 h, 3 h or 4 h of incubation. The survival rate was calculated by counting. Percentage survival was calculated using the formula 34 : where N1 is the viable count after exposure to simulated gastrointestinal fluids for specific time intervals and N0 represents cell count before treatment 35 .

The treatments of Danshen. All plant experiments complied with the IUCN Policy Statement on
Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The Danshen was divided into three different treatments, boiled, enzymolysis and fermentation. (1) Boiled Ultramicro mill was used to crush Danshen into powder above 1500 mesh. The ratio of the powder and distilled water was 1:9. After the uniform stirring, the water was extracted by boiling at 100 °C for 30 min. And then the water was filled up. The sample was centrifuged at 10,000 r/min and the supernatant was reserved.
(2) Enzymolysis The Danshen was ultra-fine pulverized and dissolved in sterile water (1:9, pH 4.8-5.0) after ultraviolet irradiation. The enzymolysis was did at 50 °C for 2 h by adding acid pectinase and acid cellulase (acid fiber and sour fruit amount accounted for 1 ‰ of solid). Then sugar (2%), peptone (5 ‰ of total volume) and protein powder (2.5 ‰ of total volume) were added. www.nature.com/scientificreports/ groups (n = 7 per group). Mice were treated with saline for 15 days in Negtive control groups (NC), DSS, Boiled groups (Boi), compound bacterium fluid groups (CBF, the mixture of F-B4-1 and F-A7-1 in 1:1 ratio) and FDS47 groups were treated with drinking water containing 2.5% DSS for 7 days 36 . On the 8th day, mice were treated daily with saline, Boiled Danshen (50 mg/Kg), CBF or FDS47 (50 mg/Kg) by gavage respectively in the DSS, Boi, CBF and FDS47 groups. The probiotics of F-B4-1 and F-A7-1 were inoculated as described in the "Antimicrobial spectrum analysis". Every mice was gavaged with 200 μL samples in the experiment. All mice were weighed every day. On the 15th day of the experiment, all mice were killed by exsanguination after deep anesthesia 37,38 .
Evaluation of colitis. The disease activity index (DAI) was determined by an investigator blinded to the protocol by scoring changes in weight, hemoccult positivity or gross bleeding, and stool consistency as the protocol previously described 36 .
Blood and tissue collection. Serum was prepared by centrifugation at 3000g for 20 min at 4 °C and stored at − 80 °C for biochemical analysis. The mice were killed by exsanguination after deep anesthesia. Colons were rapidly removed and their length were documented. Then the colons were snap-frozen in optimal cutting temperature (OCT) embedding medium (Tissue-Tek) for histology analysis.

Enzyme-linked immunosorbent assays (ELISA). The mouse intestinal tissues were homogenized on
ice with NP40 lysis buffer (Beyotime Biotechnology, China). The homogenates were quantified using the BCA assay as reported previously (Beyotime Biotechnology, China) 40 . Cell supernatants, serum and tissue homogenates were collected respectively for the determination of IL-6, IL-1β and TNF-α concentrations according to the manufacturers' instructions.

Statistical analysis.
All experiments were repeated at least 3 times independently. The normal distribution was firstly analyzed by SPSS v11.5 (SPSS Inc., Chicago, IL). All the data were expressed as mean ± SEM and analyzed with one-way ANOVA with use of SPSS v11.5 to compare the treatment means when data was normally distributed. Tukey-Kramer multiple comparison procedure was used for Post-Hoc comparisons. P values < 0.05 were considered statistically significant. Images were processed by use of Graphpad Prism 5 (GraphPad Software, La Jolla, CA, USA) and Adobe Photoshop CS6 (Adobe, San Jose, USA). The results showed that the viability of F-B12-1 and F-B25-1 were just 13.89% in the artificial intestinal juice (Fig. 1A). Probiotics need to colonize and survive in the intestine before they reach the intestine and play function. Thus probiotics must be tolerant to the effects of gastric acid, pepsin and trypsin 41,42 . Probiotics colonization and tolerance to artificial gastroenteric fluid have been used to determine the survival rate in the upper gastrointestinal tract, which is also one of the important indicators of probiotics screening 43 . The level of tolerance for choosing probiotics is higher than 70% 44 . Thus, the probiotics of F-B4-1, F-B8-1, F-B9-1, F-B16-1 and F-A7-1 were selected for the further experiment to examine the CFU (Fig. 1B). A large number of studies have shown that after probiotics enter the digestive system, their viable bacteria content must reach more than 1 × 10 6 CFU/ mL to play their health effects. Therefore, the number of viable bacteria in fresh probiotics products should not be less than 1 × 10 6 CFU /mL to compensate for the loss of viable bacteria when probiotics pass through the human gastrointestinal tract 45,46 . Our data suggested that the probiotics of F-B4-1, F-B8-1, F-B9-1, F-B16-1 and F-A7-1 were all reach this level. The CFU of these five probiotics was all higher than 1 × 10 6 CFU /mL. LPS could induce inflammatory injury in caco2 cells which is a recognized cell model for UC 47  www.nature.com/scientificreports/ supernatant. The data showed that the level of IL-6 could be increased significantly after treated with 50 μg/mL LPS for 12 h (Supplemental fig. 1). Thus, we used 50 μg/mL LPS in our next experiments. After caco2 cells were treated with LPS for 12 h, F-B8-1, F-B4-1, F-B9-1, F-B16-1 and F-A7-1 (5 mg/mL) were added to examine the IL-6 concentration. It is suggested that all of these five probiotics could decrease the IL-6 concentration induced by LPS. However, after treated for 12 h, there was a statistical significance among F-B4-1 group and other four probiotic groups. And after treated for 24 h, there was a statistical significance among F-B16-1, F-B9-1 and F-A7-1 probiotic groups. Thus, among these five probiotics, F-B4-1 and F-A7-1 had the best effects and were selected for the further experiments (Fig. 1C).

Results
In order to explore which technology has the better effect of anti-inflammation, Danshen was boiled or enzymatic hydrolyzed to examine the IL-6 concentration in caco2 cells treated with LPS. After treated for 12 h, there was no statistical significance among boiled groups and LPS group (P > 0.05). And after treated for 24 h, there was a statistical significance among 0.2 mg/mL of enzymatically hydrolyzed Danshen group and other boiled or enzymatic hydrolyzed groups (Fig. 1D). The results showed that enzymatic hydrolyzed Danshen at 0.2 mg/mL had the best effect to decrease the IL-6 concentration. Based on above results, enzymatic hydrolyzed Danshen and F-B4-1 and F-A7-1 were selected.
Effects of different fermentation methods on LPS-mediated upregulation of TNF-α, IL-6 and IL-1β. ELISA was done to measure the levels of TNF-α, IL-6 and IL-1β in caco2 cells after treatment. The www.nature.com/scientificreports/ levels of TNF-α, IL-6 and IL-1β were all upregulated after treated with LPS. All of the samples from different fermentation methods could decrease the all three inflammatory factor levels (Fig. 2). Among these different fermentation methods, FDS47 had the best effects of inflammatory factor inhibition. And the effects of inflammatory factor inhibition was better than that in the enzymolysis groups (Fig. 2). There was a statistical significance of TNF-α level among FDS47 group and other treatment groups. As the level of IL-6, there was a statistical significance among FDS47 group and FDS74/FDS-sta/Enz groups. As the level of IL-1β, there was a statistical significance among FDS47 group and FDS-sta/Enz groups (Fig. 2). Thus, together these results, FDS47 had the best effects of inflammatory factor inhibition. Meanwhile, the CFU were examined in F-B4-1 and F-A7-1 with different fermentation methods. There was a statistical significance for the CFU of F-B4-1 among FDS47 group and FDS74/FDT47-sta groups. Although there was no difference between FDS47 and FDT47-sha group in the CFU of F-B4-1 (P > 0.05), there was a statistical significance for the CFU of F-A7-1 among FDS47 group and FDT47-sha/FDT47-sta groups. It is suggested that the CFU was higher in FDS47 group (Supplemental fig. 2). Thus, fermented Danshen with F-B4-1 and then with F-A7-1 was selected to do further research.
The ELISA results showed that FDS47 at 0.2 mg/mL could inhibit the IL-6 level induced by LPS significantly. With the increased concentrations (5 mg/mL), the IL-6 level elevated in caco2 cells treated with FDS47 for 24 h (Fig. 3A,B). Furthermore, the cell viability was investigated. FDS47 at different concentrations could not cause the changes in cell viability which suggested that FDS47 did not have the cytotoxicity of caco-2 (Fig. 3C). www.nature.com/scientificreports/

FDS47 alleviated the clinical symptoms of DSS-induced colitis in mice.
We first examined the organs (heart, spleen, kidney, lung and liver) of mice in each treatment group. There was no significant changes in all organs (Supplemental fig. 3). The body weight and colon length are important indicators of colitis severity. DSS-induced colitis could decrease the body weight and shorten the colon length. The data showed that FDS47 could effectively prevented body weight loss induced by DSS (Fig. 4A). At the 15th day, only the body weight in FDS47 group was much higher than that in DSS group (P < 0.05) (Fig. 4B). The grade of UC induced by DSS was evaluated by the DAI score, which was the sum of scores given for body weight loss, stool consistency, and presence of fecal blood. A significant increase of DAI score was observed in the DSS-treated group at the 15th day compared with the normal groups (p < 0.05). In three treatment groups, the DAI scores were significantly decreased when compared with the DSS group at the 15th day (Fig. 4C). And FDS47 most effectively prevented DAI increase compared with Boi and CBF groups (Fig. 4D). In addition, mean colon length was the highest in the normal mice and lowest in the DSS-induced colitis mice (Fig. 4E, F). In all three treatment groups, the colon lengths were longer than those in the DSS group. In the FDS47 group, the colon length was longer than that in Boi and CBF groups (P < 0.05) which almost returned to the normal level (Fig. 4F). As can be seen from body weight, DAI and colon length results above, the effect of FDS47 in the treatment of colitis is better than those of Boi and CBF. H&E staining of the colon revealed the degree of inflammation and epithelial damage. The colons from all of the mice in each group were examined in H&E stained slides. According to the Fig. 5A, DSS treated mice displayed the most severe infiltration of inflammatory cells, disruption of surface epithelium, and loss of crypts. Intragastric administration of FDS47 showed the least severe colitis compared with the DSS treated group. H&E scores in all the mice were determined at the 15th day. The total lesion scores in DSS group increased compared with the normal group. And the decrease of the total lesion scores were the most remarkable in the FDS47 group All groups (Nor, Enz., FDS47) were statistically analyzed with the LPS group. Group-to-group statistical analysis was performed at 24 h between 0.2 mg/mL FDS47 group, 1 mg/ mL FDS47 group and 5 mg/mL FDS47 group. (Data are expressed as means ± S.E., *p < 0.05, **p < 0.01 and ***p < 0.001). www.nature.com/scientificreports/ The body weights of mice were detected every day. a-d represented significant differences among different groups by one-way ANOVA procedure followed (p < 0.05). (B) The statistical analysis of the body weights at the 15th day. (C) DAI (disease activity index) score of mice was examined every day. a-e represented significant differences among different groups by one-way ANOVA procedure followed (p < 0.05). (D) The statistical analysis of the DAI score at the 15th day. (E) The photos of colon length (one representative colon from each group). Images were processed by use of Adobe Photoshop SC6 (6.1, https:// www. adobe. com/ cn/ produ cts/ photo shop. html) (F) The colon lengths calculated from E. All groups (NC, Boi., CBF, FDS47) were statistically analyzed with the DSS group. The FDS47 group and other groups (Boi., CBF) were statistically analyzed from group to group. (Data are expressed as means ± S.E. # p > 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001). www.nature.com/scientificreports/ (Fig. 5B). All these above results indicated that FDS47 could significantly alleviate the clinical symptoms of DSSinduced colitis in mice. And the alleviate effects in FDS47 group were better than those in Boi and CBF groups.

FDS47 decreased pro-inflammatory cytokines in DSS-induced mice.
To understand the mechanism that underlined the alleviation of DSS-induced colitis in mice after treatment with FDS47, the levels of pro-inflammatory (TNF-α, IL-6, and IL-1β) in serum (Fig. 6A-C) and colon tissues (Fig. 6D-F) were examined. No matter in serum or colon tissues, the levels of IL-6, IL-1β and TNF-α were significantly increased in  www.nature.com/scientificreports/ DSS treatment group (Fig. 6). After intragastric administration of FDS47, a remarkable decrease of the three pro-inflammatory cytokines were observed. There were statistical differences among FDS47 group and Boi/ CBF groups in the levels of IL-1β in serum. And there were statistical differences among FDS47 group and Boi/ CBF groups in the levels of IL-6 and TNF-α in the colonic homogenate of DSS-induced UC mice (Fig. 6). Thus, FDS47 was better than Boi and CBF.

Discussion
Probiotics could relieve various inflammatory diseases by regulating the microbiota in gastrointestinal tract, especially in UC treatment 48 . Nowadays, many probiotics have been shown to have therapeutic effects on UC.
Usually, these products are multi-component probiotic mixtures, because each strain may have additive or synergistic effects 49,50 . It is reported that the supplement of L. plantarum ZDY2013 and B. bifidum WBIN03 could remit UC through modification of gut microbiota to regulate oxidative stress and inflammatory mediators 51 . By the analysis of antibacterial spectrum, gastrointestinal fluid tolerance, bacterial viability and inflammatory factor inhibition, F-B4-1 and F-A7-1 were selected in this work. They could not only effectively reduce the growth of several harmful bacteria, but also have good tolerance to the gastrointestinal environment (Fig. 1A,B). Additionally, the metabolites of these two probiotics could significantly reduce the release of inflammatory factors in caco-2 cells caused by LPS (Fig. 1C). From these results, it is suggested that F-B4-1and F-A7-1could become hopeful strains in curing UC.
In China, Herbs have been used for thousands of years to cure of diseases. Until now, a large number of biologically active substances in herbs have been proved 52 . Danshen is a very valuable herb in Chinese traditional medicine. It has been reported that Danshen has the antioxidant, anti-inflammatory and antibacterial activities 53 . The anti-inflammation efficacy of Danshen is generally played by the main biological activities in it, such as Salvianolic acid B, tanshinone IIA, Danshensu and dihydrotanshinone I 54 . Thus, Danshen was selected as fermentation material for the treatment of UC in this work. The traditional extraction methods for Danshen were boiling water extraction and enzymatic hydrolysis. Compared with boiling water extraction, enzymatic hydrolysis could better reduce the release of cellular inflammatory factors (Fig. 1D). Thus, we selected enzymatic hydrolysis to deal with Danshen to release more biological activities.
It is reported that the therapeutic effects of fermented herbs were better than that of herb itself. KIOM-MA is a specific agent for allergic and chronic inflammatory diseases, which is composed of several plants, included Glycyrrhizae radix, Polygoni cuspidati radix, Sophorae radix, Cnidii rhizoma, and Arctii fructus. Recently, it is proved that the KIOM-MA128, the probiotics fermentation product of KIOM-MA had the improved therapeutic efficacy via the absorption and bioavailability of the active ingredients 55 . FRAM, the fermented products of Rhizoma Atractylodis Macrocephalae (RAM), also exerted a better protective effects on intestinal epithelial cells (IECs) against LPS-induced perturbation of membrane resistance and permeability 56 . The metabolic processes could be improved in herbs fermented with probiotics 57 . And in the process of fermentation, the decomposition of organic matter could be promoted by microorganisms and many new micromolecules could be produced from macromolecules 58 . It is reported that fermentation could better extract the effective ingredients in Chinese herbal medicines to treat enteritis 52 . Therefore, we used the selected probiotics F-B4-1and F-A7-1to ferment Danshen for the first time. The data showed that the fermented Danshen had the better effects than boiled Danshen itself or bacteria solution itself on UC treatment, including the colon length recover, decreased DAI score, lung damage recover and anti-inflammatory activities (IL-6, IL-1β and TNF-α) in serum and colons in DSS induced UC mice (Figs. 4,5,6). It is suggested that after fermentation, Danshen and probiotics might interact synergistically. However, the synergetic mechanisms, whether effective ingredients was increased or the new biological components were produced after fermentation, need to be further investigated in the next step.
At present, although it is believed that probiotics are safe under normal circumstances, there are individual reports of local or systemic infections such as pericarditis and sepsis caused by the ingestion of certain lactobacilli, bifidobacteria and other lactic acid bacteria 59 . Especially for patients with immunodeficiency, broken bowel syndrome, central duct occlusion, heart valve disease or premature infants, the risk of adverse events might be higher if probiotics are taken indiscriminately 60 . In severely UC, due to the destruction of the integrity of the intestinal barrier, there is a danger of live probiotics from the intestines and stomach to the internal organs of the body (bacterial displacement), which may lead to bacteremia 61 . Due to the dangers of probiotics during use, we choose safer metabolic fermentation products and verify its safety in in vitro cell experiments and in vivo experiments in mice.
It has been pointed out that the reduction of inflammatory cytokines in the serum and colons represents a logical target for UC therapy 62 , such as TNF-α, IL-6 and IL-1β, which play leading roles in the formation of UC 3,63 . Our data suggested that after intragastric administration of the fermentation products, all these pro-inflammatory cytokine (TNF-α, IL-6 and IL-1β) levels were decreased in the serum and colons of the DSS-induced UC mice (Fig. 6). Also, the in vitro experiments showed it. The fermentation products inhibited the pro-inflammatory cytokine (TNF-α, IL-6 and IL-1β) levels in the caco-2 cells treated by LPS (Fig. 2). All of the results proved that the probiotics fermented Danshen products relieved the DSS-induced UC mice by blocking pro-inflammatory cytokines.

Conclusions
In summary, we screened F-B4-1and F-A7-1 from many probiotics which could reduce harmful bacteria growth, have tolerance to the gastrointestinal environment and inhibit the inflammatory factors caused by LPS to ferment enzymatic Danshen. The fermentation products has anti-colitis effects through the inhibition of pro-inflammatory factors (Fig. 7). And compared with Danshen, fermented Danshen relieved DSS-induced UC in mice more www.nature.com/scientificreports/ effectively. Thus, the fermented Danshen might be further developed as an effective treatment approach to treat intestinal inflammation.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.