2019 Volume 25 Issue 3 Pages 413-423
In this study, the preventive effects of Lactobacillus plantarum CQPC07 (LP) on dextran sodium sulfate (DSS)-induced male C57BL/6J mice colitis were investigated. The results showed that LP can increase the colon length and decrease the ratio of colon weight/colon length in DSS-induced colitis mice. Analyses of the sera of mice showed that LP can reduce the level of serum endothelin (ET), substance P (SP), and interleukin-10 (IL-10), enhance the activity of somatostatin (SS) and vasoactive intestinal peptide (VIP), and increase the levels of IL-2. Biochemical analyses of colonic tissues from mice showed that LP can increase the levels of glutathione (GSH), superoxide dismutase (SOD), and decrease the level of myeloperoxidase (MPO), malondialdehyde (MDA). A pathological tissue evaluation showed that LP can inhibit colon tissue damage in DSS-treated mice. Furthermore, LP can also increase neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), c-Kit, stem cell factor (SCF) mRNA expression and decrease inducible nitric oxide synthase (iNOS), IL-8, and C-X-C motif chemokine receptor 2 (CXCR2) mRNA levels in the colon tissues of colitis mice. Thus, LP has a colitis inhibitory effect, the higher the concentration, the better the effect, and the effect of high concentration of LP is close to the drug of sulfasalazine.
The production of Chinese Sichuan pickles involves washing the Chinese cabbage, adding salt water and cabbage into jars, and allowing fermentation to proceed for 7–10 days in a sealed container. Salt water plays a role in the exudation of vegetable juice and provides a natural environment for lactic acid bacteria to access the soluble ingredients, such as sugar and nitrogen-containing substances (Feng et al., 2012). In addition, the metabolism of acidic substances and flavor components provides these pickles the unique crisp texture and sour flavor (Pan et al., 2017). Pickles are rich in natural lactic acid bacteria that play a key role in developing the pickle flavor and quality. Specific lactic acid bacteria are also used as probiotics and have various benefits for human health, including preventing constipation, colitis, and weight loss (Chen et al., 2012; Chen et al., 2013). The differences in lactic acid bacterial species in pickles can be caused by such factors as region, climate, and method of production. Additional strains should be isolated and identified, and abundant microbiological resources should be accumulated, which could increase our repertoire of industrial and probiotic species. Lactobacillus strains in Sichuan pickles have been isolated and identified. The primary strains of Lactobacillus that have been identified are L. plantarum, L. pentosus, L. brevis, L. casei, L. fermentum, L. acidophilus, L. coryniformis, L. salivarius, and L. alimentarius, with the identified cocci primarily being L. mesenteroides, and P. pentosaceus (Yang et al., 2015). The most widely reported species are L. plantarum and L. mesenteroides, which represent the most common species in Sichuan pickles. Our lab also observed that Sichuan pickles contain Lactobacillus plantarum, in agreement with existing reports (Yang et al., 2015; Chen et al., 2012), what were isolated and identified additional fermented lactic acid bacterial strains from pickles produced in different regions of China Sichuan. Furthermore, numerous functional activities of L. plantarum (Chen et al., 2018; Zhao et al., 2018b) and specific in vitro activities of the bacteria were studied (Yang et al., 2015).
Ulcerative colitis (UC) is an inflammatory bowel disease that is primarily characterized by abdominal pain, diarrhea and bloody stool. Although the pathogenesis of UC has not been thoroughly characterized to date, it is a common disease that is well studied using animal models (Nong and Huang, 2014). Among these models, the dextran sulfate sodium (DSS)-induced colitis model is similar to clinical UC and is the most ideal UC model (Wen et al., 2011). Prolonged oxidative stress in the intestine will lead to intestinal damage. However, certain lactic acid bacteria in the intestine have antioxidant activities, eliminating free radicals to relieve UC (Li et al., 2011). When inflammation is aggravated, intestinal mucosal permeability is greatly increased (Olson et al., 2006; Arrieta et al., 2009). Investigations have shown that one way in which lactic acid bacteria alleviate DSS-induced colitis is through improving intestinal permeability (Aubry et al., 2016). Clinical studies have also reported that lactic acid bacteria can significantly facilitate the treatment of UC and effectively enhance the therapeutic effects of treatments (Nishida et al., 2017). Although Lactobacillus can alleviate UC in many ways, the associated mechanisms have not been well studied. In addition, little research has been performed on the discovery of novel probiotic lactic acid bacteria.
In this study, the lactic acid bacterium Lactobacillus plantarum CQPC07 (LP) was isolated and identified from natural fermented Sichuan pickles in Chongqing, China, its inhibitory effect on DSS-induced colitis in mice was studied. The serum and colon tissues of mice were analyzed, and the effects of LP on colitis mice were detected by molecular biological methods, particularly to assess changes in colitis-related gene expression. In this study, we evaluated the effects of LP on the inhibition of DSS-induced colitis in mice.
Identification of microbial strain One gram of pickled vegetables that had been naturally fermented for 7 days was added to 10 mL of distilled water, which was then mixed by crushing and subsequently filtered. One milliliter of the filtered pickle water was collected and serially diluted 10-fold in sterile saline to a 10−6 dilution, after which 100 µL aliquots of 10−4, 10−5 and 10−6 dilutions were spread on plates and grown at 37 °C for 48 h to observe the colony morphology. Next, single colonies were selected for streak culturing, which was repeated three times to obtain isogenic strains. Pure strains were inoculated into MRS broth and cultured at 37 °C for 24 h. Genomic DNA was extracted from cells using a DNA extraction kit (Invitrogen, Carlsbad, CA, USA), and the extracted DNA was used to PCR amplify the 16S rRNA gene. The amplified products were sequenced to identify the strains (Liu et al., 2017). The strain L. bulgaricus (CCTCC AB 200048), which was purchased from CCTCC (China Center for Type Culture Collection, Wuhan, Hubei, China), was also used in this study.
Determination of Lactobacillus tolerance to artificial gastric juice and bile salt The tolerance of LP and L. bulgaricus to artificial gastric juice and bile salt was determined using the methods of Qian et al. (2018). The formula used to calculate the tolerance of Lactobacillus to artificial gastric juice was as follows: (%) = viable count (CFU/mL) at 3 h/viable count (CFU/mL) at 0 h × 100 %. The formula used to calculate the bile salt tolerance of the lactic acid bacteria was as follows: (%) = bile salt culture medium OD600/ blank medium OD600 × 100.
Animal experiments C57BL/6J mice (male, seven-week-old) were purchased from the Experimental Animal Center of Chongqing Medical University (License No. SYXK 2012-0001). Mice were fed in environmental conditions including a temperature of 25 ± 2 °C, a relative humidity of 50 ± 5 %, and a 12 h light/dark cycle. One week after feeding (Table 1), C57BL/6J mice weighing 25 ± 2 g were selected and divided into the following 5 groups: the normal group, the model group, the LPL group (low concentration of LP group, 1 × 108 CFU/kg b.w.), the LPH group (high concentration of LP group, 1 × 109 CFU/kg b.w.) and the sulfasalazine (Sigma, Burlington, MA, USA) group (positive control group). During the 5-week experimental period, mice in the normal group were given free access to drinking water and food. The mice in the model group were given 2 % (w/v) aqueous solution of DSS at weeks 3 for adaptation to DSS, then the mice continued to give 4 % (w/v) aqueous solution of DSS at weeks 5, and were provided free access to water and food thereafter. In the 5-week experimental period, the mice in the LPL and LPH groups were gavaged with 0.2 mL of liquid containing 1 × 108 and 1 × 109 CFU/kg b.w., respectively; and the mice in the sulfasalazine group were given sulfasalazine orally every day at a concentration of 20 mg/kg b.w. and were treated with DSS aqueous solution as the model group. (Chen et al., 2017b). After 5 weeks, the mice were anesthetized, the neck of the mouse was squeezed to congest the posterior orbital venous plexus, the blood was taken from the inner canthus of mice by means of capillary puncture. After taking blood from mice, the mice were killed by cervical dislocation, and after subsequent dissections, the colon mass and length for each mouse were determined. The disease activity index (DAI) of mice was calculated as DAI = (body weight loss score + stool trait score + stool blood score)/3 (Table 2). The present study was approved by the Animal Ethics Committee of Chongqing Medical University (SYXK (Yu) 2017-0001).
| Formula | Content (%) |
|---|---|
| Wheat | 39.250 |
| Nonfat milk powder | 12.000 |
| Soybean meal | 6.950 |
| Vegetable oil | 3.450 |
| Beer yeast | 2.400 |
| Wheat germ | 33.00 |
| Salt | 1.375 |
| Calcium bicarbonate | 1.000 |
| Ferric citrate | 0.075 |
| Vitamin | 0.500 |
| Weight loss (%) | Stool trait | Stool blood | Dividing value |
|---|---|---|---|
| No loss | Normal | Occult blood (−) | 0 |
| 1–5 | Semidilute stool | Occult blood (−) | 1 |
| 6–10 | Semidilute stool | Occult blood (+) | 2 |
| 11–15 | Dilute Stool | Occult blood (+) | 3 |
| >15 | Dilute Stool | Macroscopic blood | 4 |
Determination of serum hormone and cytokines in mice The blood samples were incubated at room temperature for 30 min and then at 4 °C for 4 h (Qian et al., 2013). After centrifugation at 4 500 rpm for 15 min, the serum samples were obtained, and the serum hormone levels of endothelin (ET; No. LYD30382, Beijing Lvyuan Bode Biological, Beijing, China), somatostatin (SS; No. H092, Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), substance P (SP; No. H218, Nanjing Jiancheng Bioengineering Institute), vasoactive intestinal peptide (VIP; No. H219, Nanjing Jiancheng Bioengineering Institute) and the serum cytokine levels of interleukin-2 (IL-2; No. AD2771Mo, Beijing Chenglin Biotechnology Co., Ltd., Beijing, China), interleukin-10 (IL-10; No. AD2776Mo, Beijing Chenglin Biotechnology Co., Ltd.), interleukin 1 beta (IL-1β; No. AD3364Mo, Beijing Chenglin Biotechnology Co., Ltd.), tumor necrosis factor alpha (TNF-α; No. AD3051Mo, Beijing Chenglin Biotechnology Co., Ltd.) were measured using the indicated kits.
Determination of MPO, GSH, MDA and SOD levels in mice Colon tissue (including mucosa and muscle layer) was homogenized by ultrasonic pulverization after being mixed 1:9 with normal saline, and the levels of myeloperoxidase (MPO; No. A044, Nanjing Jiancheng Bioengineering Institute), glutathione (GSH; No. A006-1, Nanjing Jiancheng Bioengineering Institute), malondialdehyde (MDA; No. A003- 1, Nanjing Jiancheng Bioengineering Institute) and superoxide dismutase (SOD; No. A001-3, Nanjing Jiancheng Bioengineering Institute) in colon tissue were determined using the indicated kits.
Pathology experiment Colon tissues collected 2 cm from the anus of each mouse were fixed with 10 % formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). The colon tissues were subsequently observed under a microscope (Olympus BX43, Olympus Co., Tokyo, Japan) for histological assessments.
Reverse transcription-polymerase chain reaction (RT-PCR) assay After lysing the cells in the mouse colon tissue samples, the total RNA was extracted with RNAzol (Invitrogen, Carlsbad, CA, USA), and the extracted RNA was subsequently diluted to 1 µg/µL. Five microliters of the diluted total RNA was used to make cDNA following the instructions of the reverse transcription kit. RT-PCR reactions contained 2 µL of cDNA template, 10 µL of SYBR Green PCR Master Mix (MedChemExpress, Monmouth Junction, NJ, USA) and 1 µL each of specific forward and reverse primers (Thermo Fisher Scientific, New York, NY, USA) to detect the expression of neuronal nitric oxide synthase (nNOS), forward (5′-ATGTCCTCAAAGC-CATCCAG-3′) and reverse (5′-ACTCAGATCTAAGGCGGTTG-3′); endothelial nitric oxide synthase (eNOS), forward (5′-TGTCTGCGGCGATGTCACT-3′) and reverse (5′-CATGCCGCCCTCTGTTG-3′); inducible nitric oxide synthase (iNOS), forward (5′-CAGCTGGGCTGTACAAACCTT-3′) and reverse (5′-CATTGGAAGTGAAGCGTTTGG-3′); c-Kit, forward (5′-CATAGCCCAGGTAAAGCACAAT-3′) and reverse (5′-GAACACTCCAGAATCGTCAACTC-3′); stem cell factor (SCF), forward (5′-TCAGGGACTACGCTGCGAAAG-3′) and reverse (5′-AAGAGCTGGCAGACCGACTCA-3′); IL-8, forward (5′-CTAGGCATCTTCGTCCGTCC-3′) and reverse (5′-TTGGGCCAACAGTAGCCTTC-3′); and C-X-C motif chemokine receptor 2 (CXCR2), forward (5′-TCTGCTCACAAACAGCGTCGTA-3′) and reverse (5′-GAGTGGCATGGGACAGCATC-3′). The thermocycling conditions were as follows: 95 °C for 15 s and 55 °C for 60 sec, followed by 40 cycles of 95 °C for 30 s, 55 °C for 35 s and 72 °C for 35 s (Step One Plus Real-Time PCR System, Thermo Fisher Scientific, New York, NY, USA) (Zhao et al., 2018a). GAPDH expression was evaluated using specific primers (forward [5′-TGCACCACCAACTGCTTAG-3′] and reverse [5′-GATGCAGGGATGATGTTC-3′]) a reference to calculate the relative expression levels of the assayed genes according to the formula 2−ΔΔCt.
Statistical analysis The data of 10 mice in each group were measured and the data were presented as the means ± standard deviation of three individual experiments for every mouse. Significant differences between groups were evaluated by one-way analysis of variance followed using Duncan's post hoc test for multiple comparisons. The analysis was performed using SAS statistical software version 9.1 (SAS Institute Inc., Cary, NC, USA). Differences were considered significant at P < 0.05 (Chen et al., 2017a).
Isolation and identification of microorganisms The results showed that most of the isolated colonies were round and white or milky white. The edges of the colonies were distinct, and the surface of the colonies was wet and smooth (Fig. 1A). The strain was identified as a lactic acid bacterium by microscopic examination using the Gram staining method, with long and short rods observed without budding (Fig. 1B). The sequencing results showed that the strain was 99 % homologous to the known fermented lactic acid bacteria in the GenBank database (Gene Bank accession number: NZ_GL575020.1), and as this strain represented a novel strain of lactic acid bacteria, the strain was named Lactobacillus plantarum CQPC07. The Lactobacillus plantarum strain was subsequently submitted to the China General Microbiological Culture Collection Center with the preservation number CGMCC 14956.
Colony morphology (A) and Gram staining result (B) of Lactobacillus plantarum CQPC07.
Artificial gastric juice and bile salt resistance activities The observed tolerance of LP in the artificial gastric juice (pH 3.0) (91.76±7.92 %) and in 0.3 % bile salt (11.91±0.20 %) was better than that of a common L. bulgaricus strain (27.65±5.61 and 2.41±0.33 %, respectively). To be useful as probiotics, lactic acid bacteria need survive the stomach and small intestine to colonize the colon. To pass through the stomach, the bacteria must be able to tolerate the gastric acid, and in the small intestine, they must tolerate the bile salts present in the bile (Qian et al., 2018). LP exhibited better tolerance to artificial gastric juice and bile salt than did commercial L. bulgaricus. Thus, the novel strain LP should be further studied.
Effect of LP on colitis symptoms As shown in Table 3, after inducing colitis in mice using DSS, the DAI value of mice increased, with the highest values observed for the model group. DSS-induced colitis mice treated with LP exhibited decreased DAI values compared to the model group, and the LPH group showed the stronger decreasing effect than the LPL group. Furthermore the DAI values of mice in LPH group were close to those of the drug sulfasalazine group, no significant difference was observed between the two groups (P > 0.05). The DAI value is a good indicator of colitis in animal models, where the higher the DAI value, the more severe the colitis is (Sun et al., 2017). Based these results, LP was observed to have specific inhibitory effects on colitis.
| Group | 21st day (2% DSS treatment) | 35th day (4% DSS treatment) |
|---|---|---|
| Normal | 0.00±0.00d | 0.00±0.00d |
| Model | 1.83±0.32a | 2.92±0.41a |
| LPL | 1.51±0.18b | 2.03±0.28b |
| LPH | 1.37±0.26c | 1.37±0.31c |
| Sulfasalazine | 1.21±0.23c | 1.32±0.25c |
Presented values are the means ± standard deviation (N=10/group).
LPL: Lactobacillus plantarum CQPC07 low dose (1.0×108 CFU/kg b.w.); LPH: Lactobacillus plantarum CQPC07 high dose (1.0×109 CFU/kg b.w.); and sulfasalazine: 20 mg/kg b.w. sulfasalazine dose.
Colon length and weight of mice As shown in Table 4, mice in the model group exhibited the shortest colon length and the highest colon weight to colon length ratio, whereas mice in the normal group showed the longest colon length and the lowest colon weight to colon length ratio. Interestingly, the LPH group also had a longer colon length and a lower colon weight to colon length ratio than those observed the LPL group. The LPH group also exhibited slightly higher colon weight to colon length ratios than did those observed in sulfasalazine-treated group, although no significant difference was observed. The DSS-induced colitis mice showed differences in colon weight and colon length compared with those of normal group; thus, the colon weight to colon length ratio was used as an important criterion to assess the degree of experimental UC in mice in combination with UC colonic length. The colonic length values tended to decrease in mice with DSS-induced colitis compared with normal group, while the colon weight to colon length ratios were higher in DSS-induced colitis mice than in normal mice (Strober et al., 2002). The results obtained in this study supported previous observations that DSS-induced colitis can cause shorter colon and higher colon weight to weight ratios in mice and that LP can significantly reduce these changes in mice.
| Group | Colon length | Colon weight | Colon weight / Colon length |
|---|---|---|---|
| Normal | 9.4±0.4a | 162.6±6.5a | 17.3±1.4d |
| Model | 4.1±0.3d | 168.5±11.4a | 41.1±2.6a |
| LPL | 5.3±0.5c | 186.6±12.3a | 35.2±2.2b |
| LPH | 7.7±0.5b | 186.5±11.2a | 24.2±1.6c |
| Sulfasalazine | 8.1±0.3b | 183.1±10.8a | 22.6±1.8c |
Presented values are the means ± standard deviation (N=10/group).
LPL: Lactobacillus plantarum CQPC07 low dose (1.0×108 CFU/kg b.w.); LPH: Lactobacillus plantarum CQPC07 high dose (1.0×109 CFU/kg b.w.); and sulfasalazine: 20 mg/kg b.w. sulfasalazine dose.
Measurements of serum levels of ET, SS, SP and VIP in mice As shown in Table 5, the serum levels of ET and SP in normal group were the lowest among the assayed groups, whereas the levels of SS and VIP were the highest in this group. In contrast, Model group showed the opposite trends, exhibiting the lowest SS and VIP levels and the highest ET SP levels among the assayed groups. A reduction in ET, SP and increased SS, VIP levels can inhibit colitis. The levels of ET and SP in mice from the LPH group were lower than those in the LPL model groups. The colitis could raise ET, SP and reduce SS, VIP levels, from these results, LPH could reduce the colitis, and these effects were stronger than LPL. In addition, mice from the LPH group had higher SS and VIP levels than those observed in the LPL and model groups. Endothelin is a long-lasting and potent vasoactive peptide (Wang et al., 2015). Studies have shown that ET-induced vasoconstriction causes colon mucosal erosion, and in severe cases, it causes ulcers and plays a key role in the pathogenesis of UC (Utsumi et al., 2016). SS is an important gastrointestinal hormone that can inhibit the secretion of gastric acid and other gastrointestinal fluids; therefore, by reducing secretion of gastrointestinal fluid, gastrointestinal inflammation can be reduced (Legaki et al., 2016). Substance P is a neurocutaneous substance with immunomodulatory effects. The regulation of the nervous and immune systems by substance P may play a role in the regulation of colitis, and the massive accumulation of substance P may exacerbate colitis (Utsumi et al., 2016). In addition, studies have shown that substance P can induce colitis, while inflammation associated with colitis is alleviated after antagonizing substance P in animals (Fang et al., 2017). The results showed that VIP level was negatively close to severity of colitis. The decrease in VIP levels directly leads to disorders in immune regulation and the aggravation of inflammation in UC (Legaki et al., 2016). Furthermore, VIP can inhibit iNOS transcription in the body to prevent iNOS in colon tissue from leading to excessive NO production and intestinal mucosal injury (Suo et al., 2015). Thus, LP can inhibit UC by lowering ET and SP levels and by increasing the levels of SS and VIP, similar to the effects elicited by the drug sulfasalazine.
| Group | ET (pg/mL) | SS (pg/mL) | SP (pg/mL) | VIP (pg/mL) |
|---|---|---|---|---|
| Normal | 5.42±0.39e | 64.55±3.62a | 31.08±2.51e | 74.18±3.39a |
| Model | 15.20±1.32a | 33.78±2.89d | 78.15±4.32a | 34.05±2.11d |
| LPL | 12.03±1.52b | 41.97±3.02c | 61.20±3.86b | 46.31±2.26c |
| LPH | 8.39±0.47c | 52.82±3.63b | 48.55±2.80c | 61.02±3.17b |
| Sulfasalazine | 7.25±0.45d | 55.21±3.66b | 40.18±2.55d | 64.32±3.61b |
Presented values are the means ± standard deviation (N=10/group).
LPL: Lactobacillus plantarum CQPC07 low dose (1.0×108 CFU/kg b.w.); LPH: Lactobacillus plantarum CQPC07 high dose (1.0×109 CFU/kg b.w.); and sulfasalazine: 20 mg/kg b.w. sulfasalazine dose.
Serum cytokine levels in mice As shown in Table 6, mice in the model group had the lowest levels of IL-2 and the highest levels of IL-10, IL-1β, and TNF-α. After treatment with LP or sulfasalazine, the level of IL-2 level was increased, while the levels of IL-1β and TNF-α levels decreased compared to the those observed in the model group. The effects observed in the LPH group were greater than those observed in the LPL group but lower than those determined for the sulfasalazine group. IL-2 is a cytokine secreted by Th2 cells and is directly related to UC. The immediate immune response by Th2 cells affects UC, and IL-2 plays a role in suppressing inflammation by affecting Th2 cells, alleviating the degree of UC (Alex et al., 2009; Matsuoka et al., 2004; Mańkowska-Wierzbicka et al., 2015). IL-10 is a cytokine secreted by Treg cells that has immunosuppressive effects. IL-10 has a proinflammatory role in UC-associated inflammation and exacerbates UC. LP can enhance the level of IL-2 and reduce the level of IL-10, which can inhibit inflammation and play an important role in reducing the UC process (Mańkowska-Wierzbicka et al., 2015). After the onset of UC, a large amount of IL-1β is produced in the colonic tissues. IL-1β can promote the activation and degranulation of neutrophils, thereby releasing platelet-activating factor and prostaglandin from inflammatory cells, and eventually aggravating intestinal mucosal inflammation. IL-1β also promotes the activation of macrophages and the release of inflammatory cells; aggravates intestinal mucosal inflammation; causes congestion, edema, weakening of barrier function; and can also cause intestinal bacterial translocation, which is conducive to the roles of inflammatory factors (Zhang et al., 2005). TNF-α can induce endothelial cells to produce platelet activating factor and the chemokine IL-8, this situation can promote the adhesion of circulating neutrophils, lymphocytes mononuclear macrophages to vascular endothelial cells, and this leads to the migration of normal cells and infiltration of local tissues, leading to inflammatory reactions (Honda et al., 2016). In addition, TNF-α can cause small blood vessels to form micro-thrombus due to inflammation, leading to mucosal microcirculation disorders, weakening the barrier function of intestinal mucosa and aggravating colitis (Wang et al., 2010).
| Group | IL-2 (pg/mL) | IL-10 (pg/mL) | IL-1β (pg/mL) | TNF-α |
|---|---|---|---|---|
| Normal | 208.17±22.19a | 110.56±13.47e | 22.32±2.59e | 121.03±9.30e |
| Model | 78.20±12.14e | 752.34±26.52a | 57.89±4.42a | 573.61±22.36a |
| LPL | 117.83±21.08d | 548.71±22.67b | 45.62±3.06b | 422.45±18.79b |
| LPH | 159.71±19.83c | 263.17±23.97c | 33.10±2.26c | 238.79±12.03c |
| Sulfasalazine | 183.29±15.79b | 187.83±25.31d | 28.36±2.12d | 202.41±8.97d |
Presented values are the means ± standard deviation (N=10/group).
LPL: Lactobacillus plantarum CQPC07 low dose (1.0×108 CFU/kg b.w.); LPH: Lactobacillus plantarum CQPC07 high dose (1.0×109 CFU/kg b.w.); and sulfasalazine: 20 mg/kg b.w. sulfasalazine dose.
Oxidation-related molecule levels in colon tissue from mice As shown in Table 7, compared with the normal group, after inducing colitis, the levels of MPO and MDA in colon tissue from mice in the model group were increased, while those of GSH and SOD were decreased. The levels of MPO and MDA in DSS-induced colitis mice were increased, while those of GSH and SOD were increased by LP and sulfasalazine, with greater effects observed in animals treated with sulfasalazine than with LPH. When DSS causes inflammation of the colon, the aggregation of neutrophils in inflammatory tissues begins to decrease, and more neutrophils enter into the tissues. This process is manifested by a significant increase in MPO activity (Mustafa et al., 2006). DSS causes a large number of free radicals in the colon, including reactive oxygen specie (ROS) and reactive nitrogen species (RNS). These large amounts of free radicals cause damage and toxic reactions in the colon. With the increase of free radicals, the degree of inflammation of colitis also increases (Osman et al., 2008). After oxidative damage of colon tissue, the level of GSH in colon tissue decreases greatly, while the peroxide product MDA increases greatly. (Fiocchi et al., 2004). Clinical study have also shown that UC can cause colon dysfunction under oxidative stress and decrease the activity of SOD in decomposing free radicals (Chao et al., 2017). Depletion of GSH happens before tissue destruction by ROS, studies show that antioxidant substances can prevent tissue destruction under GSH-depletion conditions (You and Park, 2013; Meo et al., 2016). The results of this study also confirmed that LP may also elevate GSH levels prior to ROS-induced intestinal injury, and inhibit oxidative stress-induced intestinal injury by elevating SOD activity and lowering MPO and MDA levels after intestinal injury. LP significantly inhibited the oxidative stress response of UC in the colon and inhibited colitis.
| Group | MPO (mU/mg tissue) | GSH (µmol/mg tissue) | MDA (nmol/mg tissue) | SOD (U/mg tissue) |
|---|---|---|---|---|
| Normal | 5.53±0.31d | 7.82±0.45a | 0.31±0.06d | 95.26±4.11a |
| Model | 33.59±2.18a | 2.51±0.29d | 1.79±0.23a | 30.82±3.14d |
| LPL | 19.08±2.30b | 4.17±0.28c | 0.99±0.14b | 58.71±4.23c |
| LPH | 10.33±1.69c | 6.69±0.33b | 0.67±0.15c | 73.17±4.03b |
| Sulfasalazine | 9.36±1.08c | 6.87±0.39b | 0.62±0.12c | 76.79±3.22b |
Presented values are the means ± standard deviation (N=10/group).
LPL: Lactobacillus plantarum CQPC07 low dose (1.0×108 CFU/kg b.w.); LPH: Lactobacillus plantarum CQPC07 high dose (1.0×109 CFU/kg b.w.); and sulfasalazine: 20 mg/kg b.w. sulfasalazine dose.
Pathological observations of colon tissue from mice Colon tissues were observed after H&E staining (Fig. 2), in the model group photo, the arrow indicated that the colonic tissue of mice in model group had extensive tissue damage compared with normal mice. In the LPH group figure, under microscope, the degrees of infiltrating and necrotizing cells in colon tissue of LPL group mice were less than that of model group mice, but more than that of LPH group mice. In the sulfasalazine group photo, the degree of tissue injury was the lowest and the morphology was most close to the normal group. H&E staining can be used to directly assess the pathological changes in colitis tissue (Chen et al., 2017), and this method was also used in this study to observe the effect of LP on colonic tissue in mice with DSS-induced colitis.
H&E staining of colon tissue in DSS-induced colitis mice (100×).
LPL: Lactobacillus plantarum CQPC07 low dose (1.0×108 CFU/kg b.w.); LPH: Lactobacillus plantarum CQPC07 high dose (1.0×109 CFU/kg b.w.); and sulfasalazine: 20 mg/kg b.w. sulfasalazine dose. The arrow pointed to the place where the tissue was damaged.
nNOS, eNOS, and iNOS expression in the colon tissue of mice As shown in Fig. 3A, the mRNA expression of nNOS and eNOS in colon tissue of mice in normal group were stronger than those of mice in model group, but the expression of iNOS was lower than that of mice in model group. LP and sulfasalazine increased nNOS, eNOS and reduced iNOS expression in mice compared to that observed in the DSS-induced colitis model group mice. These effects observed in the LPH group were close to those observed in mice from the sulfasalazine group and were significantly (P < 0.05) greater than those observed in the LPL group. Studies have shown that eNOS can control the production of moderate amounts of NO to maintain the normal state of colonic tissue (Raimura et al., 2013; Piechota-Polanczyk et al., 2016). eNOS plays an important role in regulating colonic injury observed in colitis and can inhibit the damage to colon tissue. iNOS generates NO from decomposition of arginine, and excessive NO plays a role in the damage caused to colonic tissue (Van Crombruggen et al., 2008). nNOS can keep NO at a low level in tissue to inhibit excessive NO-induced tissue damage, and nNOS can also reduce iNOS overexpression and inhibit inflammation (Van Crombruggen et al., 2008; Suo et al., 2016). In this study, mice in the LPH group exhibited decreased levels of colitis-related damage. In these mice, the expression of nNOS and eNOS was upregulated in the colon, while the expression of iNOS was downregulated.
mRNA expression of LP-CQPC07 in colon tissue from mice. (A) nNOS, eNOS and iNOS expression, (B) c-Kit and SCF expression, (C) IL-8 and CXCR2 expression.
Same expression in each group is significantly different (*, #, ^P < 0.05; **, ##, ^^P < 0.01) from the normal group and according to Duncan's multiple-range test. LPL: Lactobacillus plantarum CQPC07 low dose (1.0×108 CFU/kg b.w.); LPH: Lactobacillus plantarum CQPC07 high dose (1.0×109 CFU/kg b.w.); and sulfasalazine: 20 mg/kg b.w. sulfasalazine dose.
c-Kit and SCF expression in the colon tissue of mice As shown in Fig. 3B, the levels of c-Kit and SCF mRNA expression were highest in the normal group, and these expression of model group were lowest. The c-Kit and SCF expression of mice from the LPH group was higher than that observed in mice from the LPL group but were only slightly lower than those of mice from the sulfasalazine group. Interstitial cells of Cajal (ICC) are one of the important targets for the treatment of gastrointestinal motility disorders (Sakurai, 2003). Gastrointestinal motility disorders in UC patients are directly related to ICC. The intervention of ICC can effectively inhibit UC and relieve gastrointestinal motility disorders (Ro et al., 2010). c-Kit is a transmembrane protein of ICC, and its ligand, SCF, is produced by neurons and smooth muscle cells. SCF plays an important role in promoting ICC growth, differentiation and the maintenance of normal physiological functions of ICC (Li et al., 2009). Improving the level of c-Kit and SCF in colon tissue is very useful for controlling UC (Galli et al., 1993).
IL-8 and CXCR2 expression in the colon tissue of mice As shown in Fig. 3C, the expression of IL-8 and CXCR2 in colon tissue from mice in the model group was the highest of all groups, whereas that in mice from the normal group was the lowest. LP and sulfasalazine treatment reduced the levels of IL-8 and CXCR2 expression in DSS-colitis induced mice, and the expression levels of these factors in the LPH group were higher than those observed in the sulfasalazine group but were lower than those seen in mice from the LPL group. IL-8 is a neutrophil chemotactic and activating factor that mediates various pathway-induced inflammatory responses, including mediating UC inflammation (Reddy and Naidu, 2016). CINC-1 is a neutrophil chemokine in the IL-8 family, and CXCR2 is a receptor for CINC-1. CXCR2 can regulate the levels of IL-8 and CINC-1 in the colon tissue of individuals with UC; thus, reducing the level of CXCR2 in colon tissue is helpful to reduce colon damage associated with UC (He et al., 2010; Shi et al., 2010). LP was observed to significantly downregulate the expression of IL-8 and CXCR2 mRNA in the colons of mice with colitis, thereby inhibiting colitis.
Lactobacillus can activate the phagocytosis activity of macrophages, meanwhile Lactobacillus can colonize in the intestines and enhance the body's immunity. Lactobacillus can also stimulate peritoneal macrophages, induce the production of interferon, and promote cell division, the production of antibodies and cellular immunity, thus allowing it to enhance the nonspecific and specific immune responses of the body and improving its disease resistance (Oh et al., 2018). In this study, LP was shown to be able to improve immune function, thereby decreasing the adverse effects of colitis.
In this study, a DSS-induced mouse colitis animal model was used to assess the ability of LP to inhibit colitis. Through assays of mouse sera and colon tissues, the results showed that LP can inhibit the decrease in colon length and the colon weight to colon length ratio associated with DSS-induced colitis in mice. LP can reduce the levels of ET and SP and increase the levels of SS and VIP in the sera of mice with colitis, and LP could also increase the level of IL-2 and decrease the level of IL-10 in serum. The results of colon tissue evaluations showed that LP could enhance the activities of GSH and SOD and reduce the activities of MPO and MDA in mice with DSS-induced colitis. Thus, LP can inhibit the oxidative stress in colon tissue caused by DSS. Furthermore, the quantitative PCR results showed that LP upregulated the expression of nNOS, eNOS, c-Kit, and SCF mRNA and downregulated the expression of iNOS, IL-8 and CXCR2 in the colons of mice with colitis. Thus, our results show that LP can inhibit DSS-induced colitis in mice. The concentration and effect of LP used was related, in which the higher the concentration used, the better the effect. Furthermore, the observed effects using a high concentration of LP were similar to those observed using the colitis drug sulfasalazine. Thus, LP is a lactic acid bacterium that can inhibit colitis and should be further developed and utilized.
Acknowledgments This research was supported by the Open Fund of Beijing Advanced Innovation Center for Food Nutrition and Human Health (20161001), China.
