The aim of this study was to investigate the anti-inflammatory effects by ursodeoxycholic acid (UDCA) in rats with a spinal cord injury (SCI). A moderate mechanical compression injury was imposed on adult Sprague-Dawley (SD) rats. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale and the tissue volume of the injured region was analyzed using hematoxylin and eosin staining. The pro-inflammatory factors were evaluated by immunofluorescence (IF) staining, a quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 in mitogen-activated protein kinase (MAPK) signaling pathways related to inflammatory responses were measured by Western blot assays. UDCA improved the BBB scores and promoted the recovery of the spinal cord lesions. UDCA inhibited the expression of glial fibrillary acidic protein (GFAP), tumor necrosis factor-α (TNF-α), ionized calcium-binding adapter molecule 1 (iba1), and inducible nitric oxide synthase (iNOS). UDCA decreased the pro-inflammatory cytokines of TNF-α, interleukin 1-β (IL-1β), and interleukin 6 (IL-6) in the mRNA and protein levels. UDCA increased the anti-inflammatory cytokine interleukin 10 (IL-10) in the mRNA and protein levels. UDCA suppressed the phosphorylation of ERK, JNK, and the p38 signals. UDCA reduces pro-inflammatory responses and promotes functional recovery in SCI in rats. These results suggest that UDCA is a potential therapeutic drug for SCI.
Spinal cord injuries (SCI) cause marked neuropathology and are associated with limited functional recovery . In addition, the injured spinal cord physiologically induces secondary damage such as apoptotic cell death of affected neurons and glia [2, 3] as well as the production of inflammatory mediators . With regard to these types of secondary damage, numerous researchers have attempted to suppress inflammatory responses after SCI [5,6,7].
While the extent of the primary injury is determined by the initial trauma, which is typically beyond the control of caregivers, the severity of secondary pro-inflammatory injuries can potentially be modulated using pharmacological agents such as methylprednisolone (MP). Although MP has been used as an anti-inflammatory drug , it causes adverse side effects, including wound infections, pneumonia, and acute corticosteroid myopathy [9, 10].
Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, has been used in Chinese medicine for more than 3000 years . UDCA was originally approved by the US Food and Drug Administration (FDA) for the treatment of several cholestatic liver disorders . Specifically, UDCA is widely used for the treatment of hepatic diseases, such as gallstones, chronic hepatitis, and primary biliary cirrhosis [13,14,15]. In other words, the effects of UDCA are mainly linked to efforts to remedy damage to hepatocytes.
In a recent study by the authors, we evaluated the anti-inflammatory effect of UDCA using LPS-stimulated RAW 264.7 macrophages in vitro . The inflammatory response in an injured human spinal cord is largely similar to that observed in rodents [17, 18]. Thus, we aimed to evaluate the effects of UDCA as an anti-inflammatory drug in SCI rats.
Materials and Methods
Eighty-one adult female Sprague-Dawley (SD) rats (200–220 g) as used in this study were purchased from Orient Bio Inc. (Seongnam, Korea), housed in a facility at 55–65% humidity and a controlled temperature of 24 ± 3 °C with a light/dark cycle of 12 h, and given free access to water and food. All surgical interventions and postoperative animal care procedures were performed in accordance with the Guidelines and Policies for Rodent Survival Surgery provided by the Institutional Animal Care Committee (IACUC) of CHA University (IACUC160074).
Modeling of Animals
The animals were anesthetized with a Zoletil® (50 mg/kg, Virbac Laboratories, France)/Rompun® (10 mg/kg, Bayer, Korea) solution administered intraperitoneally. Complete anesthesia was assessed using the hindlimb withdrawal reflex in response to a noxious foot pinch. After skin preparation and precise positioning of the anesthetized rats, the ninth thoracic spinal cord segment (T9) was exposed by laminectomy. The vertebral column was supported and stabilized by Allis clamps at the T7 and T11 spinous processes using a method described previously . A metal impounder (35 g) was then gently applied onto the T9 dura for 5 minutes, resulting in moderate standing weight compression. After the compression injury, the surgical site was closed by suturing the muscle and fascia and suturing the skin, followed by an external povidone-iodine application. All surgical procedures were performed by the same person (S Sohn), and the animals for the sham groups underwent a T9 laminectomy without weight compression injury.
We randomly divided all rats into three groups: the laminectomy-only group (n = 27, sham group), the normal saline (NS) injection after injury group (n = 27, injury + NS group), and the UDCA injection after injury group (n = 27, injury + UDCA group). The predetermined experimental day was 1, 3, or 7 days after the injury. Nine rats per group were sacrificed and analyzed each day for hematoxylin and eosin (H&E) and immunofluorescence (IF) staining (n = 3), quantitative real-time polymerase chain reactions (qRT-PCR, n = 3), enzyme-linked immunosorbent assay (ELISA), and Western blot assays (n = 3). The bladders of the injured animals were manually emptied twice daily until normal function returned.
Administrations of UDCA
Sodium ursodeoxycholate hydrate was obtained from TCI (Tokyo Chemical Industry Co., Ltd., Japan) and solubilized in sterilized NS for the injections. A total of 25 mg/kg/day of UDCA (in a volume of 250 μL) or 250 μL of NS was injected intraperitoneally immediately after SCI in the injury + UDCA and injury + NS groups. The solutions were then also given once a day for 3 or 7 days at the indicated time points, as described in Fig. 1. The UDCA solutions were prepared immediately before the experiments. Three rats per group on predetermined days were anesthetized and sacrificed by intra-cardiac perfusion with saline for H&E and IF staining. The other six rats per group were euthanized by CO2 asphyxiation and the spinal cords were removed for qRT-PCR and Western blot assays.
Briefly, two trained investigators who were blind to the experimental conditions performed the behavioral analyses. To test the hindlimb locomotor function, open-field locomotion was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotion scale, as previously described . BBB is a 22-point scale (with scores of 0–21) that systematically and logically follows the recovery of hindlimb function from a score of 0, indicative of no observed hindlimb movements, to a score of 21, representative of a normal ambulating rodent. Five rats per group were measured daily for 7 days.
Tissue Preparation for Histological Analysis
On predetermined days, three rats per group were perfused transcardially with PBS followed by 4% paraformaldehyde in PBS via a method previously described . The length of the spinal cord was 10 mm from the rostral to caudal sections, including the epicenter lesion. The spinal cords were post-fixed overnight in the same 4% paraformaldehyde, embedded in paraffin, and dewaxed for a histological analysis. Serial longitudinal sections through the dorsoventral axis of the spinal cord (5 μm thick) were collected at lengths of every 100 μm (20th) and 105 μm (21st) per rat. The 20th section for each rat was used for H&E staining and for quantification of the tissue volume. The 21st section for each rat was used for IF staining and for quantification of the intensity levels. At each section, the most severely injured site of the spinal cord was selected as the region of interest (ROI, 500 × 300 μm). The morphological changes of the H&E-stained tissues and IF images of glial fibrillary acidic protein (GFAP), tumor necrosis factor-α (TNF-α), ionized calcium-binding adapter molecule 1 (iba1), and inducible nitric oxide synthase (iNOS) were observed at × 40 (scale bar, 500 μm) and × 400 (scale bar, 50 μm) magnification using a light microscope and an inverted fluorescence microscope, respectively (Olympus IX71, Japan).
Quantification of Tissue Volume
The serial longitudinal sections through the dorsoventral axis of the spinal cord (5 μm thick) were collected at lengths of every 100 μm (20th), and the 20th section was stained with an H&E solution (BBC Biochemical, Mount Vernon, USA). On the 20th section for three rats per group/predetermined day, we randomly designated two rectangular areas (200 × 125 μm, for a total of six images/group/1, 3, or 7 days) in the ROI (500 × 300 μm) for quantification of the tissue volume. The randomly and blindly selected rectangular area of 200 × 125 μm in the ROI was represented 100% and the filled tissue volume was calculated and quantified using the ImageJ software (http://rsb.info.nih.gov/ij/index.html, ImageJ; National Institutes of Health (NIH), Bethesda, MD, USA).
Immunofluorescence Staining of GFAP, TNF-α, iba1, and iNOS
Serial longitudinal sections through the dorsoventral axis of the spinal cord (5 μm thick) were collected every 105 μm (21st) and the 21st section was double-stained against GFAP and TNF-α. Every 110 μm (22nd), the 22nd section was double-stained against iba1 and iNOS. The 21st sections were treated with a blocking solution to prevent a nonspecific binding reaction for 1 h and then stained by incubation overnight at 4 °C with the following primary antibodies in PBS: monoclonal mouse anti-mouse GFAP (1:200; Sigma, St. Louis, USA) and polyclonal anti-rabbit TNF-α (1:200; Abcam, Cambridge, UK). The 22nd sections were treated with polyclonal anti-goat iba1 (1:200; Abcam) and polyclonal anti-rabbit iNOS (1:200; Abcam). The 21st sections were then incubated with fluorescent secondary donkey anti-mouse Alexa 568 and donkey anti-rabbit Alexa 488 in a blocking solution (1:400; Invitrogen Life Sciences, Carlsbad, CA, USA) for 1 h at room temperature. The 22nd sections were incubated with fluorescent secondary donkey anti-goat 594 and donkey anti-rabbit Alexa 488. At the end of the process, nuclei were stained with DAPI and sections were washed with PBS and mounted with a specific medium (Dako Cytomation, Milan, Italy).
Quantification of GFAP, TNF-α, iba1, and iNOS
Serial longitudinal sections through the dorsoventral axis of the spinal cord (5 μm thick) were collected every 105 and 110 μm (21st and 22nd) from three rats per group/predetermined day. We randomly designated two rectangular areas (200 × 125 μm, for a total of six images/group/1, 3, or 7 days) in the ROI (500 × 300 μm) for the quantification of the GFAP, TNF-α, iba1, and iNOS intensities in × 400 magnifications. The area of the captured images under × 400 magnification (200 × 125 μm) was 1,392,640 (1360 × 1024) pixels. The stained immunofluorescence pixels per image were calculated and quantified using an intensity measurement method available in the ImageJ software package (NIH) .
RNA Extraction and qRT-PCR
Segments of the spinal cord (10 mm) including the lesion epicenter were collected at 1, 3, and 7 days after SCI. The segments were homogenized using a T 25 digital homogenizer (IKA, Seoul, Korea) in TRIzol reagent (Invitrogen, Life Sciences, Carlsbad, CA, USA) according to the manufacturer’s instructions for RNA extraction. Complementary DNA (cDNA) was synthesized from 1 μg of the total RNA using a synthesis kit (Takara Bio, Shiga, Japan) and qRT-PCR was conducted using an ABI StepOne Real-time PCR System (Applied Biosystems, Warrington, UK). The PCR protocol consisted of 40 cycles of denaturation at 95 °C for 15 s, followed by 60 °C for 30 s to allow for extension and amplification of the target sequence. The relative expression levels of TNF-α, IL-1β, IL-6, and IL-10 were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the 2-ΔΔCT method . The primers were obtained from Bioneer (Daejeon, Korea). The primer sequences used in this study are shown in Table 1. These experiments were repeated three times.
Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting
Segments of the spinal cord (10 mm) including the lesion epicenter at the middle were collected at 1, 3 and 7 days after SCI. The segments were collected and washed with PBS, placed at 4 °C, and homogenized using a T 25 digital homogenizer (IKA) in lysis buffer (1× RIPA lysis buffer), after which they were centrifuged at 14,000 rpm at a temperature of 4 °C for 15 min. Protein concentrations in tissue lysates were measured with the aid of a BCA protein assay kit (Thermo Scientific, Rockford, IL). The ELISA kits for TNF-α, IL-1β, IL-6, and IL-10 were from Koma Biotech (Seoul, Korea). The content of each segment was obtained according to the standard curve at 450 nm. For the Western blot assay, equal amounts of protein (40 μg) were subjected to SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with 5% skim milk for 1 h to block the nonspecific binding and then probed with the primary antibodies of the phosphorylated forms of extracellular signal-regulated kinase (p-ERK, 1:1000), c-Jun N-terminal kinase (p-JNK, 1:1000), and p38 (p-p38, 1:1000). Subsequently, equal membranes were stripped and reprobed with the total forms of ERK (t-ERK, 1:1000), JNK (t-JNK, 1:1000), and p38 (t-p38, 1:1000). All of the primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) except for β-actin (1:2000, Abcam, Cambridge, UK). As an internal control, β-actin was also probed into the membranes. All primary markers were followed by incubation with secondary antibodies (1:5000, Santa Cruz Biotechnology, Dallas, TX). The visualized signal bands were detected using an ECL solution (Amersham, Buckinghamshire, UK) through the ChemiDoc XRS System (Bio-Rad, Hercules, USA). The phosphorylated form/total form (p/t form) volumes for the predetermined days were calculated and quantified using ImageJ software (NIH). The p/t form volume of the 1-day sham group was set at onefold and the ratio of the normalized fold change was relatively calculated and quantified for 7 days. These experiments were repeated three times.
All values are presented as the mean ± standard error of the mean (SEM). Behavioral scores were analyzed by Student’s t tests. Multiple comparisons among groups were performed with a one-way analysis of variance (ANOVA), and Tukey’s multiple-comparison test was used as a post hoc analysis method. Differences with p values for which *p < 0.05 and **p < 0.01 were considered statistically significant.
UDCA Improved Functional Recovery after SCI
To assess functional recovery by UDCA, BBB scores were measured during 1 week after injury. The BBB scores for the hindlimb motor function in the injury + NS group improved from 1.20 ± 0.45 on day 1 to 7.60 ± 1.52 on day 7 (Fig. 2). Figure 2 shows that the scores in the injury + UDCA group demonstrated a significant improvement from 2.80 ± 0.84 on day 1 to 14.20 ± 2.28 on day 7 after SCI (**p < 0.01). Figure 2 also indicates that the BBB scores in the injury + UDCA group significantly increased compared to those in the injury + NS group on each day (##p < 0.01). The scores in the sham group were constant at 21 for 7 days (data not shown).
UDCA Promoted the Recovery of Tissue Cavities after SCI
We performed H&E staining whether UDCA promoted the recovery of injured tissue. On day 1 after SCI, the spinal cord tissues showed apparent areas of structural damage in both the injury + NS and injury + UDCA groups. Distinct recovery was not shown in the injury + UDCA group on day 1 (Fig. 3a). On day 3 after SCI, however, the lesion of the tissue showed obvious cavities (as indicated by “+”) in the injury + NS group, and the volume of lesion in the injury + UDCA group was reduced (Fig. 3b). On day 7 after SCI, the cavities of the lesion in the injury + UDCA group were also reduced as compared to that observed in the injury + NS group. Figure 3d demonstrates that the tissue volume of the randomly designated ROI was significantly increased in the injury + UDCA group (67.70% ± 2.30) compared to that of the injury + NS group (54.93% ± 4.22) on day 3 (**p < 0.01). In addition, the tissue volume was significantly increased in the injury + UDCA group (84.87% ± 2.07) compared to that of the injury + NS group (72.38% ± 1.77) on day 7 (**p < 0.01).
UDCA Inhibited the Expression of GFAP, TNF-α, iba1, and iNOS after SCI
In order to evaluate whether UDCA inhibited the inflammatory responses in the lesion of spinal cord, we examined the immunoreactivities for GFAP, TNF-α, iba1, and iNOS. GFAP and TNF-α fluorescence were slightly expressed in the sham groups for 7 days (Fig. 4a–c, sham images at 3 and 7 days not shown). The fluorescence of GFAP and TNF-α in the injury groups for 7 days is also presented in Fig. 4a–c. On day 3, the GFAP intensity in the injury + UDCA group (494,181 ± 18,627) was significantly decreased as compared to that in the injury + NS group (773,477 ± 41,309, **p < 0.01, Fig. S1a). TNF-α intensity in the injury + UDCA group (217,451 ± 13,285) was also significantly decreased as compared to that in the injury + NS group (616,215 ± 34,845) after 3 days (**p < 0.01, Fig. S1b). On day 7, the GFAP intensity in the injury + UDCA group (314,541 ± 17,869) was significantly decreased as compared to that in the injury + NS group (565,384 ± 32,084, **p < 0.01, Fig. S1a). The TNF-α intensity in the injury + UDCA group (74,228 ± 4519) was also significantly decreased as compared to that in the injury + NS group on day 7 (155,997 ± 9659, **p < 0.01, Fig. S1b). The fluorescence of iba1 and iNOS in the injury groups for 7 days was presented in Fig. 5a–c. The increased iba1 and iNOS intensities by injury were decreased from 3 to 7 days by UDCA treatment (Fig. S2a, b).
UDCA Reduced the mRNA and Protein Expressions of Inflammatory Cytokines and Increased the mRNA and Protein Expression of an Anti-Inflammatory Cytokine, IL-10, After SCI
We conducted qRT-PCR and ELISA to determine whether UDCA inhibited the inflammatory responses in mRNA and protein levels. As shown in Fig. 6a–c, the inflammatory cytokines of TNF-α, IL-1β, and IL-6 were significantly inhibited by UDCA for 7 days (**p < 0.05). The TNF-α level in injury + NS groups (3.842 ± 0.154, 1.259 ± 0.104, and 0.689 ± 0.040 on days 1, 3, and 7, respectively) was increased after injury as compared to those in sham groups (1.000 ± 0.000, 0.423 ± 0.062, and 0.322 ± 0.026 on days 1, 3, and 7, respectively, Fig. 6a). However, UDCA significantly suppressed the expression of TNF-α mRNA (3.170 ± 0.166, 0.807 ± 0.043, and 0.499 ± 0.024 on days 1, 3, and 7, **p < 0.01, respectively) as compared to those in the injury + NS group (3.842 ± 0.154, 1.259 ± 0.104, and 0.689 ± 0.040 on days 1, 3, and 7, respectively, Fig. 6a). This suppression tendency by UDCA was also demonstrated in IL-1β and IL-6 mRNA (Fig. 6b, c). On the other hand, an anti-inflammatory cytokine, IL-10, was recorded at the maximum level at each time point in the injury + UDCA group (6.455 ± 0.423, 6.693 ± 0.408, and 6.969 ± 0.415 on days 1, 3, and 7, respectively, Fig. 6d). Similar inhibition tendency by UDCA was demonstrated in protein levels for 7 days (Fig. 7a–c). On the other hand, UDCA increased the anti-inflammatory cytokine such as IL-10 in protein levels (Fig. 7d).
Effect of UDCA on ERK, JNK, and p38 in Mitogen-Activated Protein Kinases After SCI
To determine whether UDCA inhibited the inflammatory signal pathways such as mitogen-activated protein kinases (MAPKs) including ERK and JNK and p38, we performed Western blot assays. The p/t volume of ERK was 6.95 ± 0.24 on day 3 in the injury + NS group, and it was significantly lower at 3.72 ± 0.18 in the injury + UDCA group (**p < 0.01, Fig. 8b). The p/t volumes of JNK and p38 in the injury + UDCA group were also significantly decreased (4.96 ± 0.27 and 1.17 ± 0.04, respectively) as compared to those in sham groups (1.24 ± 0.02 and 0.96 ± 0.02, respectively) on day 3 (**p < 0.01, Fig. 8d, f). The p/t volumes of the internal control, β-actin, were 0.97 ± 0.03 (sham group), 0.96 ± 0.01 (injury + NS group), and 0.99 ± 0.02 (injury + UDCA group) for 7 days (Fig. 8h).
In this study, UDCA promoted functional recovery in SCI rats and it ameliorated histopathological damage to spinal cords. UDCA reduced the expression levels of the pro-inflammatory cytokines of TNF-α, IL-1β, and IL-6 after SCI. UDCA also increased the expression of an anti-inflammatory cytokine, IL-10, after SCI. In addition, the phosphorylation outcomes of ERK, JNK, and p38 in MAPK signal pathways were inhibited by UDCA in SCI rats.
Following SCI, resident microglia is activated and pro-inflammatory cytokines are upregulated, including TNF-α, IL-1β, IL-6, iba1, and iNOS [24, 25]. TNF-α is one of the major inflammatory cytokines and is an important component of the acute-phase injury reaction. Bethea et al. showed that the reduction of TNF-α promoted functional recovery following SCI ; their results were in accordance with the findings here (Fig. 2). The BBB scores in the injury + UDCA group steadily increased from day 2 after SCI (Fig. 2), and the expression level of TNF-α was significantly suppressed in the injury + UDCA group as compared to that in the injury + NS group on days 1, 3, and 7 (Figs. S1b, 6a, and 7a). The reduction of TNF-α by the UDCA treatment may affect the functional recovery outcomes in our study.
SCI induces an acute activation of microglia followed by a delayed activation of astrocytes . The activated astrocytes increase the production of GFAP, which results in multiple pro-inflammatory cytokines after SCI [28, 29]. We demonstrated that the expression of GFAP was significantly suppressed in the ROI using IF staining and quantification methods (Fig. 4 and Fig. S1a).
IL-10 has been reported to suppress the production of TNF and thereby exert an inhibitory influence on the activation of monocytes and other immune cells after SCI . We verified that UDCA significantly increased IL-10 as compared to those in the injury + NS group on days 1, 3, and 7 after injury (Figs. 6d and 7d).
The phosphorylation of ERK, JNK, and the p38 signals in the MAPK pathway is a principle process during the inflammatory response after SCI . On day 3 after the injury, the increased phosphorylation levels of ERK, JNK, and p38 in the injury + NS group were significantly inhibited by the UDCA treatment (Fig. 8b, d, f). However, the phosphorylation of the MAPK signals was not significantly inhibited in the injury + UDCA group on day 1 (Fig. 8b, d, f). UDCA did not inhibit the phosphorylation of the MAPK signals in 7 days as well (Fig. 8b, d, f). These phenomena are difficult to explain; however, when we consider that the pro-inflammatory cytokines were significantly decreased in the injury + UDCA group on day 7 (Figs. S1, 6a–c, and 7a–c), the inhibited phosphorylation in the MAPK signals on day 3 could have affected the reduction of the cytokines. Further studies are warranted to elucidate the exact mechanisms of the decreased cytokines.
In our current study, rats administrated UDCA showed no abnormal behaviors, such as hyper-locomotion and head-weaving behaviors  for 7 days. Cheng et al. demonstrated that 300 mg/kg/day of UDCA did not significantly affect body weight gain or daily food intake levels in rats for 10 days . To the best of our knowledge, this is the first report to investigate the effects of UDCA in SCI rats.
Our study suggests that UDCA is a valuable anti-inflammatory agent and that it may serve as a therapeutic drug in SCI.
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This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and future Planning (NRF-2016M3A9E8941668).
Conflict of Interest
The authors declare that they have no conflict of interest.
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Ko, W., Kim, S.J., Jo, M. et al. Ursodeoxycholic Acid Inhibits Inflammatory Responses and Promotes Functional Recovery After Spinal Cord Injury in Rats. Mol Neurobiol 56, 267–277 (2019). https://doi.org/10.1007/s12035-018-0994-z
- Ursodeoxycholic acid
- Spinal cord injury
- BBB score
- MAPK signaling