THERAPEUTIC ULTRASOUND SUPPRESSES NEUROPATHIC PAIN AND UPREGULATION OF SUBSTANCE P AND NEUROKININ-1 RECEPTOR IN RATS AFTER PERIPHERAL NERVE INJURY
Yu-Wen Chen1,2, Ph.D., Jann-Inn Tzeng3,4, M.S., M.D., Po-Ching Huang5, M.S., Ching-Hsia Hung5,*, Ph.D., Dong-Zi Shao6, Ph.D., and Jhi-Joung Wang2, M.D., Ph.D.
1 Department of Physical Therapy & Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan
2 Department of Medical Research, Chi-Mei Medical Center, Tainan, Taiwan
3 Department of Food Sciences and Technology, Chia Nan University of Pharmacy and Science, Jen-Te, Tainan City, Taiwan
4 Department of Anesthesiology, Chi-Mei Medical Center, Yong Kang, Tainan City, Taiwan
5 Institute & Department of Physical Therapy, National Cheng Kung University, Tainan, Taiwan
6 Department of Cosmetics Application and Management, Chung Hwa University of Medical Technology, Tainan, Taiwan
*Corresponding author:
Ching-Hsia Hung, Ph.D.
National Cheng Kung University,
Institute & Department of Physical Therapy, No.1 Ta-Hsueh Road, Tainan, Taiwan Phone: 886-6-2353535 ext 5939 FAX: 886-6-2370411
Email: [email protected]
Abstract—We studied the mechanisms and impact of therapeutic ultrasound (TU)
against pain from nerve injury. TU began on postoperative day 5 (POD5) and then daily for the next 22 days. Sensitivity to thermal and mechanical stimuli and the levels of neurokinin-1 receptor (NK-1R), substance P (SP), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) in the sciatic nerve were examined. On POD7, chronic constriction injury (CCI) rats undergoing TU with the intensity of 1 W/cm2, but 0.25 or 0.5 W/cm2 showed an increase in mechanical withdrawal threshold and thermal withdrawal latency compared to the CCI group. Moreover, CCI rats exhibited an up- regulation of NK-1R, SP, TNF-α and IL-6 in the sciatic nerve on PODs 14 and 28, whereas TU inhibited their increased expression. The efficacy of TU is dependent on its ability to limit the up-regulation of SP, NK-1R, TNF-α and IL-6 around the injured sciatic nerve.
Key Words: Therapeutic ultrasound, Neuropathic pain, Substance P, Neurokinin-1 receptor, Tumor necrosis factor α, Interleukin-6.
INTRODUCTION AND LITERATURE REVIEW
Therapeutic ultrasound (TU) has been used for multiple therapeutic application,
including treatments involving the local region, and has been identified as a highly effective and low-cost resource to therapy trigeminal neuropathic pain (Savernini et al. 2012) and to manage carpal tunnel syndrome, the most frequent compressive
neuropathy (Kapuscinska and Urbanik 2013). It has been well established that the peripheral mononeuropathy model (e.g. CCI, chronic constriction injury) elicits hyperalgesia and allodynia in rats similar to abnormal pain sensitization observed in humans (Bennett and Xie 1988). Moreover, our previous experiment revealed that a peripheral neuropathy elicited the increased expression of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in the sciatic nerve of CCI rats (Chen et al. 2012).
Increasing evidence presumed that pro-inflammatory cytokines caused pain (Wei et al. 2013; Zelenka et al. 2005), whereas treatments with anti-inflammatory cytokines
or inhibitors of pro-inflammatory cytokines attenuated pain (Arruda et al. 2000;
Milligan et al. 2005; Schafers and Sommer 2007).
The varieties of mechanisms trigger pain perception after nerve and tissue injuries, including sensitization of pain receptors, spontaneous ectopic firing in peripheral nociceptive neurons, and alterations in gene expression of receptors and ion channels within nociceptors and neurons (Campbell and Meyer 2006; Flatters 2008;
Navarro et al. 2007; Sah et al. 2003). Substance P (SP), a neurotransmitter or neuromodulater, is a pain-related neuropeptide contained in the sciatic nerve, dorsal
root ganglions, and spinal cord (Corder et al. 2010; Fuchs et al. 2010; Ma and Bisby 1998). Furthermore, Wei et al. reported that SP activation and the up-regulated
neurokinin-1 receptor (NK-1R) results in skin warmth, protein leakage, local edema,
and keratinocyte proliferation in a tibia fracture rat model (Wei et al. 2009).
Only a few studies have examined the therapeutic efficacy and mechanism of action of ultrasound in neuropathic pain models (i.e. CCI). The purpose of this study was to examine whether TU alters parameters indicative of allodynia or hyperalgesia following CCI and investigate the expression of NK-1R and the levels of SP, TNF-α and IL-6 after CCI of the sciatic nerve in response to TU.
METHODS
Animals
The experimental procedures were approved by the Institutional Animal Care
and Use Committee of National Cheng Kung University (Tainan, Taiwan) and conducted according to IASP ethical guidelines (Zimmermann 1983). Male Sprague- Dawley rats (200 to 250 g) were obtained from the Laboratory Animal Center of National Cheng Kung University and kept in the animal housing facilities at National Cheng Kung University, with controlled humidity (approximately 50% relative humidity), room temperature (22C), and a 12-hour (6:00 AM to 6:00 PM) light/dark cycle.
The animal model of CCI
Animals were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Four ligatures were tied loosely around the sciatic nerve as described by Bennett and Xie (Bennett and Xie 1988). The CCI surgery evoked significant mechanical allodynia and thermal hyperalgesia in the ipsilateral hindpaw was seen by postoperative day 3 (POD 3) and increased to maximal levels from POD10, as previously described (Chen et al. 2012). All behavioral measurements were tested between 9:00 a.m. and 11:00
a.m. Rats were assessed for mechanical withdrawal threshold and heat withdrawal latency after a period of at least 5 days of habituation to the testing environment and experimenters. For consistency, an experienced investigator, who was blinded to the groups, was responsible for handling all the animals and behavioral evaluation.
For assessment of mechanical withdrawal threshold, rats were placed individually in a clear plexiglass chamber and supported by a wire mesh floor.
Mechanical sensitivity was evaluated by von Frey filaments (Anesthesiometer, Somedic AB, Sweden). In ascending order of force, each filament was applied for 5 seconds vertically, to the lateral plantar area of the hind paw (Chen et al. 2013a; Chen et al. 2012). Withdrawal responses caused by mechanical stimulation were determined
including foot shaking, licking and squeaking.
Heat withdrawal latency was evaluated by the Hargreaves’ Method (Hargreaves et al. 1988). Briefly, rats were placed individually in a clear plexiglass chamber, and
the animals stood on a glass sheet with the temperature maintained at 30 ±1ºC to decrease the influence of the temperature in different seasons. The lateral plantar surface of rat hind paw was exposed to a constant intensity radiant heat source through the Hargreaves' plantar test apparatus (Ugo Basile, Comerio, Italy). A maximal automatic cut-off latency of 20 seconds was used to avoid tissue damage (Chen et al. 2013a; Chen et al. 2012).
Groups and design
Animals were randomly divided into six groups. The sham group (n = 8), the rats are sham-operated, except that no chromic gut sutures were applied. The CCI group
(n = 8), rats received CCI, and the CCI+TU-0 group (n = 8), CCI rats received TU with the ultrasound turned off. The CCI+TU-0.25 group (n = 8), CCI rats received TU with the intensity of 0.25 W/cm2. The CCI+TU-0.5 group (n = 8), CCI rats received TU with the intensity of 0.5 W/cm2. The CCI+TU-1group (n = 8), CCI rats received TU with the intensity of 1 W/cm2. After the behavior testing, the sciatic nerves of 4-6 animals were randomly used to NK-1R, substance P and cytokines (TNF-α and IL-6)
analyses on PODs 14 and 28.
On day 5 after CCI, the rats received TU in which they did not receive the previous day. TU was delivered to the CCI rats for 5 minutes once a day commencing immediately on day 5 after CCI and then daily for 5 minutes for the next 22 days. In our hands, significant mechanical allodynia and heat hyperalgesia in animals began 3 days after rats had been received CCI and lasted for up to 1 month (Chen et al. 2012).
All animals were evaluated twice for mechanical and heat sensitivity on the day before CCI, and the 2 measurements were averaged to obtain a single baseline mechanical and heat sensitivity for each evaluation. These rats were subsequently evaluated on days 7, 14, 21 and 28 after CCI or on the analogous day if CCI did not occur. The 4-day postsurgery evaluation was performed at 22 hours after the final ultrasound therapy and/or isoflurane anesthesia. The last ultrasound therapy occurred 27 days after CCI.
Application of therapeutic ultrasound
Ultrasound (US-750, Ito Co., Ltd; Bunkyo-ku, Tokyo, Japan) was performed right after the surgery incision was closed. Treatment performed daily, 1 MHz frequency probe was chose with 0.25, 0.5 or 1 W/cm2 intensity and 100% on-off cycle, gel was applied and the probe gently moved for 5 minutes at the level of the middle of the thigh. During the ‘‘false application’’, only massage was practiced with the ultrasound turned off (TU-0).
Cytokines and substance P analyses
Rats were anesthetized with urethane (1.67 g/kg, i.p.) and killed on PODs 14 and 28. The sciatic nerve was exposed proximal to the sciatic trifurcation and an
approximately 7-mm section of the nerve was obtained. The concentrations of substance P, TNF-α, and IL-6 were determined by the DuoSet® ELISA Development Kit (R&D Systems, Minneapolis, MN) (Chen et al. 2013b; Hung et al. 2013a; Hung et al. 2013b). All experimental procedures were practiced in accordance with the
manufacturer’s recommended protocols. Plates were individually inserted into the plate reader for reading optical density by a 450-nm filter. Data were then analyzed using Ascent Software for iEMS Reader and a four-parameter logistics curve-fit. Data were expressed in pg/mg protein of duplicate samples.
NK-1R analysis
The expression of NK-1R was analyzed using western blotting. The primary substance P receptor antibody (PA1-16713, Thermo scientific, USA) was diluted to 1:1,000 in antibody binding buffer overnight at 4ºC, incubated for 1 hour with secondary antibody (anti-rabbit, Amersham, UK) and diluted 10,000-fold in TBS buffer at 4°C. The bound antibody was detected using Luminata Western HRP Substrates (WBLUC0100, Millipore, USA) and then the membrane was quantified through a Gel-Pro Analyzer (version 4.0; Media Cybernetics, USA). For internal control, membranes were reprobed with a monoclonal anti β-actin antibody (A5441, 1:10000, Sigma-Aldrich, USA). Actin was used as a loading control, so it adjusted the NK-1R values as a ratio to actin to compensate for unequal protein that was loaded in wells.
Statistical analysis
The results are presented as the mean ± S.E.M. of N observations unless noted otherwise. Statistical significance between multiple experimental groups was
determined by one-way or two-way ANOVA with a Bonferroni multiple comparison post-hoc analysis. A statistical software, SPSS for Windows (version 17.0; SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. In each case, statistical
significance was set at P< 0.05.
RESULTS
Therapeutic ultrasound with the intensity of 1 W/cm2, but not 0.25 or 0.5 W/cm2
attenuates CCI-evoked heat hyperalgesia and mechanical allodynia
On POD 7, the CCI+TU-1 group showed an increase in mechanical withdrawal threshold (Fig. 1A) and thermal withdrawal latency (Fig. 1B) compared to the CCI group, whereas both CCI+TU-0.25 and CCI+TU-0.5 groups did not have this efficacy. When compared with the sham group, the other four groups demonstrated
lower mechanical withdrawal threshold (Fig. 1A) and thermal withdrawal latency (Fig. 1B).
Ultrasound treatment delays the development of CCI-associated mechanical
allodynia and thermal hyperalgesia
The CCI rats on day 7 after CCI showed an increased sensitivity to innocuous von Frey stimulation (2.2 ± 0.8 g, n = 8) that were maintained over the 4-week course of this study (Fig. 2A). Compared with the CCI rats, CCI+TU-0 rats displayed
similarly to von Frey hair stimulus on PODs 7, 14, 21 and 28 (P> 0.05; two-way repeated measures ANOVA). Moreover, CCI-treated rats at first week underwent TU had paw withdrawal thresholds of 5.4 ± 0.9 g (n = 8), higher than those of CCI rats
and lower than these of sham rats (Fig. 2A).
In Fig. 2B, the thermal withdrawal latency (6.0 ± 0.7 s, n = 8) obtained from CCI rats was not markedly different from those of CCI+TU-0 rats (4.8 ± 0.8 s, n = 8) on POD 7 (P> 0.05; two-way repeated measures ANOVA).When thermal withdrawal latency was evaluated on POD 7, CCI rats uniformly experienced thermal
hyperalgesia as indicated by a significant decrease in paw withdrawal latency (P<
0.05), compared with the sham rats. By comparison, CCI+TU-1 rats exhibited a significant increase in heat withdrawal latency on POD 7 as compared with CCI rats
(Fig. 2B).
Ultrasound intervention suppresses excess TNF-α and IL-6 levels in the sciatic nerve
following CCI
Figure 3 reveals the levels of TNF-α and IL-6 in the sciatic nerve of sham, CCI,
CCI+TU-1, and CCI+TU-0 rats on PODs 14 (Figs. 3A and C) and 28 (Figs. 3B and D). The TNF-α and IL-6 levels in the sciatic nerve on PODs 14 (Figs. 3A and 3C) and
28 (Figs. 3B and 3D) were significantly increased in the CCI (P < 0.01) and CCI+TU- 0 (P < 0.01) group, respectively, compared with the sham group. The CCI and
CCI+TU-0 rats showed similar cytokine levels in the sciatic nerve. By comparison, the CCI-TU-1 exhibited markedly lower TNF-α (P< 0.01, Fig. 3A; P< 0.05, Fig. 3B) and IL-6 (P< 0.05, Fig. 3C; P< 0.05, Fig. 3D) than those in CCI-treated rats without TU.
Ultrasound therapy prevents the up-regulation of SP and NK-1R in rat sciatic nerve
after CCI
The SP content and NK-1R expression of rat sciatic nerve obtained on PODs 14 and 28 are shown in Fig. 4 in sham, CCI, CCI+TU-1, and CCI+TU-0 groups. The levels of SP and NK-1R in the sciatic nerve were not significantly different between CCI and CCI+TU-0 groups (Fig. 4). The SP and NK-1R levels in the sciatic nerve
were prominently increased in the CCI (P < 0.05) and CCI+TU-0 (P < 0.05) groups on PODs 14 (Figs. 4A and 4C) and 28 (Figs. 4B and 4D), respectively, when compared with the sham group. Furthermore, the CCI rats underwent TU program displayed lower SP (P< 0.05, Fig. 4A; P< 0.05, Fig. 4B) and NK-1R (P< 0.01, Fig.
4C; P< 0.05, Fig. 4D) levels than those in CCI rats without TU.
DISCUSSION
In this current study we reported for the first time that a sustained attenuation of CCI-induced neuropathic pain when ultrasound was applied daily beginning 3 days after nerve injury through day 27. Furthermore, TU inhibited excess TNF-α and IL-6 expression in the sciatic nerve of CCI rats. Our results were in resemblance to the previous study that CCI rats showed an increased TNF-α level in the sciatic nerve (Chen et al. 2012). Another finding was that CCI rats with ultrasound treatment prevented the up-regulation of NK-1R and substance P caused by CCI in rat sciatic
nerve. Overall, we suggested that ultrasound therapy suppressed the progression of persistent neuropathic pain, in part, possibly relating to inhibit the up-regulation of
NK-1R, substance P, TNF-α, and IL-6 in the sciatic nerve.
In this present study we showed that CCI-operated rats on PODs 7, 14, 21 and 28 developed a markedly plantar responsiveness to mechanical and thermal stimuli.
These results are similar to the previous study that found that rats following CCI operation exhibited at least 2 month of hypersensitivity to paw mechanical and heat stimulation (Bennett and Xie 1988). Efficacy of TU is of particular interest as it is the physical agent commonly used by physical therapists for management of painful musculoskeletal conditions and, therefore, widely acceptable (Shanks et al. 2010; Tok et al. 2012). Although TU was able to be extensively used for effective treatment of
carpal tunnel syndrome (Kapuscinska and Urbanik 2013), it is unclear whether TU can treat CCI-evoked neuropathic pain and the role of TU in its amelioration. Our present study revealed that TU did reduce peripheral neuropathic pain evoked by CCI
in rats.
The effects of high intensity TU (1 W/cm2) are partial and do not result in complete reversal of neuropathic pain. In addition, Savernini et al. (Savernini et al.
2012) clearly showed that TU for both intensities tested, 0.3 and 0.4 W/cm2, elicited long-acting antinociceptive effect with independent and opioid-dependent
mechanisms for the two phases on an experimental rat model of trigeminal neuropathic nociception. Moreover, on the 14th day after TU treatment, the group using 1 MHz frequencies with maximum 0.5 W/cm2 intensities and 20% duty cycle recovered up to 90% rat sciatic function in mice caused by crushing their sciatic nerves (Akhlaghi et al. 2012). The low intensities (0.25 and 0.5 W/cm2) of TU
produce no antiallodynic and antihyperalgesic effects in this current study.
Results from animal experiments support definite evidence for the crucial role of cytokines in the onset and maintenance of pain (Milligan et al. 2003; Thacker et al.
2007). Our studies show that increased TNF-α and IL-6 levels are in parallel with
CCI-induced neuropathic pain. These experimental results are in agreement with the report by Chen et al. (Chen et al. 2012) who demonstrated that TNF-α and IL-1β levels increased in the sciatic nerve of rats after CCI. Although we cannot be sure the mechanisms of proinflammatory cytokines are involved, it is generally believed that TNF-α was present in mast cells and Schwann cells of the sciatic nerve on the nerve-
injured side of CCI rats (Hayashi et al. 2008).
Our observations are apparent that TU dose decrease pro-inflammatory cytokines in the sciatic nerve of CCI rats. Moreover, it has been shown that administrations of pro-inflammatory cytokines inhibitors and anti-inflammatory cytokines attenuated neuropathic pain (Arruda et al. 2000; Schafers and Sommer 2007). Additionally, we
did not measure the temperature at the site of application of TU. Because TU has a thermal effect, it has been used extensively to therapy a variety of conditions
(Bierman 1954; Cambier et al. 2001; Lehmann et al. 1966). It is well known that TU increases tissue temperature at depths up to 5 cm and accompanies only a little
increase in skin temperature (Draper et al. 1998; Lehmann et al. 1966; Lehmann et al.
1967). For instance, an increase of 2℃ to 3℃ (moderate heating) not only increases
blood flow but also decreases pain, chronic inflammation, and muscle spasm (Baker and Bell 1991; Draper et al. 1995; Lehmann et al. 1967).
It has been presumed that SP creates nociceptive sensitization after mouse paw incision (Sahbaie et al. 2009). Furthermore, SP acts on endothelial NK1 receptors to cause spontaneous protein extravasation in normal animals and that after fracture there is a local increase in keratinocyte and endothelial NK1 receptors, resulting in intensified microvascular permeability, cutaneous vasodilatation, and edema in injured hindlimbs (Wei et al. 2009). In this recent study, we manifested that CCI rats showed a significant increase in SP level and NK-1R expression in the sciatic nerve and accompanied to develop an increased plantar responsiveness to heat and
mechanical stimuli. This can be explained by the finding that SP reduced withdrawal latency and NK-1R antagonist RP67580 increased it in CCI rats (Yoshimura and Yonehara 2006).
To investigate the mechanisms of therapeutic effects of ultrasound on neuropathic pain, we explored the possible role played by SP and NK-1R in the sciatic nerve of CCI rats. Our resulting data showed that TU prevented the increased expression of SP and NK-1R in the sciatic nerve of rats following CCI. The NK1 antagonist LY303870 markedly suppressed hind paw skin temperature after distal tibial fracture, suggesting that facilitated SP signaling mediated the increase in hindpaw warmth after injury (Guo et al. 2004). It is possible that lack of SP reduces the intensity of the inflammatory reaction thereby providing a second mechanism for
the decreased thermal and mechanical sensitization (Sahbaie et al. 2009).
We concluded that application of therapeutic ultrasound results in an antiallodynic and antihyperalgesic effect in rats made neuropathic after a sciatic chronic constriction injury. Our resulting data also imply that this
antiallodynic/antihyperalgesic effect of ultrasounds may involve suppression of NK- 1R, substance P, TNF-α, and IL-6. The study highlights the fact that therapeutic ultrasound (a simple, safe, non-invasive technique) may have a great potential as a treatment for neuropathic pain, and needs further investigation in this context.
ACKNOWLEDGMENTS
We gratefully acknowledge the financial support provided by grants NSC 100- 2314-B-039-017-MY3 and NSC 101-2314-B-006-037-MY3 from the National Science Council, Taiwan. The authors report no conflict of interest.
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FIGURE LEGENDS
Fig. 1. The mechanical withdrawal threshold (A) and thermal withdrawal latency (B) on day 7 after CCI in 5 different groups of rats: sham, CCI+TU-0.25, CCI+TU-0.5, CCI+TU-1 and CCI (sham = sham-operated; CCI = chronic constriction injury;
CCI+TU-0.25 = CCI rats received therapeutic ultrasound (TU) with the intensity of 0.25 W/cm2; CCI+TU-0.5 = CCI rats received TU with the intensity of 0.5 W/cm2; CCI+TU-1 = CCI rats received TU with the intensity of 1 W/cm2). Data are presented as mean ± S.E.M. for 8 rats per group. The asterisk (**, ***) indicates P < 0.01 and P
< 0.001, respectively, when compared with the sham group; the plus symbol indicates P < 0.05 when compared with the CCI group (1-way ANOVA followed by post hoc Bonferroni’s test).
Fig. 2. The behavioral time courses of mechanical withdrawal threshold (A) and thermal withdrawal latency (B) in sham, CCI+TU-1, CCI and CCI+TU-0 rats, where sham = sham-operated; CCI = chronic constriction injury; CCI+TU-1 = CCI rats received therapeutic ultrasound with the intensity of 1 W/cm2; CCI+TU-0 = during the ‘‘false application’’ in CCI rats, only massage was practiced with the ultrasound turned off. The paw withdrawal threshold (g) and latency (s) to mechanical and heat stimuli, respectively, were not markedly different between the CCI and CCI+TU-0 groups. Data are presented as mean ± S.E.M. for 8 rats per group. The asterisk indicates P < 0.05 when compared with the sham group; the plus symbol indicates P
< 0.05 when the CCI+TU-1 group was compared with the CCI group (2-way ANOVA of repeated measures followed by post hoc Bonferroni’s test).
Fig. 3. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on days 14 (A and C) and 28 (B and D) after CCI in the sciatic nerve of 4 different groups of rats: sham, CCI+TU-1, CCI and CCI+TU-0 (sham = sham-operated; CCI = chronic constriction injury; CCI+TU-1 = CCI rats received therapeutic ultrasound (TU) with the intensity of 1 W/cm2; CCI+TU-0 = during the ‘‘false application’’ in CCI rats, only massage was practiced with the ultrasound turned off).The values are presented as mean ± S.E.M. for 6 rats per group. The asterisk (*, **, ***) indicates P < 0.05, P
< 0.01 and P < 0.001, respectively, when compared with the sham group; the plus symbols (+, ++) indicate P < 0.05 and P < 0.01, respectively, and when CCI+TU-1 group was compared with the CCI group (1-way ANOVA followed by post hoc
Bonferroni’s test).
Fig. 4. The substance P (SP) level and neurokinin-1 receptor (NK-1R) expression in the sciatic nerve on days 14 (A and C) and 28 (B and D) after CCI was quantified by the ELISA and western blotting method, respectively, in 4 different groups of rats, where sham = sham-operated; CCI = chronic constriction injury; CCI+TU-1 = CCI rats received therapeutic ultrasound (TU) with the intensity of 1 W/cm2; CCI+TU-0 = during the ‘‘false application’’ in CCI rats, only massage was practiced with the ultrasound turned off. The values are presented as mean ± S.E.M. for 4-6 rats per group. Compared to the CCI group, the CCI+TU-1 group showed a significant decrease in SP level and NK-1R expression in the sciatic nerve (P<0.05). The asterisk (*, **, ***) indicates P < 0.05, P < 0.01 and P < 0.001, respectively, when compared with the sham group; the plus symbols (+, ++) indicate P < 0.05 and P < 0.01,
respectively, and when CCI+TU-1 group was compared with the CCI group (1-way ANOVA followed by post hoc Bonferroni’s test).
