The findings showed the anti-hypersensitive efficacies of systemically administered TR, LRG or TR+LRG combination in reducing mechanical and cold allodynia caused by CG injection in the rat paw. In addition, TR, LRG and TR+LRG combination suppressed the CG-induced rat paw edema and paw temperature increase, even at different levels, indicating that these treatments have significant anti-inflammatory potential, together with their anti-edema and anti-pyretic effects. Finding also suggested that decreases in tactile and cold sensitivities by these treatments in the non-inflamed paw may indicate their anesthetic/hypoesthetic potential. To the best of our knowledge, this is the first report showing the anti-hypersensitive, anti-edema and anti-pyretic actions of LRG in rats with acute peripheral inflammation.
In this present study, development of edema, redness and inflammatory pain behaviors, which are indicators of inflammatory reaction, were observed within the first hour following CG injection. The development of allodynia, which is defined as a painful response to a stimulus that does not produce pain responses [20, 21], was observed after mechanical and cold stimuli. Sensor tests were performed 3 times within 5 hours (1 hour before, 1 hour and 5 hours after CG injection) and the results were evaluated for inflammatory pain behaviors. Mechanical and cold allodynia responses peaked 5 hours after CG injection. In addition, remarkable edema and temperature increase were detected in the paw. In addition, responses to mechanical and cold stimuli from the non-inflamed paw were used to evaluate the effects of the treatments on the tactile and cold sensitivities of rats, and to determine possible anesthetic (hypoesthetic) efficacies.
Inflammatory pain hypersensitivity is the cardinal sign of acute peripheral inflammation that occurs depending on the process [20, 21]. Consistent with previous works, decreases in mechanical threshold and cold latency values is an indication of the development of allodynia (mechanical and cold hypersensitivities) [20-22]. It has been known that sensory afferents contribute to inflammatory pain hypersensitivity. Peripheral inflammation may cause increases in the sensitivities of the peripheral nerve terminals of A-delta and C nerve fibers at the inflammation site [1, 5, 23].
The effects of TR and LRG on latency and threshold parameters were different from each other. TR enhanced the threshold and latency in inflamed rats, while mechanical and cold sensitivity in the non-inflamed paw were reduced by TR treatment. Findings, as expected, showed that TR may has anti-hypersensitive and anesthetic effects. These results are consistent with the data obtained from previous studies [9, 10]. It has been previously reported that TR may produce analgesic/anesthetic activities due to its high affinity for µ-opioid receptors as well as inhibition of both serotonin and norepinephrine reuptake [7-9]. There is also lots of evidence that TR inhibits the activity of voltage-gated Na channels, delayed rectifier K channels, N-methyl-d-aspartate receptors, and substance P receptors in vitro [8, 10, 24, 25].
LRG (compared to TR) further increased the reduced latency and threshold values of rats with paw inflammation, revealing its potent anti-hypersensitive (anti-allodynic) activity. Moreover, LRG treatment also resulted in greater increases in both threshold and latency compared to TR in non-inflamed rats. In addition to the anti-hypersensitive effects of LRG in inflamed rats, the anesthetic/ hypoesthetic effects of LRG in non-inflamed rats showed that LRG may also modulate the conduction and function of sensory nerves which relay the peripheral information to the spinal cord. Previous studies have been reported that in addition to neuroprotective effect in diabetic neuropathy LRG treatments enhance the neurogenesis, recover the cognitive function, and decrease the amyloid plaque deposition in Alzheimer’s disease models [26-28]. The present study reports for the first time the anti-hypersensitive potential of LRG in rats with peripheral acute inflammation.
When two drugs co-administered, the treatment can produce independent effects or additive effects (equal to the sum of the effects of each). Combined treatment can also inhibit or decrease each other's effects (antagonism), or, the effect can be greater than the expected effect (synergy). In this present study, combined administration of an opioid, TR, and an anti-diabetic, LRG, resulted in further reduction of sensitivity in both the inflamed and non-inflamed paw. The remarkable suppression of cold and mechanical allodynia with anti-hypersensitive activity may suggest that this combination treatment may be very effective in inflammatory pain.
Paw masses were measured at the end of the experiments (5 hours after CG application) to determine the anti-edema effects of the treatments. Increased edema activity following CG administration resulted in an increase in paw mass up to approximately 2 times after 5 hours. TR, LRG or TR+LRG combination treatments showed significant decreases on the paw masses at approximately the same levels. These results can show the anti-edema effects of TR, LRG and TR+LRG combination.
One of the most important signs of inflammation after CG was a significant increase in the temperature of the inflammation area [1-3]. The increase in temperature in inflammation is an indication that the cellular immune defense mechanisms are activated against the harmful factor, which is caused by different mediators released in the inflammation area. This increase in temperature of the inflammation area was significantly reduced by all drug treatments. However, compared to TR, LRG caused a greater reduction in the temperature of the inflammation site. These results may clearly demonstrate the anti-pyretic effects of TR, LRG or TR+LRG combination treatments.
The presented findings may imply that the anti-hypersensitive, antipyretic and anti-edema effects of treatments may be closely related with the inflammatory environments includes various mediators such as cytokines, chemokines, and growth factor proteins which, alone or altogether. In order to obtain more detailed information about the molecular action mechanisms of treatments, the changes of biomarkers in the inflammation site were determined.
Findings showed that the levels of both pro-inflammatory and anti-inflammatory cytokines may increase in inflammation area. It has been known that pro-inflammatory cytokines released from inflammatory cells coming to the inflammation area after the inflammatory factor may cause more leukocyte migration to the area and cause an increase in pro-inflammatory cytokines [3,4]. The main function of anti-inflammatory cytokines is to inhibit or suppress the effect produced by pro-inflammatory cytokines and thus to maintain the balance between pro- and anti-inflammatory cytokines [3]. While TR produced the anti-inflammatory activity by suppressing the increase of the pro-inflammatory cytokine TNF-α, the treatments of LRG or LRG+TR combination showed anti-inflammatory activity by increasing the level of anti-inflammatory cytokine IL-10.
Angiogenesis is a biological event that plays a role in many physiological and pathological processes [29]. VEGF is one of the most important factors stimulating angiogenesis, and its inhibition can also be selected as a therapeutic target to reduce angiogenesis [29, 30]. It is known that TGF-β1 increases the release of VEGF angiogenic factors and is important in the occurrence of complications. In this present study, the suppression of VEGF-A and TGF-β1 levels by LRG suggests that the anti-angiogenic property of LRG may be an important mechanism contributing to its anti-inflammatory activity.
Many previous studies have shown that oxidative stress can play important roles in the inflammatory process [31, 32]. In this study, it was found that TAS did not change in inflamed paw tissues, while TOS increased significantly. While TR did not cause any change, LRG and LRG +TR increased the TAS level in inflamed tissue. The findings may suggest that LRG treatment may be important for the prevention of inflammation induced oxidative stress due to its anti-oxidant properties.
Consequently, compared with TR, which known effects, reduction of sensitivities to sensory stimuli of the non-inflamed paw by LRG treatment may indicate that it has anesthetic/hypoesthetic effects, which are described by reduced or slowing of sensory nerve conduction and functions. LRG may also ameliorate the pain hypersensitivity, edema and fewer. Anti-hypersensitive, anti-edema and antipyretic effects of LRG in rats with peripheral acute inflammation may be due to its anti-inflammatory, anti-angiogenic and anti-oxidative actions tissues (Fig. 7). Furthermore, LRG, when combined with TR, may be an important treatment option for controlling inflammation and inflammatory signs. However, more experimental and molecular studies are needed, especially to examine the anti-inflammatory potential of LRG and its mechanisms of action in the underlying inflammatory processes. In addition, studies on the pharmacological mechanisms of the additive/synergistic effect occurring in the combined application of LRG and TR are required.