Levo-tetrahydropalmatine attenuates oxaliplatin-induced mechanical hyperalgesia in mice

ABSTRACT Common chemotherapeutic agents such as oxaliplatin often cause neuropathic pain during cancer treatment in patients. Such neuropathic pain is difficult to treat and responds poorly

to common analgesics, which represents a challenging clinical issue. _Corydalis yanhusuo_ is an old traditional Chinese medicine with demonstrated analgesic efficacy in humans. However, the

potential analgesic effect of its active component, levo-tetrahydropalmatine (_l_-THP), has not been reported in conditions of neuropathic pain. This study found that _l_-THP (1–4 mg/kg,

i.p.) produced a dose-dependent anti-hyperalgesic effect in a mouse model of chemotherapeutic agent oxaliplatin-induced neuropathic pain. In addition, we found that the anti-hyperalgesic

effect of _l_-THP was significantly blocked by a dopamine D1 receptor antagonist SCH23390 (0.02 mg/kg), suggesting a dopamine D1 receptor mechanism. In contrast, _l_-THP did not

significantly alter the general locomotor activity in mice at the dose that produced significant anti-hyperalgesic action. In summary, this study reported that _l_-THP possesses robust

analgesic efficacy in mice with neuropathic pain and may be a useful analgesic in the management of neuropathic pain. SIMILAR CONTENT BEING VIEWED BY OTHERS ORAL ADMINISTRATION OF CYSTINE

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ALLEVIATES CHEMOTHERAPY-INDUCED PERIPHERAL NEUROPATHY IN RATS Article 25 April 2022 INTRODUCTION Chemotherapy for cancer treatment often induces a form of unique peripheral neuropathy

characterized by provoked and ongoing pain, which is increasingly considered as a serious side effect associated with some chemotherapeutic agents, including taxanes, platinum agents (e.g.,

oxaliplatin) and vinca alkaloids. The occurrence rate of chemotherapy-induced neuropathic pain varies substantially from 30–75% in the cancer patients, depending on the treatment regimens.

Besides pain, other common peripheral sensory symptoms include paresthesias and dysesthesias, numbness and tingling and sensitivity to touch and temperature. In addition, motor symptoms are

also reported, including weakness and gait and balance disturbances1. Unfortunately, this kind of neuropathic pain is only partially reversible even long after the cessation of treatment and

in some rare cases damage can be permanent. Currently, there is no effective pharmacotherapies that is considered safe and widely useful for the clinical control of chemotherapy-induced

neuropathic pain. Thus, the development of alternative effective analgesics has been considered a crucial clinical need. _Corydalis yanhusuo_ is a perennial herb in the Papaveraceae family

and has long been used in traditional Chinese medicine. Contemporary phytochemistry studies of _Corydalis yanhusuo_ started in 1960s and Hsu and Kin were the first to isolate

_l_-tetrahydropalmatine (_l_-THP) from _Corydalis yanhusuo_ and did the first pharmacological characterization of the compound2,3. _l_-THP has been identified as one of the major active

components of _Corydalis yanhusuo_ and it has been used clinically in China for more than 40 years as an analgesic with sedative/hypnotic properties4. However, although _l_-THP has been used

for the treatment of headache and other mild pain in China, relatively few preclinical studies support these use and there is no study to examine the potential effectiveness of _l_-THP for

the treatment of chronic neuropathic pain. Given the long clinical use of _l_-THP which has proved its human safety, exploring novel applications of this compound may expand its clinical

usage. In this study, we described the potent anti-hyperalgesic effect of _l_-THP in a mice model of oxaliplatin-induced neuropathic pain. We also conducted antagonist studies to understand

the receptor mechanisms of the anti-hyperalgesic actions. Our results revealed a primary dopamine D1 receptor mediated effect. RESULTS Chronic oxaliplatin treatment (3 mg/kg) led to marked

mechanical hyperalgesia in mice as measured by von Frey filament (Fig. 1). Paired t-test revealed that oxaliplatin treatment produced a significant decrease in the paw withdrawal threshold

(t (7) = 13.99, P < 0.0001). In addition, repeated test every 10 min over a period of 100 min did not alter the hyperalgesic condition, which remained significantly lower than the

baseline measurement prior to oxaliplatin treatment (Fig. 2). Two-way ANOVA revealed significant main effects of oxaliplatin treatment (F [3, 21] = 102.3, P < 0.0001) and time (F [9, 63]

= 44.44, P < 0.0001). _l_-THP dose-dependently increased the paw withdrawal threshold in mice (Fig. 2). A smaller dose of _l_-THP (1 mg/kg) only slightly elevated the paw withdrawal

threshold and _post hoc_ multiple analysis showed that at a dose of 1 mg/kg _l_-THP significantly increased the paw withdrawal threshold during the time points of 20 and 30 min. A larger

dose of _l_-THP (2 mg/kg) markedly and significantly increased the paw withdrawal threshold. Two-way ANOVA revealed a significant main effect of _l_-THP treatment (F [1, 63] = 27.36, P <

0.0001). Multiple comparison analysis found that the paw withdrawal threshold was significantly increased throughout the 10–50 min time period. When the dose of _l_-THP was further increased

to 4 mg/kg, the paw withdrawal threshold was significantly increased to the pre-oxaliplatin treatment level (Fig. 2).Two-way ANOVA revealed a significant main effect of _l_-THP treatment (F

[1, 63] = 76.45, P < 0.0001). Multiple comparison analysis found that the paw withdrawal threshold was significantly increased throughout the 10–80 min time period. In order to

understand the receptor mechanism underlying the anti-hyperalgesic actions of _l_-THP, a dose of the selective dopamine D1 receptor antagonist SCH23390 (0.02 mg/kg) was studied in

combination with 4 mg/kg _l_-THP (Fig. 3). SCH23390 significantly attenuated the anti-hyperalgesic effects of _l_-THP. Two-way ANOVA revealed that there were significant main effects of

SCH23390 treatment (F [1, 14] = 158.0, P < 0.0001) and time (F [9, 126] = 60.13, P < 0.0001). Post hoc analysis found that the anti-hyperalgesic effect of _l_-THP was significantly

decreased across the 10–70 min time period. We also studied the anti-hyperalgesic effects of repeated daily _l_-THP treatment (Fig. 4). Daily treatment with 4 mg/kg _l_-THP, a dose that

completely reversed mechanical hyperalgesia, maintained its anti-hyperalgesic effect and no significant antinociceptive tolerance was observed over a period of 10 days of daily treatment.

Two-way ANOVA revealed a significant main effect of _l_-THP treatment (F [1, 7] = 103.5, P < 0.0001), but no significant main effects of time or interaction were found. Post hoc analysis

found that the paw withdrawal threshold after 4 mg/kg _l_-THP treatment was significantly higher as compared to the daily pre-drug treatment baseline. In addition, the anti-hyperalgesic

effect among the 10 daily treatments was not significantly different. The potential effect of _l_-THP treatment on the general locomotor activity in naïve mice was examined with different

doses of _l_-THP (Fig. 5). It was found that _l_-THP had a dose-dependent effect in decreasing the general locomotor activity in mice. One-way ANOVA found a significant effect of _l_-THP

dose (F [4, 35] = 11.48, P < 0.05). Post hoc analysis revealed that 8 mg/kg _l_-THP treatment significantly decreased the locomotor activity. DISCUSSION In this study, we reported that an

active component from the plant _Corydalis yanhusuo_, _l_-THP, produced robust anti-hyperalgesic effect in a mouse model of chemotherapy-induced neuropathic pain. We also reported that the

anti-hyperalgesic effect of _l_-THP was primarily mediated by dopamine D1 receptors and the effect was not due to general behavioral impairment. Although _l_-THP has been used in China for

the treatment of mild to moderate pain including headache, this is the first study that identified the antinociceptive effects of _l_-THP in a mouse model of chemotherapeutic agent-induced

neuropathic pain. In summary, although preliminary, these results support the expanding use of _l_-THP in the clinical control of neuropathic pain. Many microtubule-targeting cancer

chemotherapeutic agents including oxaliplatin has long been recognized to cause peripheral and cranial neuropathy5,6,7. In an effort to better understand this form of neuropathy and develop

novel treatment for its management, several animal models of chemotherapeutic agent-induced neuropathy was developed8,9,10. Rodents treated with chemotherapeutic agents typically develop

thermal and mechanical hyperalgesia. In consistency with the literature, we found that mice treated with 3 mg/kg intermittently for 10 injections developed a reliable mechanical hyperalgesia

as measured by von Frey filament test. Repeated measures within a short period of time (100 min) did not significantly change the test results, which offers an opportunity to determine the

duration of actions of the study drug. We found that _l_-THP produced a robust effect in decreasing mechanical hyperalgesia. This effect was both dose-dependent and time-dependent and at

larger doses it completely reversed the mechanical hyperalgesia. This represents the first study that clearly demonstrated that _l_-THP has very robust antinociceptive effect in a mouse

model of chronic neuropathic pain. In addition, the observed anti-hyperalgesic effects were not due to general behavioral suppression because within the dose range studied, _l_-THP did not

significantly alter the locomotor activity in mice. More importantly, repeated treatment with _l_-THP did not show the development of antinociceptive tolerance. Considering the long-term

therapeutic need to treat neuropathic pain, this lack of tolerance development is significant and clearly supports the use of _l_-THP in the treatment of chronic neuropathic painful

conditions. Early receptor binding studies suggest that _l_-THP binds to dopamine D1 and D2 receptors at relatively high affinity and binds to serotonergic 5-HT1A receptors at lower

affinity2. In a rat radiant tail flick test, _l_-THP produced a dopamine D2 receptor mediated antinociception11. This study found that a selective dopamine D1 receptor antagonist, SCH23390,

significantly blocked the anti-hyperalgesic effect of _l_-THP, suggesting that the anti-hyperalgesic action of _l_-THP is primarily mediated by activating dopamine D1 receptors. This

discrepancy may be due to the pain model used (radiant tail flick versus neuropathic pain), species (rats versus mice) and the doses studied (10–40 mg/kg versus 1–4 mg/kg). In summary, this

study for the first time demonstrated that _l_-THP has a potent antinociceptive effect in a mouse model of chemotherapeutic agent-induced neuropathic pain, without apparent adverse effects

(motor impairment). Although more studies are needed to examine the generality of these findings, because _l_-THP has been used in China for over 40 years and its human safety has been

clearly demonstrated, the current data suggest to explore the expansion of _l_-THP for the treatment of neuropathic pain. METHODS ANIMALS Male C57BL/6 mice weighing 16–22 g (Weitong Lihua,

Beijing, China) were acclimated to the temperature, humidity and lighting (12 h light/dark cycle, lights on at 7:00 AM) controlled vivarium and housed in groups of four for at least one week

before behavioral studies began. The animals had free access to dietary food and water except during the test sessions. All animal experimental protocols were approved by the local

Institutional Animal Care and Use Committee, The First Hospital Affiliated to the Chinese PLA Hospital. Animals were maintained and experiments were conducted in accordance with the _Guide

for the Care and Use of Laboratory Animals_ (8th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC).

All efforts were made to minimize animal suffering and to reduce the number of animals used. DRUGS Oxaliplatin (Sigma-Aldrich, St. Louis, MO) was dissolved in 5% dextrose (1 mg/ml) and

prepared fresh for daily use. Levo-tetrahydropalmatine (_l_-THP) was purchased from Shanghai Lei Yun Shang Pharmaceutical Co. (>95% purity, Shanghai, China). SCH23390 was purchased from

Sigma-Aldrich (St. Louis, MO, USA) and dissolved in saline. _l_-THP was dissolved in saline with one drop of acetic acid. Except otherwise noted, all injections were given intraperitoneally

in a volume of 1 ml/100 g of body weight. After habituation to the test environment and baseline measurements of pain sensitivity, mice were randomized to two treatment conditions of either

oxaliplatin (3.0 mg/kg) or vehicle (0.9% saline). Using injection volume of 10 ml/kg, mice were treated with daily administration for 5 days, followed by 5 days of rest, for two weekly

cycles. Total cumulative dose of 30 mg/kg oxaliplatin over a total of ten injections was used. MECHANICAL HYPERALGESIA MEASUREMENT Mechanical hyperalgesia was assessed prior to and 1 day

after the last oxaliplatin treatment using Von Frey filaments of varying forces (0.07–4.0 g) applied to the mid-plantar surface of the right hind paw, with each application held until curved

for 6 s using the up-down method2. Mice were placed in individual Plexiglas compartments atop of a wire grid floor suspended 50 cm above the laboratory bench top and acclimated to the

environment for 30 min prior to each test session. For the time course studies, baseline von Frey filament measurement was immediately followed by an injection of _l_-THP and then the paw

withdrawal threshold was measured every 10 min until the drug effect dissipated to a point that the paw withdrawal threshold was not significantly different from the pre-drug data. In

studies that test the effect of the antagonist SCH23390, drug was administered 10 min prior to _l_-THP treatment and a time course measurement was followed. For repeated treatment studies,

mice were measured daily before drug treatment and 30 min after drug treatment for 10 days. LOCOMOTOR ACTIVITY TEST The locomotor activity of naïve mice treated with vehicle or _l_-THP was

measured automatically with a Small Animal Locomotion Recording Apparatus (Shandong Academy of Medical Sciences, China), which consisted of six acrylic boxes and in each box there was one

pyroelectric infrared sensor 4 cm above the floor. The sensor could detect the movements of the mice through infrared radiation. The apparatus recorded only gross movements of the mice,

whereas small movements such as gnawing or grooming could not be differentiated and recorded. DATA ANALYSES For the mechanical hyperalgesia test prior to and 1 day after the last oxaliplatin

treatment, data were analyzed using paired t-test. For the antinociceptive studies, data were presented as paw withdrawal threshold (grams) plotted as a function of time (min or days),

respectively. Data were analyzed by two-way repeated measures analysis of variance (ANOVA) (time × _l_-THP treatment or time × oxaliplatin treatment) followed by post hoc Bonferroni test.

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equally to this work. AUTHORS AND AFFILIATIONS * Department of Anesthesiology, The First Hospital Affiliated to the Chinese PLA Hospital, Beijing, 100048, China Zhenggang Guo, Xiaoyan Wang, 

Heng Jin, Xuefeng Sun, Xiaojun Su & Jianhua Hao * Anesthesia and Operation Center, Chinese PLA General Hospital, Beijing, 100853, China Yuanyuan Man & Weidong Mi Authors * Zhenggang

Guo View author publications You can also search for this author inPubMed Google Scholar * Yuanyuan Man View author publications You can also search for this author inPubMed Google Scholar *

Xiaoyan Wang View author publications You can also search for this author inPubMed Google Scholar * Heng Jin View author publications You can also search for this author inPubMed Google

Scholar * Xuefeng Sun View author publications You can also search for this author inPubMed Google Scholar * Xiaojun Su View author publications You can also search for this author inPubMed 

Google Scholar * Jianhua Hao View author publications You can also search for this author inPubMed Google Scholar * Weidong Mi View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS Z.G., J.H. and W.M. designed the experiments; Z.G., Y.M., X.W., H.J. and X.S. conducted the experiments; Z.G., X.S., J.H. and W.M. wrote the main

manuscript text; Y.M. and X.W. conducted the statistical analyses and prepared the figures. All authors reviewed and approved the manuscript. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing financial interests. RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license,

visit http://creativecommons.org/licenses/by/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Guo, Z., Man, Y., Wang, X. _et al._ Levo-tetrahydropalmatine attenuates

oxaliplatin-induced mechanical hyperalgesia in mice. _Sci Rep_ 4, 3905 (2014). https://doi.org/10.1038/srep03905 Download citation * Received: 02 January 2014 * Accepted: 09 January 2014 *

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