Acupuncture Inhibits Morphine Induced-Immune Suppress via Antioxidant System

Objectives A powerful analgesic called Morphine causes addiction behaviors and immune suppression as a potential oxidative stressor. Acupuncture showed to inhibit oxidative stress-induced hepatic damage, regulate reactive oxygen species, and attenuate morphine addiction behaviors. Therefore, we investigated the potential effects of acupuncture on morphine-induced immune suppression. Materials and Methods Rats received morphine intravenously through implanted catheters for 3, 7, or 21 days to determine the optimal condition for morphine-induced immune suppression. Second, we examined whether intravenous (iv.) or intraperitoneal (ip.) administration produced different results. Third, the effects of acupuncture in rats who received morphine for 21 days were investigated. Spleen and submandibular lymph node (S-LN) weights and natural killer (NK) cell activity were measured, and the white pulp diameter, total and cortical spleen thicknesses, and the number of lymphoid follicles in S-LNs were examined. The number of immunoreactive cells was also measured. Results Decreased organ weights and increased atrophic changes were observed as morphine-induced immune suppression. However, dose-dependent increased immune suppression was not observed between 5.0 mg/kg and 10.0 mg/kg of morphine. And, 3-day withdrawal did not affect. Similar histopathological findings were observed in 5.0 and 10.0 ip. rats when compared to equal dosages of iv., respectively. The morphine induced-immune suppression evidenced by spleen and left S-LN weights, splenic NK cell activities, histopathological findings, and the immunoreactive cell number were normalized by acupuncture. Conclusion These results indicate that acupuncture inhibits morphine-induced immune suppression, maybe via antioxidative action.


Introduction
Addiction is a serious problem and has negative health, social, economic, and cultural effects [1][2][3]. e most common drugs abused are opiates including opium, codeine, morphine, and heroin [1]. Morphine, a natural alkaloid found in opium poppy [4], has been frequently used to treat severe pain due to its powerful analgesics and sedative effects [5,6]. Morphine suppresses the affective reaction to pain by inhibiting transmission of pain impulses, especially in the spinal cord, and through modulation of central neural circuits in the brain. However, morphine causes adverse effects when improperly prescribed [7], including respiratory, cardiovascular, gastrointestinal, or psychiatric problems [6,[8][9][10]. In addition, morphine acts as a potential oxidative stress-causing agent [7], and induces oxidative stress-related hepatic damage [11][12][13]. Some studies with innate immune cells from animals and humans and animal in vivo studies have shown that opiate abuse impairs innate immunity and is responsible for increased susceptibility to bacterial infection [14,15]. Morphine weakens immune system activity and inhibits immune response in the spleen, thymus, and lymph nodes [1,4]. Opioid causes spleen atrophy [16,17] and reduces the number of natural killer (NK) cells [18,19] B and T lymphocytes [20,21].
Immune functions are indispensable because they are the host defenses against infection and cancer and play a crucial role in maintaining health [22]. Declining immune function that occurs due to aging, chronic illnesses, physical and mental stress, or unhealthy lifestyles has been a major health problem [22][23][24], and therefore, modulation of the immune function has attracted great interest [25,26]. However, many of the available immunomodulators such as levamisole, glucans, telerones, L-fucose, and Corynebacterium parvum, showed side effects such as fever, neutropenia, leucopenia, and allergic reactions [26,27]. Hence, efforts to find better agents and evaluate their immunomodulatory potential have been performed [26].
Certain nutrients play a crucial role in the maintenance of optimum immune responses, and both deficiency and excessive intake can adversely affect the number and activity of the immune cells [26]. e underlying mechanism by which nutrients support the immune system is via the provision of antioxidants. Immune cells such as T-cells, NK cells, and T-helper cells are characterized by excessive levels of reactive oxygen species (ROS), which kill ingested pathogens. In addition, immune cell membranes are enriched with polyunsaturated fatty acids that are susceptible to ROS-mediated damage [26,28]. erefore, supplementation of nutrients with antioxidant properties such as carotenes, vitamin E, vitamin C, zinc, selenium, and polyphenols may quench these free radicals and influence several components of the immune system [26,29].
Notably, experimental and clinical studies confirmed that acupuncture influence the immune system [40,41]. In addition, acupuncture showed antioxidant properties [42][43][44], especially in morphine-induced liver injury [13], and regulated ROS levels [45]. us, we hypothesized that acupuncture could regulate morphine-induced immune suppression and investigated possible mechanisms, based on the previous studies.

Animals.
Male Sprague-Dawley rats (Daehan Animal, Seoul, Korea) weighed 270-300 g were used when the study began. Housing conditions were temperature (22 ± 2°C), humidity (60 ± 5%), and 12 h light-dark cycle (turn on at 7 : 00 pm). ey freely accessed food and water and were acclimated to the experimental environment before the experiment. Experimental procedures were approved by the Institutional Animal Care and Use Committee at Daegu Haany University.

Surgery.
To mimic the same condition with humans of intravenous morphine administration, animals were given catheter implantation surgery. Chronic silastic catheters (Dow Corning, Midland, MI, USA; 0.02 inch ID × 0.037 inches OD) were implanted into the right jugular vein under anesthesia with pentobarbital (50 ㎎/㎏, i. p.) and fixed using Mersilene mesh (Ethicon Inc., Somerville, NJ, USA) [46]. e catheter was exteriorized at the back of the animals using a 22 gauge guide cannula (Palstics One, Roanoke, VA, USA) through the skin incision. Silastic tubing and guide cannulae were embedded in dental cement and secured with Prolene mesh. 0.2 ㎖ solution of saline containing heparin (30 U/㎖) was flushed into the catheter daily to maintain patency during recovery from surgery.   [36][37][38][39], and we also assigned lower dose of 0.1 mg/kg to examine if morphine induces immune decrease at a low dose Each group n � 5.

Comparison of the Effects of Administration Route.
To investigate if there is a significant difference between the effects of intravenous (i. v.) and intraperitoneal (i. p.) administrations on immunity, other animals were subjected to morphine for 21D after assignment to the following 4 groups.  Evidence-Based Complementary and Alternative Medicine

Acupuncture Effects on Immune Suppression by
Morphine. e different rats were assigned to the following 5 groups to investigate the effects of acupuncture on the decrease of immunity induced by morphine treatment. Normal group (n � 9): vehicle; Control group (n � 7): morphine; HT7 group (n � 9): morphine + acupuncture at HT7; SI5 group (n � 9): morphine + acupuncture at SI5; LI5 group (n � 9): morphine + acupuncture at LI5. Animals were given morphine or vehicle for 21 days intravenously.
2.5. Acupuncture. Acupuncture groups received acupuncture treatment at each acupoint bilaterally. HT7 is located on the transverse crease of the wrist of the forepaw, radial to the tendon of the muscle flexor carpi ulnaris [46,47]. SI5 is located on the posteromedial aspect of the wrist in the depression between the triquetral bone and the styloid process of the ulna [47,48]. LI5 is located on the posterolateral aspect of the wrist, at the radial side of the dorsal wrist crease, distal to the radial styloid process, in the depression of the anatomical snuffbox [46,47]. e locations of acupoints followed the anatomical structures and were equivalent to those in human as described in the previous studies [38,46].
Acupuncture was performed once a day for 1 min immediately after morphine by one researcher. A stainless-steel needle (diameter 0.18 mm, length 8 mm, Dongbang Acupuncture Inc., Chingdao, China) was inserted vertically into a depth of 2-3 mm, and was bidirectionally twisted for stimulation [38]. Rats received acupuncture in awaken state with a slight movement restriction. Daily handling was given for 2-3 min to minimize the stress from the movement restriction. e normal and control groups received the same treatment with acupuncture groups without needle stimulation.

Organ Weight Measurements.
At sacrifice, the weights of individual spleen and left submandibular lymph node (S-LN) were measured at g levels (absolute wet-weights) using an automatic electronic balance (XB320 M, Precisa Instrument, Zuerich, Switzerland), and to reduce the differences between individual body weights, the relative weights (% of body weights) were also calculated using body weight at sacrifice and absolute weight as Relative Organ Weights (%) � [(Absolute spleen or S-LN wet-weights/Body weight at sacrifice) × 100].
2.8. Histopathology. At sacrifice, samples from spleen and left S-LN were fixed in 10% neutral buffered formalin (NBF). Equal regions of individual spleen and S-LN were crossly trimmed as one part in each organ, and all crossly trimmed spleen and S-LN parts were re-fixated in 10% NBF for 24 h. en paraffin embedding blocks were prepared using an automated tissue processor (Shandon Citadel 2000, ermo Scientific, Waltham, MA, USA) and embedding center (Shandon Histocentre 3, ermo Scientific, Waltham, MA, USA), and 3-4 μm sections were prepared using automated microtome (RM2255, Leica Biosystems, Nussloch, Germany). Representative sections were stained with HE for general histopathology [50,51], and individual crosstrimmed spleen and S-LN tissues were light microscopically observed (Model Eclipse 80i, Nikon, Tokyo, Japan). To more detail changes, the total splenic thicknesses (mm/ central regions), white pulp thickness (μm/white pulps) and numbers (white pulps/mm 2 of spleen), total and cortex thicknesses of S-LN (μm/central regions), lymphoid follicle numbers (Follicles/mm 2 of S-LN) were calculated using an automated image analyzer (iSolution FL ver 9.1, IMT isolution Inc., Vancouver, Canada), respectively [50,51].
e debris was allowed to settle, and the cell suspension was pelleted by centrifugation. RBCs were lysed by resuspending the pellet in cold 1% ammonium oxalate and incubating on ice for 10 min. e cells were pelleted and washed twice with HBSS (Hanks Balanced Salt Solution; Gibco BRL, Grand Island, NY, USA). Splenocytes were cultured overnight in Dulbecco's Modified Eagle Medium (Invitrogen, Grand Island, NY, USA) in the absence or presence of recombinant IL-2 (1000 IU/mL; Proleukin Chiron, Emeryville, CA, USA). e HTLA-230 neuroblastoma target cells were labeled for 2 hrs with Na 2 51 CrO 4 (100 μCi/1 × 106 cells) (ICN Biomedicals, Asse, Belgium). Target cells were then incubated for 6 h at 37°C with splenocytes as effector cells. e effector target cell ratio was 100 : 1. Supernatants were collected, and the amount of radioactivity released into the supernatants were counted with a gamma counter (Cobra 5002; Canberra Packard, Meriden, CT, USA). e percentage of specific target cell lysis was calculated as follows: e changes of immunoreactivities in the spleen and S-LN against markers of T cell subsets-CD3, CD4, CD8, and Foxp3, general and hematopoietic stem cells-CD34 and CD45, and immunerelatedcytokines-iNOS, TNF-α, IFN-c, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-12A were observed using purified primary antibodies (Table 1) with ABC (Avidin-biotinperoxidase complex) and peroxidase substrate kit (Vector Labs, Burlingame, CA, USA). Briefly, endogenous peroxidase activity was blocked by incubating in methanol and 0.3% H 2 O 2 for 30 min and nonspecific binding of immunoglobulin was blocked with normal horse serum blocking solution for 1 h in humidity chamber after heating (95-100°C) based epitope retrievals in 10 mM citrate buffers (pH 6.0) [50,53]. Primary antisera were treated overnight at 4°C in a humidity chamber, and then incubated with biotinylated universal secondary antibody and ABC reagents for 1 h. Finally, sections were reacted with peroxidase substrate kit for 3 min. All sections were rinsed in 0.01 M phosphatebuffered saline 3 times, between each step.

Statistical Analyses.
All numerical data were expressed as mean ± standard deviation (SD). Multiple comparison tests for different dose groups were conducted. Variance homogeneity was examined using the Levene test [54]. If the Levene test indicated no significant deviations from variance homogeneity, the data were analyzed by one-way ANOVA test followed by the least-significant differences (LSD) multicomparison test to determine which pairs of group comparisons were significantly different. In case of significant deviations from variance, homogeneity was observed at the Levene test, a nonparametric comparison test, Kruskal-Wallis H test was conducted. When a significant difference is observed in the Kruskal-Wallis H test, the Mann-Whitney U (MW) test was conducted to determine the specific pairs of group comparison, which are significantly different. Statistical analyses were conducted using SPSS for Windows (Release 14.0 K, IBM SPSS Inc., Armonk, NY, USA) [55]. In addition, the percent changes between normal and control groups at each sacrifice time were calculated to observe the severities of immune suppresses induced by treatment of morphine at dose levels of 0.1, 1.0, 5.0, and 10.0 mg/kg at each sacrifice times 3D, 7D, 21D, and 21D3DW, according to the previous studies [51,56], respectively. In addition, the percent changes between equal dosages of i. v. and i. p. treatments were calculated to observe the differences along with the administration route, according to the previously established methods [51,57], respectively.
Also, the percent changes between normal and control groups were calculated to observe the severity of immune decrease induced by morphine, and between control and acupuncture groups to observe immunomodulatory effects of acupuncture as following equations (3) and (4), according to the previous studies [56,57].
Equation ( Tables 3-7). However, no dose-dependent increases of immunosuppress signs were demonstrated between M 5.0 and M 10.0, also similar spleen and S-LN atrophic changes were observed in 21D3DW as compared to 21D.

Comparison between Administration Routes.
According to the results of the first experiment, we selected M 5.0 and M 10.0 and 21D as proper conditions to induce immune suppression and investigated if administration routes of i.v. and i. p. results in difference. After i. v. or i. p. treatment with morphine for 21D, the body weights at sacrifice, spleen and S-LN weights, and gross and histopathological findings were evaluated. Total thickness, white pulp diameter and numbers of spleen, total and cortex thicknesses, and lymphoid follicle numbers of S-LN were measured as histomorphometric items. e results were compared between equal dosages of i. v. or i. p. administration. e changes in body and organ weights showed no significant difference between i. v. and i. p. (Figure 12, Table 8). e gross and histopathological findings showed similar results (Figures 13-15, Tables 9-11), suggesting that morphine-induced immunosuppress occurred regardless of administration routes of i.v. or i.p.

Acupuncture Effects on Morphine-Induced Immune
Suppression. In the present study, we have observed a possibility of immune modulation by acupuncture. Following the first and second experiments, M 10 was administered for 21D intravenously and the body weight, spleen and left S-LN weights, and splenic NK cell activities were observed with histopathological findings (total thickness, white pulp diameter of spleen, total and cortex thicknesses, and lymphoid follicle numbers of S-LN), and CD3, CD4, CD8, Foxp3, CD34, CD45, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12A, iNOS, TNF-α, and IFN-c immunoreactive cell numbers in the spleen and S-LN parenchyma (positive cells/mm 2 ) were measured.
In morphine control rats, significant decreases of body weights, spleen and left S-LN absolute and relative weights, splenic NK cell activities, total spleen thickness, white pulp diameter and numbers of spleen, total and cortex S-LN thicknesses, lymphoid follicle numbers of S-LN, CD3, CD4, CD8, CD34, CD45, iNOS, TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12A, and IFN-c immunolabeled cell numbers, and increases of Foxp3 and IL-10 immunoreactivity were demonstrated compared to normal group, showing morphine-induced immunosuppresses. However, these morphine-induced immunosuppresses were obviously and significantly

Discussion
e present study confirmed the morphine-induced immune suppression in Sprague-Dawley rats. Vehicle or Morphine was treated, and rats were sacrificed at 3, 7, 21D including 21D3DW. e body weights at sacrifice, spleen and left S-LN weights, gross and histopathological findings were observed. Total thickness, white pulp diameter and numbers in spleen, total and cortex thicknesses, and lymphoid follicle numbers of S-LN were used as histomorphometric measures.
Decreased absolute and relative spleen and S-LN weights and increased gross and histopathological atrophic changes (increases of gross semiquantitative scores, decrease of the total thickness, white pulp diameter and numbers in spleen, total and cortex thicknesses, and lymphoid follicle numbers in S-LN at histomorphometric analysis) regarded as immunosuppress signs [50,51] were induced by morphine, in parallel with other studies [1,15]. ese signs were demonstrated obviously and significantly, in particular, by M 5.0 and 10.0 at 21D in the first experiment. However, no dosedependent increases in immunosuppress signs were demonstrated between M 5.0 and 10.0. Also, similar spleen and S-LN atrophic changes were observed at 21D3DW compared to those of 21D. ese findings were considered as direct evidence that an appropriate morphine inducedimmunosuppress rat model was produced by 21D treatment at a dose level of M 5.0 or M 10.0 and that 3D of withdrawal did not deteriorate morphine-induced immunosuppress, at least in the condition of the present experiment (Figures 2-11, Tables 3-7).
Morphine was reported to inhibit body weight increase in rats [58], and more seriously after short withdrawal [59,60]. is is parallel with our results that more severe decreases in body weights were demonstrated at 21D3DW in In the second experiment, we compared the morphine induced-immunosuppress between administration routes, (i. v. and i. p.) with M 5.0 and 10.0 treatment for 21D. After treatment, the body, spleen, and S-LN were weighed, and gross and histopathological findings were observed. Total thickness, white pulp diameter and numbers in spleen, total  Evidence-Based Complementary and Alternative Medicine e decrease in body weight observed in the control group was not shown in the acupunctured rats ( Figure 16). Decreases of absolute and relative spleen and S-LN weights, total thickness, white pulp diameter and numbers in spleen, total and cortex thicknesses, and lymphoid follicle numbers of S-LN at histomorphometric analysis are regarded as classic immunosuppress characteristics [50,51] were demonstrated as morphine-induced immunosuppress in the control group, in parallel with other studies [1,15,17]. However, these morphine-induced immunosuppress signs were obviously and significantly normalized by HT7, SI5, and LI5 acupunctures, in that order. ese findings are considered clear evidence that HT7, SI5, and LI5 acupuncture have potent and favorable inhibitory activities against morphine-induced atrophic changes in lymphoid organs (Figures 18 and 24, Tables 12-14).
NK cells are representative immune cells, and activation of NK cells has been highlighted as new treatment regimen for cancer and other immunosuppressive diseases [61,62]. In this study, significant decreases in splenic NK cell activities were observed in control rats however they were normalized by HT7, SI5, and LI5 acupunctures (Figure 17), suggesting definitive immunomodulatory effects of acupuncture through splenic NK cell activations on morphineinduced immune suppression.
is result is parallel with a previous study demonstrating that decreased postoperative NK cell activity induced by morphine was reversed by electro-acupuncture [63].
T cell antigen receptors are always membrane-bound and noncovalently associated with a set of four invariant glycoproteins collectively called CD3. us, CD3 has been regarded as a marker of T-cells [64]. CD4 is a single-chain glycoprotein of 55 kDa, and CD8 is a disulfide-linked heterodimer of a 34 kDa subunit. Either CD4 or CD8 is found on mature T cells, although immature T cells may express both. eir function is to determine the class MHC molecule that is recognized by a T cell. Generally, CD4+ cells are called helper T cell and CD8+ cells are cytotoxic T cell [65]. Foxp3, a protein involved in immune responses [66], is a member of the FOX protein family and appears to function as a master regulator of the regulatory pathway in the development and function of Treg cells [67]. Treg cells generally decrease the immune response. In cancer, an excess of regulatory T cell activity can prevent the immune system from destroying cancer cells. In autoimmune disease, a deficiency in regulatory T cell activity can allow other autoimmune cells to attack the body's own tissues [68,69]. Foxp3 has been used as a valuable marker for Treg cell activity, and increments in Foxp3+ cells represent immune suppression [66,67,70]. In our immune-histochemistric analysis, significant decreases in CD3, CD4 and CD8+ cells, and increases in Foxp3+ cells were demonstrated in the control group, suggesting morphine-induced immunosuppresses. However, these morphine-inducedimmunosuppression-related changes in T cell subsets were normalized by acupunctures (Figures 19,  20, 25, and 26). ese findings confirm that acupunctures have potent and favorable immunomodulatory activities against morphine induced-immune suppression through modulation of T cell subset. e cytokine TNF-α, produced by a variety of cell types including splenocytes, is associated with critical events leading to T-lineage commitment and differentiation [71]. TNF-α can enhance the in vivo immune response at doses much lower than those that cause weight loss or tissue toxicity. It enhances the proliferation of B and T cells and promotes the generation of cytotoxic T cells. In addition, it enhances IL-2-induced immunoglobulin production and augments IL-2stimulated-natural killer cell activity and proliferation of monocytes [72]. IL-1 is another cytokine released to various cell types such as macrophages, dendritic cells, lymphocytes, endothelial cells, fibroblasts and keratocytes, and is necessary for the successful initiation of some forms of immune response [73]. IL-2 is a type of cytokine signaling molecule in the immune system. It is a 15.5-16 kDa protein [74] that regulates the activities of white blood cells (leukocytes, often lymphocytes) that are responsible for immunity [75]. IL-4 is a cytokine that induces the differentiation of naive helper T cells ( 0 cells) to 2 cells. Upon activation by IL-4, 2 cells subsequently produce additional IL-4 in a positive feedback loop. e cell that initially produces IL-4, thus inducing 2 differentiation, has not been identified, however basophils may be the effector cell      [76]. IL-10 is an immunosuppressive glycoprotein of 19-21 kDa that is secreted by 2 cells, some B cells, and activated macrophages. IL-10 primarily acts on activated macrophages to suppress the secretion of IL-1, IL-12, TNFα, and ROS [72]. IL-6 is secreted by 2 cells and macrophages to stimulate immune response during infection and after trauma, leading to inflammation [77]. IL-6 also plays a role in fighting infection, as IL-6 has been shown to be required for resistance against the bacterium Streptococcus pneumonia [78]. IL-12 is naturally produced by dendritic cells, macrophages, neutrophils, and human Blymphoblastoid cells in response to antigenic stimulation [79], and is involved in the differentiation of naive T cells into 1 cells through stimulating the production of IFN-c and TNF-α [80]. In addition, IFN-c is a glycoprotein of 20 to 25 kDa produced by CD8+ T cells, 1 cells, and NK cells in response to IL-2. It has complex effect on B and T cell functions and enhances the NK cell and macrophage activities [72]. In this study, significant decreases of spleen and S-LN iNOS, TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12A and IFN-c+ cells, and increases of IL-10+ cells in the spleen and S-LN were demonstrated in the control group, suggesting morphine-induced immunosuppression. However, these morphine-inducedimmunosuppression-related cytokine changes were significantly normalized by HT7, SI5 and LI5 acupunctures, in those orders (Figures 21-23, Tables 12-14). ese findings are considered reliable evidence that HT7, SI5, and LI5 acupunctures have potent and favorable immunomodulatory activities against morphine-induced immunosuppression, through cytokine normalization, at least in a condition of the present experiment.

Evidence-Based Complementary and Alternative Medicine
CD34+ cells have been regarded as general stem cells, and CD45+ cells are considered hematopoietic stem cells [81,82]. CD45 is a pan-leukocyte protein with tyrosine phosphatase activity involved in the regulation of signal transduction in hematopoiesis [83]. CD45 has been used as a valuable pan-leukocyte marker [84]. In our results, significant decreases of CD34 and CD45+ cells were demonstrated in the spleen and S-LN of control rats, as courses of immune suppression. However, these morphineinducedimmunosuppression-related decreases of the CD35 and CD45+ cells were significantly inhibited by HT7, SI5, and LI5 acupuncture (Figures 20 and 26). ese findings are considered direct evidence that HT7, SI5, and LI5 acupuncture have immunomodulatory activities against morphine-induced immunosuppression, through stem cell migration and differentiation to immune cells.
Taken together, the histopathological changes and the abnormal cytokines and immune cell activities induced by morphine were normalized by acupuncture.
Morphine weakens the immune system and suppresses immune response in the spleen, thymus, and lymph nodes [1,4]. Opioid decreases the number of NK cells [18,19] and B and T lymphocytes [20,21]. A possible mechanism through which morphine induces immune decrease is that morphine acts as a potential oxidative stress inducer [7,[11][12][13]. Nutrients supporting the immune system are antioxidant providers and immune cells such as T-cells, NK cells, and T-helper cells have high levels of ROS. Also, immune cell membranes have a lot of polyunsaturated fatty  acids, susceptible to ROS-related damage [26,28]. erefore, nutrients with antioxidant property extinguish the free radicals and regulate the immune system [26,29].
In previous studies, acupuncture showed antioxidant properties [42][43][44], especially in morphine-induced liver injury [13], and regulated ROS levels [45]. Given that                     morphine is a potential oxidative stressor and weakens the immune system [1,4,15] and that antioxidation is important to support immune system, we suggest that acupunctures' inhibitory action against morphine induced-immune suppression probably is mediated, at least in part, via antioxidation.

Conclusion
Our results showed that the appropriate morphine inducedimmunosuppress rat model was confirmed by 21D treatment with doses of 5.0 and 10.0. Also, no dose-dependent increases of immunosuppress signs were demonstrated between M 5.0 and 10.0, and 3 days of withdrawal was not influenceable. In addition, the immunosuppress by morphine was induced regardless of administration routes (i. v. or i. p.). Most importantly, the key immune parameters-spleen and S-LN weights, splenic NK cell activities, and the T cell subsets (CD3, CD4, CD8, and Foxp3), general and hematopoietic stem cells (CD34 and CD45), and major immunerelated cytokines (iNOS, TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12A, and IFN-c) immunopositive cells showed immunosuppress signs by morphine, however, these morphineinducedimmunosuppress-related signs were normalized by acupunctures, suggesting that acupuncture can be a new potent alternative immunomodulatory remedy for immune disorders by morphine. Further studies are needed to elucidate a more dedicated mechanism underlying acupuncture effects.
Data Availability e data supporting this study can be obtained from the corresponding author upon request.

Conflicts of Interest
ere are no conflicts of interest.

Authors' Contributions
Rong Jie Zhao, Dae Geon Lee, and Chan Sik Park contributed equally to this research.