## 4 Discussion  
The immune system protects the body from harmful agents and external invasion; therefore, immune function is important to maintaining a healthy physiological condition. The immune system responds to, recognizes, and removes antigenic foreign substances and removes them to maintain physiological homeostasis [13; 14]. Therefore, compounds that modify immune responses have attracted considerable interest. In this study, for the first time, we evaluated the immunomodulatory effects of LJFE in CPA-induced immunosuppressed mice. In this study, 70% ethanol extracts of \(L\). _japonica_ were selected which are approved effective raw materials for health-functional food in certain indications and it was considered that these extracts could be extended to immunostaining effects. Regarding active substances of \(L\). _japonica_ fruit extract, previous studies have reported lactones, terpenoids, and flavonoids [1; 2; 3]. Although full analysis of bioactive substances was not conducted for our extract, we selected Hamabiwalactone B as the indicator bioactive substance and its presence was confirmed using the high-performance liquid chromatography (HPLC) method. Regarding mechanism of action, 70% ethanol extract \(L\). _japonica_ may promote immunostimulatory activity potentially through proinflammatory transcription factors such as NF-\(\kappa\)B as was reported to increase its target gene cytokines such as TNF-\(\alpha\), IL-6, and IL-1\(\beta\)[10]. However, an additional underlying mechanism study using Hamabiwalactone B will be subject to our further study. The spleen is one of the largest lymphoid organs in the body, and it mediates immunological functions. Therefore, the spleen injury score was used to measure the function of the immune organ [25]. In this study, LJFE significantly reduced the spleen injury score at 25, 50, and 100 mg/kg, suggesting that LJFE recovered damages to the spleen structure and function (Figure 2). In the spleen, RP extracts aged cells from the blood circulation and detects pathogens, and WP is made up of immune cells. The marginal zones are regions between RP and WP capture antigens from the blood [25; 26]. LJFE treatment recovered the architecture and these immune regions of the spleen, which were damaged by CPA treatment. T-lymphocytes play an important role in immune response. T-cells are distinguished from other lymphocytes by the presence of T-cell receptors on their surfaces. CD4+ cells are T-helper cells, which are functional in adaptive immune response [27; 28]. For example, CD4+ cells release cytokines and help in activating other immune cells such as CD8+ cells or cytotoxic T-cells [29]. In our study, LJFE significantly increased the numbers of CD4+- and CD8+-positive cells at 100 mg/kg, suggesting that LJFE notably recovered the cellular immune function of lymphocytes, which was suppressed by CPA treatment (Figure 3). However, the CD4+/CD8+ ratio improved slightly and did not significantly recover. This may be because of the short treatment duration and low dosage of LJFE. Lymphocyte activation and proliferation are adaptive immune responses [30]. Based on our results, the mechanisms by which LJFE stimulates the immune system involve the enhancement of T- and B-cell proliferation and NK-cell activation. T-helper cells help B-cells to multiply and produce antibodies and release cytokines. T-helper cells promote the activation of NK cells, another key cytotoxic lymphocyte that protects the body from pathogens and foreign substances [31; 32]. LJFE promoted the proliferation of T- and B-lymphocyte and NK-cell activity at 100 mg/kg (Figure 4). NK-cell-derived cytokines and their cytotoxic functions can regulate immune responses and pathogenesis of many immune-related diseases. The activation of cytotoxic T- and NK cells promotes the production of cytokines such as IFN-\(\gamma\) and TNF-\(\alpha\). In our study, the serum levels of IFN-\(\gamma\) and TNF-\(\alpha\) were significantly increased by LJFE treatment.