Immunopotentiating effects of herb-partitioned moxibustion on the spleens of cyclophosphamide-induced immunosuppressed rats.

BACKGROUND: To investigate the effec of the herb-partitioned moxibustion on T-lymphocyte activity in immunosuppressed rats through differential modulation of the immune checkpoint molecules CD28 and CTLA-4. METHODS: Forty-eight Sprague‒Dawley rats were randomly divided into the normal group (NG), the cyclophosphamide model group (CTX), the herb-partitioned moxibustion group (HPM), the CD28 inhibitor + herb-partitioned moxibustion group (aCD28 + HPM), the CTLA-4 inhibitor + herb-partitioned moxibustion group (aCTLA-4 + HPM), and the levamisole group (LEV) (8 rats per group). The immunosuppression model was prepared using cyclophosphamide. HPM treatments was performed via herb-partitioned moxibustion at 4 acupoints, Zhongwan (CV12), Shenque (CV8), Guanyuan (CV4), and Zusanli (ST36). Subsequently, the moxa floss was made into a conical moxa cone, which was then placed on the herbal cake and ignited. Five consecutive moxibustion strokes were performed daily for 10 consecutive days. In addition to the same moxibustion, each rat in the aCD28 + HPM group was injected intraperitoneally with 0.5 mg/kg of CD28 inhibitor per rat on the first day of treatment, and 100 µL of CTLA-4 inhibitor was injected into the aCTLA-4 + HPM group on Days 1, 4, and 7. For the positive control, levamisole (LEV) was administered by gavage at a dose of 2 mg/kg once daily for 10 days. RESULTS: Compared with those in CTX model rats, the WBC counts in the HPM and other groups were significantly higher. The immobility time of EPM in the HPM group was significantly lower than that of the CTX group. The HE stainin results also showed that after treatment, the the marginal zone area of the spleen tissue in the HPM increased, the number of lymphatic sheath lymphocytes around the small central artery of the spleen increased, and the amount of red pulp containing a small amount of pigmentation was partially reduced. Compared with those in the CTX group, the serum levels of CD28, CTLA-4, B7-1, and B7-2 were significantly lower, and the levels of α-MSH, TrkB, and BDNF were significantly greater in the HPM group. The results of the flow cytometry assay showed a significant increase in the number of CD8 + T lymphocytes after treatment with HPM or other agents compared to that in the CTX group. The immunofluorescence results showed that the levels of CD28 and CTLA-4 lower in spleen tissues than in control tissues, and the binding ability of CD28 to B7-1 and B7-2 was weakened after treatment with HPM and other treatments compared with CTX rats, PCR for CD28, CTLA-4 and B7-1 showed similar results. CONCLUSION: In the immunosuppressive rat model induced by cyclophosphamide, HPM upregulated the expression of α-MSH, TrkB, and BDNF, and downregulated the expression of CD28 and CTLA-4, thereby enhancing the activity of CD8+ T lymphocytes, restoring spleen function, improving the immunosuppressive state, restoring immune function, and effectively alleviating depressive symptoms.

Application and Effectiveness of Chinese Medicine in Regulating Immune Checkpoint Pathways.

Immunotherapy targeting immune checkpoint molecules has emerged as a key approach in cancer treatment, representing the forefront of antitumor research. However, studies on immune checkpoint molecules have mainly focused on targeted therapies. Chinese medicine (CM) research as a complementary medicine has revealed that immune checkpoint molecules also undergo disease-specific changes in the context of autoimmune diseases. This review article presents a comprehensive analysis of CM studies on immune checkpoint molecules in the last 5 years, with a focus on their role in different diseases and treatment modalities. CM research predominantly utilizes oral administration of herbal plant extracts or acupuncture techniques, which stimulate the immune system by activating specific acupoints through temperature and needling. In this study, we analyzed the modulation and mechanisms of immune checkpoint molecules associated with different coinhibitory and costimulatory molecules, and reviewed the immune functions of related molecules and CM studies in treating autoimmune diseases and tumors. By summarizing the characteristics and research value of CM in regulating immune checkpoint molecules, this review aims to provide a useful reference for future studies in this field.

[Effects of herbal cake separated moxibustion on Tim-4 and ubiquitination of PD-1 in rabbits with immunosuppression].

OBJECTIVE: To observe the effects of herbal cake separated moxibustion on macrophage effector molecule T-cell immunoglobulin and mucin-domain containing-4 (Tim-4) and ubiquitination of programmed cell death protein 1 (PD-1) in rabbits with immunosuppression, and to explore the possible mechanism on herbal cake separated moxibustion in improving immunosuppression. METHODS: Thirty-two big-ear white rabbits were randomly divided into a normal group, a model group, a moxa stick moxibustion group and a herbal cake separated moxibustion group, 8 rabbits in each group. Except the normal group, the immunosuppression model was established by intraperitoneal injection of cyclophosphamide of60 mg/kg in the other 3 groups. "Shenque" (CV 8), "Shenshu" (BL 23), "Zusanli" (ST 36), etc. were selected in both the moxa stick moxibustion group and the herbal cake separated moxibustion group. Moxa stick moxibustion was applied in the moxa stick moxibustion group, one cone at each acupoint; herbal cake separated moxibustion was applied in the herbal cake separated moxibustion group, 5 cones at each acupoint. The intervention was given once every other day for 10 times in both groups. Leukocyte content in peripheral blood was detected by blood cell analyzer; the positive expression of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD8, CD68 and Tim-4 were detected by ELISA, and the expression of Tim-4 and F-box only protein 38 (FBXO38) in the liver and spleen tissues was measured by immunohistochemistry. RESULTS: Compared with the normal group, in the model group, white blood cell count (WBC) and percentage of neutrophils (NEU%) were decreased while percentage of lymphocyte (LYM%) was increased (P<0.01) in peripheral blood; the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were increased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were increased (P<0.01), the serum level of CD8 was decreased (P<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (P<0.01). Compared with the model group, in the moxa stick moxibustion group and the herbal cake separated moxibustion group, WBC and NEU% were increased (P<0.01); the positive expression rates of PD-1 in CD+4 T lymphocytes, CD+8T lymphocytes and CD+68 macrophages in peripheral blood were decreased (P<0.01); the serum levels of IL-2, CD68 and Tim-4 were decreased (P<0.01), the serum levels of CD8 were increased (P<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (P<0.01, P<0.05). Compared with the moxa stick moxibustion group, in the herbal cake separated moxibustion group, the positive expression rate of PD-1 in CD+68 macrophages in peripheral blood was increased (P<0.05); serum level of Tim-4 was increased (P<0.01); AOD of Tim-4 in the liver tissue was decreased (P<0.05). CONCLUSION: Herbal cake separated moxibustion can improve immunosuppression by regulating the expression of macrophage effector molecule Tim-4 and the FBXO38 mediated ubiquitination of PD-1, Tim-4 may be one of the specific indexes of immunomodulation involving with herbal cake separated moxibustion.

[Effect of herbal cake-separated moxibustion on behavioral stress reactions and blood lactic acid level and muscular AMPK/PGC-1α signaling in rats with chronic fatigue syndrome].

OBJECTIVE: To observe the effect of herbal cake-separated moxibustion (HCSM) on serum lactic acid (BLA) level and AMPK/PGC-1α signaling pathway in the quadriceps femoris in chronic fatigue syndrome (CFS) rats, so as to explore its mechanisms underlying improvement of CFS. METHODS: According to the random number table, 50 SD rats were divided into blank control, model, HCSM, sham HCSM and medication (herbal medicine gavage) groups, with 10 rats in each group. The CFS model was established by using chronic restraint and exhaustive swimming, alternately, once daily for 21 days. The herbal cake was made of Xiaoyao Powder (Mental Ease Powder, composed of [Danggui (Radix Angelicae Sinensis), Baishao (Radix Paeoniae Alba), Chaihu (Radix Bupleuri), Fuling (Poria), Baizhu (Rhizoma Atractylodis, Macrocephalae), etc.]. The HCSM was applied to "Shenque" (CV8), "Guanyuan "(CV4), bilateral "Zusanli" (ST36) and "Qimen" (LR14), 5 moxa-cones for each acupoint, once daily for 10 days. For sham HCSM, the excipient was instead of herbal cake, and the same 5 moxa-cones was given as the HCSM group. Rats of the medication group received gavage of Xiaoyao Powder suspension (60 mg·kg-1), once daily for 10 days. The open field test and tail suspension test were conducted for determining the animals' locomotor activity. The blood sample was taken from the abdominal aorta under anesthesia for assaying the levels of serum BLA, chemokine ligand CXCL9 and ß-endorphin (EP) by ELISA. Bilateral quadriceps femoris were sampled for observing histopathological changes after staining with conventional H.E. technique, and for detecting the expression levels of phosphorylated AMP-activated protein kinase (p-AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by using immunohistochemistry. RESULTS: Compared with the blank control group, the number of rearing and horizontal grid-crossing times, struggling times of tail suspension test were significantly decreased (P<0.05), and the immobility time was obviously prolonged (P<0.05) in the model group. Compared with the model group, both HCSM and medication groups had a significant increase of rearing, horizontal grid-crossing times and struggling times (P<0.05), and the immobility time had a significant decrease (P<0.05). But there were no significant differences in the total movement distance among the 5 groups (P>0.05), and in the 5 indexes of behavioral measurements between the HCSM and medication groups (P>0.05). The sham HCSM could also evidently increase the struggling times and reduce the immobility time (P<0.05). The contents of serum BLA, CXCL9 and ß-EP were obviously higher in the model group than in the blank control group (P<0.05), as well as remarkably lower in the HCSM and medication groups than in the model group (P<0.05). Whereas the expression levels of muscular p-AMPK and PGC-1α were considerably lower in the model group than in the blank control group (P<0.05), and significantly increased in both HCSM and medication groups relevant to the model group (P<0.05). Compared with the sham HCSM group, the contents of BLA, CXCL9 and ß-EP in serum of the HCSM group and contents of CXCL9, ß-EP in medication group were significantly decreased (P<0.05), and the protein expressions of p-AMPK and PGC-1α in quadriceps femoris in both HCSM and medication groups were significantly increased (P<0.05). H.E. staining showed smaller intercellular space, uneven cytoplasmic staining in some muscle fibers, nucleus pyknosis and condensation, and inflammatory cell infiltration in the model group, which was milder in both HCSM and medication groups. CONCLUSION: HCSM can mitigate the stress behavioral state in CFS rats, which may be related with its functions in lowering the levels of serum BLA, CXCL9 and ß-EP, and activating AMPK/PGC-1α signaling pathway (balancing energy metabolism) in the quadriceps femoris.