ObjectiveThis study aims to investigate the action mechanism by which Xiaozheng Zhitong paste （XZP） alleviates bone cancer pain （BCP） by regulating programmed death protein 1 （PD-1）/programmed death-ligand 1 （PD-L1） pathway-induced osteoclast formation. MethodsThirty female C57BL/6 mice were randomly allocated into the following groups （n=6 per group）： normal control group， model group， low‑dose XZP group （31.5 g·kg^-1）， high‑dose XZP group （63 g·kg^-1）， and PD‑1 inhibitor （Niv） group. A bone cancer pain （BCP） model was established by injecting Lewis lung carcinoma cells. Mice in the normal control and model groups received topical application of a blank paste matrix at the wound site. Mice in the low‑ and high‑dose XZP groups were treated with XZP applied topically twice daily. Mice in the Niv group were topically administered the blank paste matrix and additionally received Niv via tail‑vein injection every two days. All interventions were continued for 21 days. During this period， behavioral tests were performed to assess mechanical， motor， and thermal nociceptive sensitivities. After 21 days， all mice were euthanized， and bone tissue from the operated side was collected for sectioning and preservation. Tartrate‑resistant acid phosphatase （TRAP） staining was used to evaluate osteoclast expression in the lesioned bone tissue. Immunohistochemistry was performed to detect the expression of Runt‑related transcription factor 2 （Runx2） in the lesioned bone tissue. Immunofluorescence was employed to assess the expression of PD‑1 and PD‑L1 in the lesioned bone tissue. ResultsCompared with the normal group， the model group showed significantly decreased limb mechanical withdrawal threshold， spontaneous paw flinching， and thermal withdrawal latency （P<0.01）， increased number of osteoclasts in the lesioned bone tissue （P<0.01）， and reduced expression of Runx2 （P<0.01）. Compared with the model group， the BCP mice in the XZP low-dose group， XZP high-dose group， and Niv group exhibited increased limb mechanical withdrawal threshold， movement scores， and thermal withdrawal latency （P<0.01）. The XZP low-dose group showed no significant changes in osteoclast number or Runx2 expression， while the XZP high-dose group and Niv group demonstrated significantly reduced osteoclast numbers （P<0.01） and significantly increased Runx2 expression （P<0.01）. In the lesioned bone tissue of BCP mice， the XZP low-dose group showed no significant decrease in the percentage of PD-1 expression， but a decrease in the percentage of PD-L1 expression （P<0.05）. In contrast， both the XZP high-dose group and the Niv group exhibited significant reductions in the percentages of PD-1 and PD-L1 expression （P<0.01）. ConclusionXZP alleviates the pain of mice with BCP by blocking the PD-1/PD-L1 pathway to inhibit osteoclastogenesis.

ObjectiveTo investigate the effects and underlying mechanisms of Xiaozheng Zhitong Paste （XZP） on bone cancer pain （BCP）. MethodsThirty female BALB/c mice were randomly divided into five groups： a Sham group， a BCP group， a BCP+low-dose XZP group， a BCP+high-dose XZP group， and a BCP+high-dose XZP + protease-activated receptor 2 （PAR2） agonist GB-110 group. BCP mice model was constructed by injecting Lewis lung carcinoma cells into the femoral cavity of the right leg， which was followed by being treated with XZP for 21 d. After 21 d， the mice were sacrificed. Nissl staining was used to evaluate the survival of spinal cord neurons. Immunofluorescence staining was conducted to localize ionized calcium-binding adapter molecule 1 （Iba1） and neuronal nuclear antigen （NeuN） in spinal cord tissue， thereby assessing microglial activation and neuronal survival. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， transforming growth factor-β （TGF-β）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） in spinal cord tissue. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect mRNA expression levels associated with M1/M2 polarization of microglia. Western blot analysis was performed to examine the expression of proteins related to microglial polarization as well as those involved in the PAR2/nuclear factor kappa B （NF-κB）/NOD-like receptor protein 3 （NLRP3） signaling pathway in the spinal cord. ResultsCompared with the Sham group， the spinal cord neurons were damaged， the number of Nissl-positive spinal cord neurons in the spinal cord tissue was significantly reduced （P<0.01）， and the rate of NeuN-positive cells was significantly decreased （P<0.01）. The spinal cord microglia were activated， the inflammatory level of the spinal cord tissue was enhanced， and Iba1 staining was significantly enhanced （P<0.01）. The levels of IL-1β， TNF-α， IL-6， TGF-β， IL-4 and IL-10 were significantly increased （P<0.01）. The mRNA expressions of IL-1β， TNF-α and inducible nitric oxide synthase （iNOS） were significantly increased （P<0.01）， and the expression of PAR2， NLRP3， ASC and NF-κB p65 proteins in the spinal cord tissue of the BCP mice was significantly enhanced （P<0.01）. Compared with the BCP group， high-dose XZP treatment significantly increased the number of Nissl-positive spinal cord neurons in the BCP mice （P<0.01）， significantly enhanced the rate of NeuN-positive cells in the spinal cord tissue， and significantly weakened Iba1 staining （P<0.01）. In addition， the levels of IL-1β， TNF-α， and IL-6 were significantly decreased， while the levels of TGF-β， IL-4， and IL-10 were significantly increased （P<0.05， P<0.01）. The mRNA expression levels of IL-1β， TNF-α， and iNOS were decreased， whereas those of cluster of differentiation 206 （CD206）， arginase-1 （Arg-1）， and YM1/2 were significantly increased （P<0.05， P<0.01）. Low-dose and high-dose XZP treatment significantly decreased the expression of PAR2， NLRP3， ASC， and NF-κB p65 proteins in the spinal cord tissue （P<0.05， P<0.01）. These effects could all be significantly eliminated by the PAR2 agonist GB-110. ConclusionXZP can mitigate BCP in mice， which may be achieved through blocking the activated PAR2/NF-κB/NLRP3 pathway.

ObjectiveThe paper aims to investigate the action mechanism by which the Xiaozheng Zhitong paste （XZP） relieves bone cancer pain （BCP）. MethodsA model of mice with BCP was established by using Lewis tumor cells. The therapeutic effects of XZP， the ferroptosis inhibitor Ferrostatin-1 （Fer-1）， and the nuclear factor erythroid 2-related factor 2 （Nrf2） inhibitor Brusatol （Bru） on BCP were examined. Mice were randomly divided into the Sham operation group， BCP group， BCP+XZP-L group， BCP+XZP-H group， BCP+Fer-1 group， and BCP+XZP-H+Bru group， with six mice in each group. Pain behavior tests were conducted on the mice to assess pain levels. Colorimetric assays were employed to measure ferroptosis-related factors in serum and spinal cord tissue including Fe， malondialdehyde （MDA）， reactive oxygen species （ROS）， and superoxide dismutase （SOD）. Immunofluorescence staining was used to assess ROS production in spinal cord tissue. Transmission electron microscopy was used to observe the ultrastructure of mitochondria in lumbar spinal cord tissue. Quantitative real-time polymerase chain reaction （Real-time PCR） was employed to detect mRNA expression of Nrf2， heme oxygenase-1 （HO-1）， glutathione peroxidase 4 （GPX4）， and solute carrier family 7 member 11 （SLC7A11） in spinal cord neuron tissue. The protein expression of Nrf2， HO-1， GPX4， and SLC7A11 in spinal cord neurons was measured by Western blot. ResultsCompared with the Sham group， mice in the BCP group exhibited significantly reduced limb usage scores， mechanical foot withdrawal thresholds， and thermal foot withdrawal thresholds （P<0.01）. Serum and lumbar spinal cord tissue levels of Fe， MDA， and reactive oxygen species （ROS） were significantly elevated （P<0.05）， while superoxide dismutase （SOD） levels were significantly decreased （P<0.05）. Lumbar spinal cord mitochondrial structural damage was observed， and mRNA and protein expression of Nrf2， HO-1， GPX4， and SLC7A11 were significantly downregulated （P<0.01）. Compared with the BCP group， both low- and high-dose XZP groups improved the aforementioned pain behavioral indicators （P<0.05，P<0.01）， reduced ferroptosis-related biomarkers including Fe， MDA， and ROS levels （P<0.05）， increased SOD levels （P<0.05，P<0.01）， alleviated mitochondrial damage， and upregulated Nrf2， HO-1， GPX4， SLC7A11 mRNA and protein expression （P<0.05，P<0.01）. The high-dose XZP group exhibited comparable efficacy to Fer-1 in alleviating pain and inhibiting ferroptosis. Following Bru administration， XZP's effects on pain behavioral indicators， regulation of ferroptosis-related markers， mitochondrial structural protection， and activation of the Nrf2/HO-1/GPX4/SLC7A11 pathway were significantly reversed （P<0.05，P<0.01）. ConclusionExternal application of XZP alleviates pain symptoms in BCP mice by activating the Nrf2/HO-1/GPX4/SLC7A11 pathway， thereby inhibiting ferroptosis and neuronal damage in spinal cord neurons.

Pain， as one of the most common symptoms in cancer patients， seriously affects the survival quality of patients. The three-step pain relief program currently used in clinical practice cannot completely relieve pain in cancer patients and is accompanied by many problems. From the perspective of Jueyin syndrome in traditional Chinese medicine （TCM）， this paper believed that the core pathogenesis of cancer pain was declined healthy Qi and cold and heat in complexity， and used Wumeiwan as the main formula with modification according to syndrome for clearing the upper， warming the lower part of the body， and harmonizing the cold and heat. It can regulate the pathological environment of deficiency， cold， stasis， toxicity， and heat， and restore the physiological function of Yang transforming Qi while Yin constituting form， so as to prevent， relieve， and even eliminate cancer pain， having achieved good clinical efficacy. It can not only help cancer patients relieve pain， but also control tumor and eliminate tumor， achieving a dual benefit of pain relief and tumor suppression. It gives full play to the characteristics and advantages of syndrome differentiation and treatment in TCM， and expands the scope of ZHANG Zhongjing's treatment for Jueyin syndrome， which provides ideas for the clinical diagnosis and treatment of cancer pain from the perspective of deficiency-excess in complexity and cold and heat in complexity.

Objective:To compare the activity difference of the high affinity humanized CD19 chimeric antigen receptor (CAR)-T cells and murine CD19 CAR-T cells.Methods:Peripheral venous blood T cells from 8 healthy volunteers were collected and infected with humanized and murine CD19 CAR lentivirus. Human and murine CD19 CAR-T cells were prepared and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The cytotoxicity of CD3 + T cells, humanized and murine CD19 CAR-T cells to NALM-6 cells was detected by lactate dehydrogenase assay. Thirty BAL B/c nude mice transplanted with NALM-6 cells were randomly divided into 3 groups with 10 mice in each group and injected humanized CD19 CAR-T cells, mouse CD19 CAR-T cells and control CD3 + T cell via tail vein, respectively. The proportion of NALM-6 cells in peripheral blood and the proportion of CD19 CAR-T cells in T cells from the vein of the inner canthus were detected by flow cytometry. The overall survival of BAL B/c nude mice was observed. Results:The proliferation of mouse and humanized CD19 CAR-T cells were (68.50±0.93)% and (80.63±1.41)%, respectively ( t=20.353, P<0.001) after cultured in vitro for 24 hours, and were (91.38±1.41)% and (148.13±1.25)%, respectively ( t=85.364, P<0.001) after cultured for 48 hours. When the effect to target ratio was 1∶1, there was no difference between the humanized and murine CD19 CAR-T cell group after co-culture for 24 hours ( P=0.169), while the killing activity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells ( P<0.01) after 48 hours of co-culture. When the effect to target ratio was 4∶1, the cytotoxicity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells in co-culture for 24 and 48 hours ( P<0.01). On the seventh day of CD19 CAR-T cell therapy, the proportion of NALM-6 cells in the peripheral blood of BAL B/c nude mice decreased to the lowest level in the humanized CD19 CAR-T cell group and the murine CD19 CAR-T cell group. After 21 days, the proportion of NALM-6 cells in the murine CD19 CAR-T cell group was higher than that in the humanized CD19 CAR-T cell group ( P21 d=0.001, P28 d<0.001, P35 d<0.001). The proportion of humanized and murine CD19 CAR-T cells in the peripheral blood reached the peaks after 7 days of therapy, and the proportion of humanized CD19 CAR-T cells was higher than that of murine CAR-T cells ( P7 d=0.002). The CD19 CAR-T cells disappeared in the peripheral blood in the murine CD19 CAR-T cell group after 14 days of therapy, while in the humanized CD19 CAR-T cell group it disappeared after 21 days of therapy. The median survival of BAL B/c nude mice in the murine CD19 CAR-T cell group and the humanized CD19 CAR-T cell group was 42 days and 63 days, respectively ( χ2=15.382, P<0.001). Conclusions:High affinity humanized CD19 CAR-T cells have stronger proliferation, higher cytotoxicity and longer survival time compared with those of murine CD19 CAR-T cells. The results indicate that the clinical efficacy of humanized CD19 CAR-T cells would be better than that of murine CD19 CAR-T cells.

Objective:To compare the activity difference of the high affinity humanized CD19 chimeric antigen receptor (CAR)-T cells and murine CD19 CAR-T cells.Methods:Peripheral venous blood T cells from 8 healthy volunteers were collected and infected with humanized and murine CD19 CAR lentivirus. Human and murine CD19 CAR-T cells were prepared and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The cytotoxicity of CD3 + T cells, humanized and murine CD19 CAR-T cells to NALM-6 cells was detected by lactate dehydrogenase assay. Thirty BAL B/c nude mice transplanted with NALM-6 cells were randomly divided into 3 groups with 10 mice in each group and injected humanized CD19 CAR-T cells, mouse CD19 CAR-T cells and control CD3 + T cell via tail vein, respectively. The proportion of NALM-6 cells in peripheral blood and the proportion of CD19 CAR-T cells in T cells from the vein of the inner canthus were detected by flow cytometry. The overall survival of BAL B/c nude mice was observed. Results:The proliferation of mouse and humanized CD19 CAR-T cells were (68.50±0.93)% and (80.63±1.41)%, respectively ( t=20.353, P<0.001) after cultured in vitro for 24 hours, and were (91.38±1.41)% and (148.13±1.25)%, respectively ( t=85.364, P<0.001) after cultured for 48 hours. When the effect to target ratio was 1∶1, there was no difference between the humanized and murine CD19 CAR-T cell group after co-culture for 24 hours ( P=0.169), while the killing activity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells ( P<0.01) after 48 hours of co-culture. When the effect to target ratio was 4∶1, the cytotoxicity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells in co-culture for 24 and 48 hours ( P<0.01). On the seventh day of CD19 CAR-T cell therapy, the proportion of NALM-6 cells in the peripheral blood of BAL B/c nude mice decreased to the lowest level in the humanized CD19 CAR-T cell group and the murine CD19 CAR-T cell group. After 21 days, the proportion of NALM-6 cells in the murine CD19 CAR-T cell group was higher than that in the humanized CD19 CAR-T cell group ( P21 d=0.001, P28 d<0.001, P35 d<0.001). The proportion of humanized and murine CD19 CAR-T cells in the peripheral blood reached the peaks after 7 days of therapy, and the proportion of humanized CD19 CAR-T cells was higher than that of murine CAR-T cells ( P7 d=0.002). The CD19 CAR-T cells disappeared in the peripheral blood in the murine CD19 CAR-T cell group after 14 days of therapy, while in the humanized CD19 CAR-T cell group it disappeared after 21 days of therapy. The median survival of BAL B/c nude mice in the murine CD19 CAR-T cell group and the humanized CD19 CAR-T cell group was 42 days and 63 days, respectively ( χ2=15.382, P<0.001). Conclusions:High affinity humanized CD19 CAR-T cells have stronger proliferation, higher cytotoxicity and longer survival time compared with those of murine CD19 CAR-T cells. The results indicate that the clinical efficacy of humanized CD19 CAR-T cells would be better than that of murine CD19 CAR-T cells.

Objective:To investigate the characteristics and cytotoxicity in vitro of the residual leukemia cells in the culture system that caused the accidental transfer of CD19 chimeric antigen receptor (CAR) into leukemia cells during the preparation of autologous CD19 CAR-T cells of relapsed/refractory B-cell acute lymphoblastic leukemia.Methods:①Peripheral blood mononuclear cells (PBMC) of 30 patients with relapsed/refractory B-cell acute lymphoblastic anemia (R/R B-ALL) who accepted CD19 CAR-T cell therapy and six healthy volunteers were collected. ②The residual leukemia cells were analyzed by flow cytometry in the system after the PBMCs of R/R B-ALL patients were sorted by CD3 magnetic beads. ③ CD3 + T cells from patients and healthy volunteers were transfected with CD19 CAR and CD22 CAR lentivirus to prepare CD19 CAR-T and CD22 CAR-T cells. ④The Nalm-6 cell line was resuscitated and the Nalm-6 cells with CD19 CAR lentivirus were transfected to prepare CD19 CAR-Nalm-6 cells. The patient's primary ALL cells were transfected with CD19 CAR lentivirus at the same time. ⑤The transfection rates were analyzed by flow cytometer, the cell proliferation was analyzed by the CCK-8 method, and the cell-killing activities were detected by the lactate dehydrogenase method. Results:① Among the 30 R/R B-ALL patients who received CD19 CAR-T cell therapy, two patients had 2.04% and 3.32% residual leukemia cells in CD3 + T cells. After 4 days in culture, the residual leukemia cells disappeared and could not be detected by a flow cytometer with prolonged cultivation in vitro. ② The proliferation of CD19 CAR-Nalm-6 cells was higher than that of the Nalm-6 cells. ③ The killing activity of the CD19 CAR-T cells on Nalm-6 cells was higher than that of the CD19 CAR-Nalm6 cells at a target ratio of 1∶1 on 24, 48, 72 h, respectively. The cytotoxicity of CD22 CAR-T cells on CD19 CAR-Nalm-6 cells was significantly higher than that of CD19 CAR-T cells. ④ The cytotoxicity of CD22 CAR-T alone on CD19 CAR-Nalm-6 cells was higher than that of CD19 CAR-T combined with CD22 CAR-T at the same target ratio. Conclusion:The residual leukemia cells in the culture system in the preparation of CD19 CAR-T cells may lead to the introduction of CD19 CAR into leukemia cells and results in the failure of the CD19 CAR-T cell therapy. Detecting the residual leukemia cells in the culture system via flow cytometry before transfection with CD19 CAR lentivirus is needed. Thus, CD22 CAR-T cell therapy could be used as one of the salvage treatments.

Objective: To retrospectively analyze the efficacy and safety of modified cell infusion method in reducing the incidence of febrile non-hemolytic transfusion reaction (FNHTR). Methods: A total of 69 patients were enrolled in the clinical trial of CD₁₉ chimeric antigen receptor T (CAR-T) cell treatment from February 2017 to October 2018. Study group received the modified cell infusion method, that 1×10⁶ CAR-T cells were re-suspended in 2 mg human serum albumin with total volume of 20 ml and injected intravenously. The control group was intravenously administrated with CAR-T cell in 100 ml normal saline. The incidence of FNHTR, cytokine releasing syndrome (CRS) grade, cytokine level and efficacy were compared. Results: (1)The incidence of FNHTR in the study group was 21.1%, significantly lower than that in the control group (71%)(P=0.000). (2)There was no statistical difference in cell proliferation between the study group and the control group on day 4, 7, 14 and 21 after CAR-T cell infusion (P=10.223, 3.254, 5.551, 7.605). (3)There was no statistical difference in CRS grading between the study group and the control group (P=0.767). There was no statistical difference in the levels of interleukin 2 receptor (IL-2R), IL-6, tumor necrosis factor (TNF)-α between the two groups. (4)The C-reaction protein (CRP) level of the study group was lower than that of the control group on day 4 and 7 (P=0.026, 0.007). (5)There was no statistical difference of response rates in acute lymphocytic leukemia (ALL) and non-Hodgkin lymphoma (NHL) patients between the two groups (P_ALL=0.842; P_NHL=0.866). Conclusion The modified cell infusion method in CD₁₉ CAR-T cell treatment reduces the incidence of treatment-related FNHTR. It does not affect the proliferation of CAR-T cells in vivo, the grading of CRS and the response rates.

Objective: To investigate the different functions of humanized and murinized CD19 chimeric antigen receptor (CAR)-T cells against Raji cell line in vitro and in vivo. Methods: Peripheral blood samples were collected from eight patients with lymphoma who were going to receive CD19 CAR-T cell therapy and used for the preparation of peripheral blood mononuclear cells (PBMC) as well as humanized and murinized CAR-T cells. Cell proliferation and cytotoxicity were detected with CCK-8 and LDH assays, respectively. A tumor-bearing mouse model was established by injecting BALB/c female nude mice with fluorescent Raji cells. Changes in tumor volume in these mice were observed by in vivo imaging technology. The transfection efficiency and amount of CAR-T cells in the mice were detected with flow cytometry. Results: No statistical difference in transfection efficiency was found between humanized and murinized CAR-T cells, nor in cell proliferation at 24 h of culture in vitro(P=0.104). The proliferation of humanized CAR-T cells showed a significant increase compared with that of murinized CAR-T cells at 48 h of culture (P=0.009). Similarly, the cytotoxicity of the two types of CAR-T cells against Raji cells showed no significant difference at 24 h at any effector/target (E/T) ratio (1∶1 or 4∶1), and that of humanized CAR-T cells was higher than that of murinized CAR-T cells at both E/T ratios at 48 h (E/T ratio=1∶1, P=0.005; E/T ratio=4∶1, P=0.008). Moreover, the cytotoxicity of CAR-T cells was higher than that of PBMC in any case. Tumor volumes in mice were reduced 14 d after humanized or murinized CAR-T cell therapy, while the mice in the PBMC control group suffered tumor progression. Tumor volume began to increase in mice 21 d after murinized CAR-T cell therapy, while no significant change was observed in the mice treated with humanized CAR-T cells. All of the mice died 25 d after murinized CAR-T cell therapy, while the deaths among those under humanized CAR-T cell therapy occurred on 31 d. The proportion of CAR-T cells in mice reached the peak 7 d after receiving humanized or murinized CAR-T cell therapy, while that in the humanized group was significantly higher than that in the murinized group at any time point (P_{4 d}=0.001, P_{7 d}=0.000, P_{14 d}=0.003). Murinized CAR-T cells became undetectable on 21 d, while humanized CAR-T cells on 35 d. The maximum survival time for mice in the PBMC and murinized and humanized CAR-T cell groups was 20 d, 25 d and 53 d, respectively. Conclusions Compared with murinized CD19 CAR-T cells, humanized CD19 CAR-T cells showed stronger proliferation potential and cytotoxicity and remained in vivo detectable for a longer period of time. This study indicated that humanized CD19 CAR-T cells were superior to murinized CD19 CAR-T cells for the treatment of B cell lymphoma.