Natural Killer Cells and Immunotherapy.

Natural killer (NK) cells are innate immune cells that demonstrate cytolytic activity against tumor cells, virus-infected cells and other physiologically stressed cells, such as senescent cells [...].

Megakaryopoiesis and Platelet Biology: Roles of Transcription Factors and Emerging Clinical Implications.

Platelets play a critical role in hemostasis and thrombus formation. Platelets are small, anucleate, and short-lived blood cells that are produced by the large, polyploid, and hematopoietic stem cell (HSC)-derived megakaryocytes in bone marrow. Approximately 3000 platelets are released from one megakaryocyte, and thus, it is important to understand the physiologically relevant mechanism of development of mature megakaryocytes. Many genes, including several key transcription factors, have been shown to be crucial for platelet biogenesis. Mutations in these genes can perturb megakaryopoiesis or thrombopoiesis, resulting in thrombocytopenia. Metabolic changes owing to inflammation, ageing, or diseases such as cancer, in which platelets play crucial roles in disease development, can also affect platelet biogenesis. In this review, I describe the characteristics of platelets and megakaryocytes in terms of their differentiation processes. The role of several critical transcription factors have been discussed to better understand the changes in platelet biogenesis that occur during disease or ageing.

Emerging Approaches for Solid Tumor Treatment Using CAR-T Cell Therapy.

Cancer immunotherapy is becoming more important in the clinical setting, especially for cancers resistant to conventional chemotherapy, including targeted therapy. Chimeric antigen receptor (CAR)-T cell therapy, which uses patient's autologous T cells, combined with engineered T cell receptors, has shown remarkable results, with five US Food and Drug Administration (FDA) approvals to date. CAR-T cells have been very effective in hematologic malignancies, such as diffuse large B cell lymphoma (DLBCL), B cell acute lymphoblastic leukemia (B-ALL), and multiple myeloma (MM); however, its effectiveness in treating solid tumors has not been evaluated clearly. Therefore, many studies and clinical investigations are emerging to improve the CAR-T cell efficacy in solid tumors. The novel therapeutic approaches include modifying CARs in multiple ways or developing a combination therapy with immune checkpoint inhibitors and chemotherapies. In this review, we focus on the challenges and recent advancements in CAR-T cell therapy for solid tumors.

The Gata1low murine megakaryocyte-erythroid progenitor cells expand robustly and alter differentiation potential.

GATA1 is a master transcription factor of megakaryopoiesis and erythropoiesis, and loss-of-function mutation can induce accumulation of megakaryocyte-erythroid progenitors (MEPs) in mice and humans. Accordingly, the murine MEP cell line (termed G1ME2 cells) encoding doxycycline (dox)-inducible anti-Gata1 shRNA on Hprt locus has been developed. The cells were CD41+CD71+KIT+, expand under dox, stem cell factor, and thrombopoietin (TPO), and terminally differentiate into erythroid cells or megakaryocytes upon removal of dox. Surprisingly, in this study, these Gata1low murine MEPs displayed accelerated growth from around 90-100 days after cell culture, impeded megakaryocytic potential, and maintained erythropoiesis. We specified them as late G1ME2 cells and discovered that increased CD41-KIT+ population during long-term culture was the main reason for the delayed megakaryopoiesis. The CD41 expression level was partially de-repressed by PI3K/AKT inhibitors, suggesting that TPO-mediated cell survival signaling pathway might have impacted on CD41 in the late G1ME2 cells. Nevertheless, among the late cells, the CD41+KIT+ cells could still generate megakaryocytes on dox withdrawal. Taken together, G1ME2 cells could provide a good model to study molecular mechanism of hematopoiesis because of their ability to expand excessively without artificial immortalization.

Molecular Mechanisms and Emerging Therapeutics for Osteoporosis.

Osteoporosis is the most common chronic metabolic bone disease. It has been estimated that more than 10 million people in the United States and 200 million men and women worldwide have osteoporosis. Given that the aging population is rapidly increasing in many countries, osteoporosis could become a global challenge with an impact on the quality of life of the affected individuals. Osteoporosis can be defined as a condition characterized by low bone density and increased risk of fractures due to the deterioration of the bone architecture. Thus, the major goal of treatment is to reduce the risk for fractures. There are several treatment options, mostly medications that can control disease progression in risk groups, such as postmenopausal women and elderly men. Recent studies on the basic molecular mechanisms and clinical implications of osteoporosis have identified novel therapeutic targets. Emerging therapies targeting novel disease mechanisms could provide powerful approaches for osteoporosis management in the future. Here, we review the etiology of osteoporosis and the molecular mechanism of bone remodeling, present current pharmacological options, and discuss emerging therapies targeting novel mechanisms, investigational treatments, and new promising therapeutic approaches.

Immunotherapy in Hematologic Malignancies: Emerging Therapies and Novel Approaches.

Immunotherapy is extensively investigated for almost all types of hematologic tumors, from preleukemic to relapse/refractory malignancies. Due to the emergence of technologies for target cell characterization, antibody design and manufacturing, as well as genome editing, immunotherapies including gene and cell therapies are becoming increasingly elaborate and diversified. Understanding the tumor immune microenvironment of the target disease is critical, as is reducing toxicity. Although there have been many successes and newly FDA-approved immunotherapies for hematologic malignancies, we have learned that insufficient efficacy due to disease relapse following treatment is one of the key obstacles for developing successful therapeutic regimens. Thus, combination therapies are also being explored. In this review, immunotherapies for each type of hematologic malignancy will be introduced, and novel targets that are under investigation will be described.

TXNIP Regulates Natural Killer Cell-Mediated Innate Immunity by Inhibiting IFN-γ Production during Bacterial Infection.

The function of natural killer (NK) cell-derived interferon-γ (IFN-γ) expands to remove pathogens by increasing the ability of innate immune cells. Here, we identified the critical role of thioredoxin-interacting protein (TXNIP) in the production of IFN-γ in NK cells during bacterial infection. TXNIP inhibited the production of IFN-γ and the activation of transforming growth factor ß-activated kinase 1 (TAK1) activity in primary mouse and human NK cells. TXNIP directly interacted with TAK1 and inhibited TAK1 activity by interfering with the complex formation between TAK1 and TAK1 binding protein 1 (TAB1). Txnip-/- (KO) NK cells enhanced the activation of macrophages by inducing IFN-γ production during Pam3CSK4 stimulation or Staphylococcus aureus (S. aureus) infection and contributed to expedite the bacterial clearance. Our findings suggest that NK cell-derived IFN-γ is critical for host defense and that TXNIP plays an important role as an inhibitor of NK cell-mediated macrophage activation by inhibiting the production of IFN-γ during bacterial infection.

TLR4/NF-κB axis induces fludarabine resistance by suppressing TXNIP expression in acute myeloid leukemia cells.

Overcoming drug resistance is one of key issues in treating refractory acute myeloid leukemia (AML). The Toll-like receptor 4 (TLR4) signaling pathway is involved in many aspects of biological functions of AML cells, including the regulation of pro-inflammatory cytokine products, myeloid differentiation, and survival of AML cells. Thus, targeting TLR4 of AML patients for therapeutic purposes should be carefully addressed. In this regard, we investigated the possible role of TLR4 as a regulatory factor against fludarabine (FA) cytotoxicity activity. Here, we identified the differential expression of TLR4 and CD14 receptors in AML cell lines and examined their relationship to FA sensitivity. We found that the stimulation of TLR4 with lipopolysaccharide (LPS) in a TLR4-expressing cell line, THP-1, increased cell viability under FA treatment condition and showed that TLR4 stimulation overcame FA sensitivity through the activation of NF-κB, which subsequently upregulated several anti-apoptotic genes. The inhibition of TLR4/NF-κB signaling could partially or completely reverse LPS-induced cell survival under FA treatment conditions. Interestingly, we found that the expression of thioredoxin-interacting protein (TXNIP), a well-known tumor suppressor, was induced by FA treatment; however, it was suppressed by LPS treatment. Furthermore, the expression level of TXNIP was critical for FA-induced cytotoxicity or LPS-induced FA resistance of THP-1 cells. Our data suggest that TXNIP plays an important role in FA-induced cytotoxicity and TLR4/NF-κB-mediated FA resistance of AML cells. Therefore, TXNIP may be a potential therapeutic target for AML treatment.

Cyclocurcumin, an Antivasoconstrictive Constituent of Curcuma longa (Turmeric).

Despite the increasing attention on the therapeutic potential of Curcuma longa (turmeric), the biological activities of curcuminoids other than curcumin are not well understood. Here, we investigated antivasoconstrictive activities of C. longa extract and its ingredients using freshly isolated rat aortic rings. C. longa extract significantly suppressed agonist-stimulated vasoconstriction, and cyclocurcumin was found to be the most potent (IC50 against phenylephrine-induced vasoconstriction: 14.9 ± 1.0 µM) among the 10 tested ingredients including four curcuminoids. Cyclocurcumin significantly inhibited contraction of vascular smooth muscle, which was mediated by the suppression of myosin-light-chain phosphorylation and calcium influx via the L-type calcium channel. The inhibitory effect of cyclocurcumin was observed to be reversible and without cytotoxicity. Taken together, we demonstrated that cyclocurcumin, a bioactive ingredient in C. longa, may have a therapeutic potential as a novel antivasoconstrictive natural product.

Deficiency of thioredoxin-interacting protein results in age-related thrombocytopenia due to megakaryocyte oxidative stress.

BACKGROUND: Platelets are generated from megakaryocytes (MKs), mainly located in the bone marrow (BM). Megakaryopoiesis can be affected by genetic disorders, metabolic diseases, and aging. The molecular mechanisms underlying platelet count regulation have not been fully elucidated. OBJECTIVES: In the present study, we investigated the role of thioredoxin-interacting protein (TXNIP), a protein that regulates cellular metabolism in megakaryopoiesis, using a Txnip-/- mouse model. METHODS: Wild-type (WT) and Txnip-/- mice (2-27-month-old) were studied. BM-derived MKs were analyzed to investigate the role of TXNIP in megakaryopoiesis with age. The global transcriptome of BM-derived CD41+ megakaryocyte precursors (MkPs) of WT and Txnip-/- mice were compared. The CD34+ hematopoietic stem cells isolated from human cord blood were differentiated into MKs. RESULTS: Txnip-/- mice developed thrombocytopenia at 4 to 5 months that worsened with age. During ex vivo megakaryopoiesis, Txnip-/- MkPs remained small, with decreased levels of MK-specific markers. Critically, Txnip-/- MkPs exhibited reduced mitochondrial reactive oxygen species, which was related to AKT activity. Txnip-/- MkPs also showed elevated glycolysis alongside increased glucose uptake for ATP production. Total RNA sequencing revealed enrichment for oxidative stress- and apoptosis-related genes in differentially expressed genes between Txnip-/- and WT MkPs. The effects of TXNIP on MKs were recapitulated during the differentiation of human cord blood-derived CD34+ hematopoietic stem cells. CONCLUSION: We provide evidence that the megakaryopoiesis pathway becomes exhausted with age in Txnip-/- mice with a decrease in terminal, mature MKs that response to thrombocytopenic challenge. Overall, this study demonstrates the role of TXNIP in megakaryopoiesis, regulating mitochondrial metabolism.

Emerging role of Hippo pathway in the regulation of hematopoiesis.

In various organisms, the Hippo signaling pathway has been identified as a master regulator of organ size determination and tissue homeostasis. The Hippo signaling coordinates embryonic development, tissue regeneration and differentiation, through regulating cell proliferation and survival. The YAP and TAZ (YAP/TAZ) act as core transducers of the Hippo pathway, and they are tightly and exquisitely regulated in response to various intrinsic and extrinsic stimuli. Abnormal regulation or genetic variation of the Hippo pathway causes a wide range of human diseases, including cancer. Recent studies have revealed that Hippo signaling plays a pivotal role in the immune system and cancer immunity. Due to pathophysiological importance, the emerging role of Hippo signaling in blood cell differentiation, known as hematopoiesis, is receiving much attention. A number of elegant studies using a genetically engineered mouse (GEM) model have shed light on the mechanistic and physiological insights into the Hippo pathway in the regulation of hematopoiesis. Here, we briefly review the function of Hippo signaling in the regulation of hematopoiesis and immune cell differentiation. [BMB Reports 2023; 56(8): 417-425].

Understanding NK cell biology for harnessing NK cell therapies: targeting cancer and beyond.

Gene-engineered immune cell therapies have partially transformed cancer treatment, as exemplified by the use of chimeric antigen receptor (CAR)-T cells in certain hematologic malignancies. However, there are several limitations that need to be addressed to target more cancer types. Natural killer (NK) cells are a type of innate immune cells that represent a unique biology in cancer immune surveillance. In particular, NK cells obtained from heathy donors can serve as a source for genetically engineered immune cell therapies. Therefore, NK-based therapies, including NK cells, CAR-NK cells, and antibodies that induce antibody-dependent cellular cytotoxicity of NK cells, have emerged. With recent advances in genetic engineering and cell biology techniques, NK cell-based therapies have become promising approaches for a wide range of cancers, viral infections, and senescence. This review provides a brief overview of NK cell characteristics and summarizes diseases that could benefit from NK-based therapies. In addition, we discuss recent preclinical and clinical investigations on the use of adoptive NK cell transfer and agents that can modulate NK cell activity.

Novel antiplatelet activity of protocatechuic acid through the inhibition of high shear stress-induced platelet aggregation.

Bleeding is the most common and serious adverse effect of currently available antiplatelet drugs. Many efforts are being made to develop novel antithrombotic agents without bleeding risks. Shear stress-induced platelet aggregation (SIPA), which occurs under abnormally high shear stress, plays a crucial role in the development of arterial thrombotic diseases. Here, we demonstrate that protocatechuic acid (PCA), a bioactive phytochemical from Lonicera (honeysuckle) flowers, selectively and potently inhibits high shear (>10,000 s(-1))-induced platelet aggregation. In isolated human platelets, PCA decreased SIPA and attenuated accompanying platelet activation, including intracellular calcium mobilization, granule secretion, and adhesion receptor expression. The anti-SIPA effect of PCA was mediated through blockade of von Willebrand factor binding to activated glycoprotein Ib, a primary and initial event for the accomplishment of SIPA. Conspicuously, PCA did not inhibit platelet aggregation induced by other endogenous agonists like collagen, thrombin, or ADP that are important in both pathological thrombosis and normal hemostasis. Antithrombotic effects of PCA were confirmed in vivo in a rat arterial thrombosis model, where PCA significantly delayed the arterial occlusion induced by FeCl(3). Of particular note, PCA did not increase bleeding times in a rat tail transection model, whereas conventional antiplatelet drugs, aspirin, and clopidogrel substantially prolonged it. Collectively, these results suggest that PCA may be a novel antiplatelet agent that can prevent thrombosis without increasing bleeding risks.

Biomimetic engineering of a generic cell-on-membrane architecture by microfluidic engraving for on-chip bioassays.

We develop a biomimetic cell-on-membrane architecture in close-volume format which allows the interfacial biocompatibility and the reagent delivery capability for on-chip bioassays. The key concept lies in the microfluidic engraving of lipid membranes together with biological cells on a supported substrate with topographic patterns. The simultaneous engraving process of a different class of fluids is promoted by the front propagation of an air-water interface inside a flow-cell. This highly parallel, microfluidic cell-on-membrane approach opens a door to the natural biocompatibility in mimicking cellular stimuli-response behavior essential for diverse on-chip bioassays that can be precisely controlled in the spatial and temporal manner.

Ginsenoside 20(R)-Rg3 enhances natural killer cell activity by increasing activating receptor expression through the MAPK/ERK signaling pathway.

Ginseng is one of the most widely used herbal remedies for various diseases worldwide. Ginsenoside Rg3 (G-Rg3), the main component of ginseng, possesses several pharmacological properties, including anti-inflammatory, anti-tumor, antioxidant, anti-obesity, and immunomodulatory activities. However, the effect of G-Rg3 on natural killer (NK) cells in humans is not fully understood. Here, we investigated the effect of G-Rg3 on NK cell function and differentiation and elucidated the underlying mechanism. G-Rg3 increased NK cell cytotoxicity and simultaneously increased the expression of NK-activating receptors, NKp44, NKp46, and NKp30. Additionally, G-Rg3 increased the mRNA expression of NK cytolytic molecules, granzyme B and perforin. The expression of CD107a, a marker of NK cell degranulation, also increased in G-Rg3-treated NK cells. We therefore proceeded to identify which MAPK signaling pathway was involved in G-Rg3-mediated cytolytic activity. Treatment with G-Rg3 increased the phosphorylation levels of extracellular signal-regulated kinase (ERK), whereas ERK inhibition eliminated G-Rg3-induced NK cell cytotoxicity, suggesting the involvement of the ERK pathway. G-Rg3 did not affect the rate of differentiation of human cord-blood-derived NK cells; however, it increased the functional maturation of differentiated NK cells and promoted their cytotoxicity. The G-Rg3 isomer, 20(R)-Rg3, effectively activated NK cells via the extracellular signal-regulated kinase (ERK) signaling pathway, whereas 20(S)-Rg3 had no effect on NK cell activity. Altogether, the results demonstrated that 20(R)-Rg3 promoted NK cell activity via activation of the MAPK/ERK pathway, suggesting that 20(R)-Rg3 may be used as an activator of NK cell cytotoxicity for the treatment of diverse types of cancers.

Aurantii Fructus Immaturus enhances natural killer cytolytic activity and anticancer efficacy in vitro and in vivo.

Aurantii Fructus Immaturus (AFI), extensively used in traditional herbal medicine, is known to have diverse physiological effects against various diseases, including obesity, diabetes, and cardiovascular disease. However, the effects of AFI on the immune system, especially natural killer (NK) cells, remain largely unknown. We aimed to investigate the effect of AFI on NK cell activity in vitro and in vivo and to elucidate the underlying mechanisms. Further, we verified the anticancer efficacy of AFI in a mouse lung metastasis model, underscoring the therapeutic potential of AFI in cancer therapy. Our results revealed that AFI significantly enhanced the cytolytic activity of NK cells in a dose-dependent manner, accompanied by an increase in the expression of NK cell-activating receptors, especially NKp30 and NKp46. AFI treatment also increased the expression of cytolytic granules, including granzyme B and perforin. Furthermore, the expression of CD107a, a degranulation marker, was increased upon treatment with AFI. A signaling study using western blot analysis demonstrated that the phosphorylation of extracellular signal-regulated kinase (ERK) was involved in increasing the NK cell activity following AFI treatment. In the in vivo study performed in mice, oral administration of AFI markedly enhanced the cytotoxic activity of spleen mononuclear cells against YAC-1 cells, which was accompanied by NKp46 upregulation. In addition, we confirmed that cancer metastasis was inhibited in a mouse cancer metastasis model, established using the mouse melanoma B16F10 cell line, by the administration of AFI in vivo. Collectively, these results indicate that AFI enhances NK cell-mediated cytotoxicity in vitro and in vivo via activation of the ERK signaling pathway and suggest that AFI could be a potential supplement for cancer immunotherapy.

Additive Effect of CD73 Inhibitor in Colorectal Cancer Treatment With CDK4/6 Inhibitor Through Regulation of PD-L1.

BACKGROUND & AIMS: Although cancer immunotherapies are effective for advanced-stage cancers, there are no clinically approved immunotherapies for colon cancers (CRCs). Therefore, there is a high demand for the development of novel therapies. Extracellular adenosine-mediated signaling is considered a promising target for advanced-stage cancers that are nonresponsive to programmed death 1 (PD-1)-/programmed death-ligand 1 (PD-L1)-targeted immunotherapies. In this study, we aimed to elucidate novel tumorigenic mechanisms of extracellular adenosine. METHODS: To investigate the effects of extracellular adenosine on tumor-associated macrophages, peripheral blood-derived human macrophages were treated with adenosine and analyzed using flow cytometry and Western blot. Changes in adenosine-treated macrophages were further assessed using multi-omics analysis, including total RNA sequencing and proteomics. Colon cancer mouse models were used to measure the therapeutic efficacy of AB680 and palbociclib. We also used tissue microarrays of patients with CRC, to evaluate their clinical relevance. RESULTS: Extracellular adenosine-mediated reduction of cyclin D1 (CCND1) was found to be critical for the regulation of immune checkpoint molecules and PD-L1 levels in human macrophages, indicating that post-translational modification of PD-L1 is affected by adenosine. A potent CD73 selective inhibitor, AB680, reversed the effects of adenosine on CCND1 and PD-L1. This result strongly suggests that AB680 is a combinatory therapeutic option to overcome the undesired side effects of the cyclin-dependent kinase 4/6 inhibitor, palbociclib, which increases PD-L1 expression in tumors. Because palbociclib is undergoing clinical trials for metastatic CRC in combination with cetuximab (clinical trial number: NCT03446157), we validated that the combination of AB680 and palbociclib significantly improved anti-tumor efficacy in CRC animal models, thereby highlighting it as a novel immunotherapeutic strategy. We further assessed whether the level of CCND1 in tumor-associated macrophages was indeed reduced in tumor sections obtained from patients with CRC, for evaluating the clinical relevance of this strategy. CONCLUSIONS: In this study, we demonstrated that a novel combination therapy of AB680 and palbociclib may be advantageous for the treatment of CRC.

Procoagulant and prothrombotic activation of human erythrocytes by phosphatidic acid.

Increased phosphatidic acid (PA) and phospholipase D (PLD) activity are frequently observed in various disease states including cancers, diabetes, sepsis, and thrombosis. Previously, PA has been regarded as just a precursor for lysophosphatidic acid (LPA) and diacylglycerol (DAG). However, increasing evidence has suggested independent biological activities of PA itself. In the present study, we demonstrated that PA can enhance thrombogenic activities in human erythrocytes through phosphatidylserine (PS) exposure in a Ca(2+)-dependent manner. In freshly isolated human erythrocytes, treatment of PA or PLD induced PS exposure. PA-induced PS exposure was not attenuated by inhibitors of phospholipase A(2) or phosphatidate phosphatase, which converts PA to LPA or DAG. An intracellular Ca(2+) increase and the resultant activation of Ca(2+)-dependent PKC-alpha appeared to underlie the PA-induced PS exposure through the activation of scramblase. A marginal decrease in flippase activity was also noted, contributing further to the maintenance of exposed PS on the outer membrane. PA-treated erythrocytes showed strong thrombogenic activities, as demonstrated by increased thrombin generation, endothelial cell adhesion, and erythrocyte aggregation. Importantly, these procoagulant activations by PA were confirmed in a rat in vivo venous thrombosis model, where PA significantly enhanced thrombus formation. In conclusion, these results suggest that PA can induce thrombogenic activities in erythrocytes through PS exposure, which can increase thrombus formation and ultimately contribute to the development of cardiovascular diseases.

A naphthoquinone derivative can induce anemia through phosphatidylserine exposure-mediated erythrophagocytosis.

A naphthoquinone derivative, beta-lapachone (betaL; 3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione), is receiving huge attention for its potent therapeutic effects against various diseases. However, during the preclinical safety evaluation, repeated oral treatment of betaL in rats induced anemia, i.e., a significantly decreased erythrocyte count. In this study, in an effort to elucidate the mechanism underlying the betaL-induced anemia, we investigated the effects of betaL on erythrocytes with freshly isolated human erythrocytes in vitro and rat in vivo. betaL did not induce erythrocyte hemolysis, indicating that direct hemotoxicity was not involved in betaL-associated anemia. Meanwhile, phosphatidylserine (PS) exposure along with spherocytic shape change and microvesicle generation, important factors in the facilitation of erythrophagocytosis, were increased significantly by betaL. The PS exposure on erythrocytes was from betaL-induced reactive oxygen species generation and subsequent depletion of reduced glutathione and protein thiol, which culminated in the modified activities of phospholipid translocases, i.e., inhibition of flippase and activation of scramblase. It is important to note that coincubation of macrophage with betaL-treated erythrocyte in vitro showed increased erythrophagocytosis, demonstrating that the removal of erythrocyte by macrophage can be facilitated by betaL-induced PS exposure. In good accordance with these in vitro results, after oral administration of betaL in rats, increased PS exposure and depletion of glutathione were observed along with enhanced splenic sequestration of erythrocytes. In conclusion, these results suggest that betaL-induced anemia might be mediated through the PS exposure and subsequent erythrophagocytosis, providing novel insight into the drug-induced anemia.

Anti-inflammatory and analgesic activities of SKLJI, a highly purified and injectable herbal extract of Lonicera japonica.

The parenteral route has many merits over the oral route, including greater predictability, reproducibility of absorption, and rapid drug action, but injectable phytomedicines are uncommon due to protein precipitating tannin and hemolytic saponin components. In this study, in an effort to develop a safe injectable analgesic phytomedicine, we prepared a tannin and saponin-free Lonicera japonica extract, SKLJI, through fractionation and column purification, and evaluated its anti-inflammatory and analgesic activities in in vivo experimental models of inflammation and pain. The removal of tannin and saponin resulted in loganin and sweroside-enriched SKLJI and it showed reduced hemolysis and protein precipitation. In efficacy tests, SKLJI inhibited croton oil- and arachidonic acid-induced ear edema, acetic acid-induced writhing, and carrageenan-induced rat hind paw hyperalgesia. Inhibition of cylcooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and 5-lipoxyfenase (5-LO) activities by SKLJI appeared to be the mechanism underlying anti-inflammatory and analgesic efficacy. Loganin and sweroside also showed anti-inflammatory and analgesic activities, suggesting that they might be active principles in the efficacy of SKLJI. These results suggest that SKLJI is a viable candidate for a new anti-inflammatory and analgesic phytomedicine that can be administered by the parenteral route.