A novel standard-free detection of adulteration method for sildenafil derivatives in dietary supplements.

The rapid and accurate detection of illegal adulteration of chemical drugs into dietary supplements is a big challenge in the food chemistry field. Detection of compounds without a standard reference is even more difficult; however, this is a common situation. Here in this study, a novel "standard-free detection of adulteration" (SFDA) method was proposed and phosphodiesterase-5 inhibitor derivatives were used as an example to figure out the possibility and reliability of this SFDA method. After analysis by quadrupole coupled time of flight-tandem mass spectrometry detection and multivariable statistics, six common fragment ions were chosen to indicate whether adulteration was present or not, while 20 characteristic fragment ions indicated whether adulteration was by nitrogen-containing heterocycles or by anilines. Furthermore, the quantitative methods were conducted by high-performance liquid chromatography-tandem mass spectrometry. In a word, this strategy allows for a quick determination of dietary supplement adulteration without any need for standard materials, improving the efficacy of food safety testing.

Reconstituted CD74+ NK cells trigger chronic graft versus host disease after allogeneic bone marrow transplantation.

Chronic graft-versus-host disease (cGVHD) is the most common long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The patients with pulmonary cGVHD in particular have a very poor prognosis. NK cells are the first reconstituted lymphocyte subset after allo-HSCT; however, the impact of reconstituted NK cells on cGVHD is unclear. Here, we found allogeneic recipients showed obvious pulmonary cGVHD. Surprisingly, deletion of reconstituted NK cells resulted in maximal relief of pulmonary cGVHD. Mechanistically, reconstituted NK cells with donor profiles modulated the pulmonary inflammatory microenvironment to trigger cGVHD. Reconstituted NK cells secreted IFN-γ and TNF-α to induce CXCL10 production by epithelial cells, which recruited macrophages and CD4+ T cells to the lungs. Then macrophages and CD4+ T cells were activated by the inflammatory microenvironment, thereby mediating lung injury. Through assessment of differences in cellular energy, we found that CD74+ NK cells with high mitochondrial potential and pro-inflammatory activity triggered pulmonary cGVHD. Furthermore, targeted elimination of CD74+ NK cells using the anti-CD74 antibody significantly alleviated pulmonary cGVHD but preserved the CD74- NK cells to exert graft-versus-leukemia (GVL) effects. Data from human samples corroborated our findings in mouse models. Collectively, our results reveal that reconstituted CD74+ NK cells trigger pulmonary cGVHD and suggest that administration of CD74 antibody was a potential therapeutic for patients with cGVHD.

Cytokine Storm Related to CD4+ T Cells in Influenza Virus-Associated Acute Necrotizing Encephalopathy.

Acute necrotizing encephalopathy (ANE) is a rare but deadly complication with an unclear pathogenesis. We aimed to elucidate the immune characteristics of H1N1 influenza virus-associated ANE (IANE) and provide a potential therapeutic approach for IANE. Seven pediatric cases from a concentrated outbreak of H1N1 influenza were included in this study. The patients' CD4+ T cells from peripheral blood decreased sharply in number but highly expressed Eomesodermin (Eomes), CD69 and PD-1, companied with extremely high levels of IL-6, IL-8 in the cerebrospinal fluid and plasma. Patient 2, who showed high fever and seizures and was admitted to the hospital very early in the disease course, received intravenous tocilizumab and subsequently showed a reduction in temperature and a stable conscious state 24 h later. In conclusion, a proinflammatory cytokine storm associated with activated CD4+ T cells may cause severe brain pathology in IANE. Tocilizumab may be helpful in treating IANE.

A new surface plasmon resonance-based immunoassay for rapid and sensitive quantification of D-dimer in human plasma for thrombus screening.

D-dimer is a protein fragment generated during the fibrin breakdown by plasmin, and it serves as a mature biomarker for diagnosing thrombotic disorders. A novel immunoassay method based on surface plasmon resonance (SPR) has been developed, validated, and successfully applied for the quantification of D-dimer in human plasma with high sensitivity and rapidity. In this methodological study, we investigated the activity and stability of the SPR biosensor, sample pre-processing, washing conditions, intra-day and inter-day precision and accuracy and detection parameters, including a limit of detection of 8.3 ng/mL, a detection range spanning from 31.25 to 4000 ng/mL, and a detection time of 20 min. We compared D-dimer plasma concentration determination results using SPR with a classical latex-enhanced immunoturbidimetric immunoassay in 29 healthy individuals and thrombotic patients, and both methods exhibited consistency. Furthermore, we propose a hypothesis about the relationship between the concentration of D-dimer and its molecular weight. With an increase in the D-dimer concentration in plasma, the D-dimer approaches its simplest form (190 kDa).

A dual-target SPR screening system for simultaneous ligand discovery of SARS-CoV-2 spike protein and its receptor ACE2 from Chinese herbs.

Traditional Chinese Medicine (TCM) is a supremely valuable resource for the development of drug discovery. Few methods are capable of hunting for potential molecule ligands from TCM towards more than one single protein target. In this study, a novel dual-target surface plasmon resonance (SPR) biosensor was developed to perform targeted compound screening of two key proteins involved in the cellular invasion process of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): the spike (S) protein receptor binding domain (RBD) and the angiotensin-converting enzyme 2 (ACE2). The screening and identification of active compounds from six Chinese herbs were conducted taking into consideration the multi-component and multi-target nature of Traditional Chinese Medicine (TCM). Puerarin from Radix Puerariae Lobatae was discovered to exhibit specific binding affinity to both S protein RBD and ACE2. The results highlight the efficiency of the dual-target SPR system in drug screening and provide a novel approach for exploring the targeted mechanisms of active components from Chinese herbs for disease treatment.

Reconstitution of double-negative T cells after cord blood transplantation and its predictive value for acute graft-versus-host disease.

BACKGROUND: With an increasing number of patients with hematological malignancies being treated with umbilical cord blood transplantation (UCBT), the correlation between immune reconstitution (IR) after UCBT and graft-versus-host disease (GVHD) has been reported successively, but reports on double-negative T (DNT) cell reconstitution and its association with acute GVHD (aGVHD) after UCBT are lacking. METHODS: A population-based observational study was conducted among 131 patients with hematological malignancies who underwent single-unit UCBT as their first transplant at the Department of Hematology, the First Affiliated Hospital of USTC, between August 2018 and June 2021. IR differences were compared between the patients with and without aGVHD. RESULTS: The absolute number of DNT cells in the healthy Chinese population was 109 (70-157)/µL, accounting for 5.82 (3.98-8.19)% of lymphocytes. DNT cells showed delayed recovery and could not reach their normal levels even one year after transplantation. Importantly, the absolute number and percentage of DNT cells were significantly higher in UCBT patients without aGVHD than in those with aGVHD within one year (F = 4.684, P = 0.039 and F = 5.583, P = 0.026, respectively). In addition, the number of DNT cells in the first month after transplantation decreased significantly with the degree of aGVHD increased, and faster DNT cell reconstitution in the first month after UCBT was an independent protective factor for aGVHD (HR = 0.46, 95% confidence interval [CI]: 0.23-0.93; P = 0.031). CONCLUSIONS: Compared to the number of DNT cells in Chinese healthy people, the reconstitution of DNT cells in adults with hematological malignancies after UCBT was slow. In addition, the faster reconstitution of DNT cells in the early stage after transplantation was associated with a lower incidence of aGVHD.

Dynamic comparison of early immune reactions and immune cell reconstitution after umbilical cord blood transplantation and peripheral blood stem cell transplantation.

Umbilical cord blood transplantation (UCBT) and peripheral blood stem cell transplantation (PBSCT) are effective allogeneic treatments for patients with malignant and non-malignant refractory hematological diseases. However, the differences in the immune cell reconstitution and the immune reactions during initial stages post-transplantation are not well established between UCBT and PBSCT. Therefore, in this study, we analyzed the differences in the immune reactions during the early stages (days 7-100 post-transplantation) such as pre-engraftment syndrome (PES), engraftment syndrome (ES), and acute graft-versus-host disease (aGVHD) and the immune cell reconstitution between the UCBT and the PBSCT group of patients. We enrolled a cohort of patients that underwent UCBT or PBSCT and healthy controls (n=25 each) and evaluated their peripheral blood mononuclear cell (PBMC) samples and plasma cytokine (IL-10 and GM-CSF) levels using flow cytometry and ELISA, respectively. Our results showed that the incidences of early immune reactions such as PES, ES, and aGVHD were significantly higher in the UCBT group compared to the PBSCT group. Furthermore, in comparison with the PBSCT group, the UCBT group showed higher proportion and numbers of naïve CD4+ T cells, lower proportion and numbers of Tregs, higher proportion of CD8+ T cells with increased activity, and higher proportion of mature CD56dim CD16+ NK cells during the early stages post-transplantation. Moreover, the plasma levels of GM-CSF were significantly higher in the UCBT group compared to the PBSCT group in the third week after transplantation. Overall, our findings demonstrated significant differences in the post-transplantation immune cell reconstitution between the UCBT and the PBSCT group of patients. These characteristics were associated with significant differences between the UCBT and the PBSCT groups regarding the incidences of immune reactions during the early stages post transplantation.

Iron deficiency in hepatocellular carcinoma cells induced sorafenib resistance by upregulating HIF-1α to inhibit apoptosis.

Sorafenib is the first-line therapeutic agent for hepatocellular carcinoma (HCC), but the drug resistance has become a major impediment. Previously we found that the abnormal iron metabolism in HCC led to iron deficiency, whether it induces sorafenib resistance during the treatment of HCC is not yet disclosed. In this study, we observed the effects of iron deficiency on sorafenib resistance and explored the underlying mechanisms. The results revealed that the killing effects of sorafenib on HCC cells were weakened by iron deficiency but effectively restored by iron re-supplementation. The ferroptosis indicators, including the contents of lipid hydroperoxide (LPO) and malondialdehyde (MDA), the level of intracellular reactive oxygen species (ROS), and the expression of glutathione peroxidase 4 (GPX4), were not significantly changed by iron deficiency in sorafenib-treated HCC cells. However, the sorafenib-induced apoptosis of HCC cells was inhibited by iron deficiency. Notably, the expression of anti-apoptotic protein B-cell lymphoma-2 (BCL-2) was elevated, and the expressions of other apoptotic proteins, BCL2-associated X (Bax), caspase-3, and caspase-9, were inhibited by iron deficiency. Mechanistically, iron deficiency upregulated hypoxia-inducible factor 1 alpha (HIF-1α) to increase BCL-2. Inhibition of HIF-1α suppressed the iron deficiency-induced BCL-2 and sorafenib resistance. In summary, iron deficiency in HCC cells generated sorafenib resistance by increasing HIF-1α and BCL-2, which therefore inhibited the sorafenib-induced apoptosis of HCC cells. These results identified iron deficiency as a new factor of sorafenib resistance in HCC cells, which would be an effective target to alleviate sorafenib resistance.

Iron deficiency downregulates ENPEP to promote angiogenesis in liver tumors.

The abnormal iron metabolism in liver cancer leads to iron deficiency in tumor tissues. We previously found that iron deficiency promoted liver cancer metastasis, but the mechanisms were not fully understood. In the present study, we identified that the angiogenesis-associated glutamyl aminopeptidase (ENPEP) was consistently decreased in iron-deficient liver tissues, iron-deficient liver tumors, and iron-deprived liver cancer cells. Interestingly, the lower expression of ENPEP was correlated with the poor prognosis of liver cancer patients, while the biomarkers of angiogenesis, CD31 and CD34, were increased in tumor tissues. In vivo imaging of liver-orthotopically implanted and tail vein-injected liver cancer cells showed that iron deficiency increased the pulmonary metastasis of liver cancer. The angiogenesis in iron-deficient tumors was enhanced, and the expression of ENPEP was decreased. Silencing ENPEP expression increased the migration of liver cancer cells and the proliferation of cocultured HUVECs. By sequence analysis, we found that the transcription factor SP1 possessed abundant binding sites in the ENPEP promoter region. Its combination with ENPEP promoters was verified by chromatin immunoprecipitation. The inhibition of SP1 by mithramycin A effectively restored the expression of ENPEP, which was decreased by iron deficiency. In conclusion, these results revealed that iron deficiency in liver tumors decreased the expression of ENPEP by SP1 and increased the angiogenesis and metastasis of liver tumors, which further explained the mechanism by which iron deficiency promoted liver cancer metastasis.

SIRT3-dependent delactylation of cyclin E2 prevents hepatocellular carcinoma growth.

Lysine lactylation (Kla) is a recently discovered histone mark derived from metabolic lactate. The NAD+ -dependent deacetylase SIRT3, which can also catalyze removal of the lactyl moiety from lysine, is expressed at low levels in hepatocellular carcinoma (HCC) and has been suggested to be an HCC tumor suppressor. Here we report that SIRT3 can delactylate non-histone proteins and suppress HCC development. Using SILAC-based quantitative proteomics, we identify cyclin E2 (CCNE2) as one of the lactylated substrates of SIRT3 in HCC cells. Furthermore, our crystallographic study elucidates the mechanism of CCNE2 K348la delactylation by SIRT3. Our results further suggest that lactylated CCNE2 promotes HCC cell growth, while SIRT3 activation by Honokiol induces HCC cell apoptosis and prevents HCC outgrowth in vivo by regulating Kla levels of CCNE2. Together, our results establish a physiological function of SIRT3 as a delactylase that is important for suppressing HCC, and our structural data could be useful for the future design of activators.

Iron Deficiency Increases Phosphorylation of SP1 to Upregulate SPNS2 Expression in Hepatocellular Carcinoma.

The sphingosine-1-phosphate (S1P) transporter spinster homolog 2 (SPNS2) promotes tumor progression by modulating tumor immunity and enhancing tumor cells migration and invasion. Previously we found that iron deficiency in hepatocellular carcinoma upregulated SPNS2 expression to increase tumor metastasis. The present study aimed to identify the underlying mechanism of SPNS2 upregulation. Since the mRNA of SPNS2 was significantly increased, we used a transcription factor activity microarray to find the transcription factor responsible for this. The results showed that iron deprivation in hepatoma cells increased the transcriptional activities of 14 transcription factors while only 2 were decreased. Among these, 3 transcription factors, HIF1α, SP1, and YY1, were predicted to bind with the transcription promoter region of SPNS2. But only HIF1α and SP1 transcriptional activities on SPNS2 were increased by iron deficiency, and the increase of SP1 transcriptional activity was stronger than HIF1α. The protein level of HIF1α was increased by iron deficiency, while SP1 was not changed at the protein level but the phosphorylation level was increased. The inhibitor of HIF1α, PX478, and the inhibitor of SP1, Mithramycin A, reversed the increased mRNA and protein expressions of SPNS2 by iron deficiency, with a more significant effect by Mithramycin A. These results provided a comprehensive view of changes in transcriptional activities by iron deficiency and identified that SP1 was the main regulator of iron deficiency-inducing SPNS2 expression in hepatoma cells.

An iron-deficient diet prevents alcohol- or diethylnitrosamine-induced acute hepatotoxicity in mice by inhibiting ferroptosis.

The liver is easily injured by exogenous chemicals through reactive oxygen species (ROS), which lead to ferroptosis, a ROS-dependent programmed cell death characterized by iron accumulation and lipid peroxidation. However, whether iron restriction has a positive role in chemicals-induced liver injuries is unknown. The present study investigated the effects of an iron-deficient diet on liver injuries induced by alcohol or diethylnitrosamine (DEN). Mice were fed an iron-deficient diet for four weeks, then treated with three doses of alcohol (5 g/kg, 24 h interval, gavage) to mimic mild liver injury or five doses of DEN (50 mg/kg, 24 h interval, i. p.) to mimic severe liver failure. The results showed that mice were iron-deficient after four weeks of feeding. Interestingly, as evaluated by H&E staining of liver slices, liver/body weight ratio, serum ALT and AST, iron deficiency significantly alleviated liver injuries triggered by alcohol or DEN. The activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), and the expression of CYP2E1 were increased by iron deficiency. Mechanistically, iron deficiency prevented the decrease of glutathione peroxidase 4 (GPX4), which eliminated malondialdehyde (MDA) by utilizing glutathione (GSH). In summary, alcohol- or DEN-induced liver injuries were mitigated by the iron-deficient diet by inhibiting ferroptosis, which might be a promising measure for preventing liver injuries induced by alcohol, DEN, or other exogenous chemicals.

Advances in Immunotherapy for Hepatitis B.

Hepatitis B virus (HBV) is a hepatotropic virus with the potential to cause chronic infection, and it is one of the common causes of liver disease worldwide. Chronic HBV infection leads to liver cirrhosis and, ultimately, hepatocellular carcinoma (HCC). The persistence of covalently closed circular DNA (cccDNA) and the impaired immune response in patients with chronic hepatitis B (CHB) has been studied over the past few decades. Despite advances in the etiology of HBV and the development of potent virus-suppressing regimens, a cure for HBV has not been found. Both the innate and adaptive branches of immunity contribute to viral eradication. However, immune exhaustion and evasion have been demonstrated during CHB infection, although our understanding of the mechanism is still evolving. Recently, the successful use of an antiviral drug for hepatitis C has greatly encouraged the search for a cure for hepatitis B, which likely requires an approach focused on improving the antiviral immune response. In this review, we discuss our current knowledge of the immunopathogenic mechanisms and immunobiology of HBV infection. In addition, we touch upon why the existing therapeutic approaches may not achieve the goal of a functional cure. We also propose how combinations of new drugs, and especially novel immunotherapies, contribute to HBV clearance.

Palbociclib promotes the antitumor activity of Venetoclax plus Azacitidine against acute myeloid leukemia.

Around 70 % of patients diagnosed with acute myeloid leukemia (AML) survive less than 5 years due to drug resistance and disease relapse. Consequently, improved clinical treatments are urgently needed. Some but not all AML patients benefit from the combination of the BCL-2 inhibitor Venetoclax with the hypomethylation agent Azacitidine. Here we investigated the utility of employing the cyclin dependent kinase (CDK6) inhibitor Palbociclib to improve the efficacy of Venetoclax/Azacitidine combination therapy. Our analysis of publicly available RNA sequencing datasets showed CDK6 was highly expressed in the major acute forms of leukemia including AML. Consistently, using qPCR and flow cytometry we found that CDK6 was overexpressed in bone marrow mononuclear cells from AML patients compared to healthy controls. Subsequent in vitro testing of Palbociclib, Venetoclax and Azacitidine, alone and in combination against CDK6-overexpressing AML cells lines THP-1 and KG-1 and primary AML cells showed that the Palbociclib/Venetoclax/Azacitidine combination improved treatment efficacy compared to Venetoclax/Azacitidine treatment alone. Additional investigations in a subcutaneous KG-1 mouse model showed similarly the three-drug combination produced the most significant reductions in tumor load together with the least amount of spleen infiltration. We established Palbociclib functioned in combination with Venetoclax/Azacitidine by increasing the rates of apoptosis in AML cells. Further investigations revealed that Palbociclib does not affect BCL-2 activity but downregulated the anti-apoptotic proteins MCL-1 and BCL-XL, making AML cells more sensitive to Venetoclax/Azacitidine treatment. Our results propose that the Palbociclib/Venetoclax/Azacitidine regimen warrants further preclinical research for clinical application in AML patients.

Cytomegalovirus-specific neutralizing antibodies effectively prevent uncontrolled infection after allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains one of the most frequent and life-threatening infectious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Herein, we comprehensively compared the immune cells of patients with uncontrolled and controlled CMV infection post-allo-HSCT and found that B-cells were extraordinarily insufficient because of impaired B-cells reconstitution in the uncontrolled infection group. Furthermore, in the controlled infection group, reconstructed B-cells showed signatures of mature B-cells, high expression of CXCR4 and IFITM1, and enrichment of CMV-associated B-cell receptors, which were lacking in the uncontrolled infection group. Consistently, sera from the uncontrolled infection group failed to inhibit CMV infection via neutralizing virus in vitro because of its lower content of anti-CMV-specific immunoglobulin G (IgG) than the controlled infection group. Overall, these results highlighted the contribution of B cells and anti-CMV-specific neutralizing IgGs to the restraint of CMV infection post-allo-HSCT, suggesting their potential as a supplementary treatment to improve outcomes.

GARP-mediated active TGF-ß1 induces bone marrow NK cell dysfunction in AML patients with early relapse post-allo-HSCT.

Relapse is a leading cause of death after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia (AML). However, the underlying mechanisms remain poorly understood. Natural killer (NK) cells play a crucial role in tumor surveillance and cancer immunotherapy, and NK cell dysfunction has been observed in various tumors. Here, we performed ex vivo experiments to systematically characterize the mechanisms underlying the dysfunction of bone marrow-derived NK (BMNK) cells isolated from AML patients experiencing early relapse after allo-HSCT. We demonstrated that higher levels of active transforming growth factor ß1 (TGF-ß1) were associated with impaired effector function of BMNK cells in these AML patients. TGF-ß1 activation was induced by the overexpression of glycoprotein A repetitions predominant on the surface of CD4+ T cells. Active TGF-ß1 significantly suppressed mTORC1 activity, mitochondrial oxidative phosphorylation, the proliferation, and cytotoxicity of BMNK cells. Furthermore, pretreatment with the clinical stage TGF-ß1 pathway inhibitor, galunisertib, significantly restored mTORC1 activity, mitochondrial homeostasis, and cytotoxicity. Importantly, the blockade of the TGF-ß1 signaling improved the antitumor activity of NK cells in a leukemia xenograft mouse model. Thus, our findings reveal a mechanism explaining BMNK cell dysfunction and suggest that targeted inhibition of TGF-ß1 signaling may represent a potential therapeutic intervention to improve outcomes in AML patients undergoing allo-HSCT or NK cell-based immunotherapy.

A multi-omics study of the anti-cancer effect of a ferulic acid derivative FA-30.

The active ingredients of Traditional Chinese Medicine are an important source of bioactive molecules and play an important role in the research and development of innovative drugs. FA-30, which is a derivative of natural product ferulic acid, inhibited cervical cancer cell proliferation and induced apoptosis as well. To understand the underlying mechanisms of FA-30, a complementary multi-omics study was conducted. Cysteine and methionine metabolism and aminoacyl-tRNA biosynthesis pathways were significantly changed both at the metabolic level and proteomic level. This may help us to get a better understanding of cervical cancer and FA-30 at the same time.

Loss of SLC46A1 decreases tumor iron content in hepatocellular carcinoma.

It is interesting that high iron is an independent inducer or cofactor of hepatocellular carcinoma (HCC) while the amount of iron is decreased in the liver tumor tissues. Due to the previous findings that iron deficiency promoted HCC metastasis, it is of significance to identify the underlying mechanism of iron deficiency in HCC. The tumor iron content and expressions of iron-metabolic molecules were observed in the primary liver cancers of rats and mice. The molecules that changed independently of iron were identified by comparing the expression profiles in the human HCC tissues and iron-deprived HCC cells. The downstream effects of these molecules on regulating intracellular iron content were investigated in vitro and further validated in vivo. Both in primary liver cancers of rats and mice, we confirmed the decreased iron content in tumor tissues and the altered expressions of iron-metabolic molecules, including transferrin receptor 1 (TfR1), six-transmembrane epithelial antigen of prostate 3 (STEAP3), divalent metal transporter 1 (DMT1), SLC46A1, ferroportin, hepcidin, and ferritin. Among these, STEAP3, DMT1, and SLC46A1 were altered free of iron deficiency. However, only silence or overexpression of SLC46A1 controlled the intracellular iron content of HCC cells. The interventions of STEAP3 or DMT1 could not change the intracellular iron content. Lentivirus-mediated regain of SLC46A1 expression restored the iron content in orthotopically implanted tumors, with correspondingly changes in the iron-metabolic molecules as iron increasing. Conclusion: Taken together, these results suggest that the loss of SLC46A1 expression leads to iron deficiency in liver tumor tissues, which would be an effective target to manage iron homeostasis in HCC.

A strategy of screening and binding analysis of bioactive components from traditional Chinese medicine based on surface plasmon resonance biosensor.

Elucidating the active components of traditional Chinese medicine (TCM) is essential for understanding the mechanisms of TCM and promote its rational use as well as TCM-derived drug development. Recent studies have shown that surface plasmon resonance (SPR) technology is promising in this field. In the present study, we propose an SPR-based integrated strategy to screen and analyze the major active components of TCM. We used Radix Paeoniae Alba (RPA) as an example to identify the compounds that can account for its anti-inflammatory mechanism via tumor necrosis factor receptor type 1 (TNF-R1). First, RPA extraction was analyzed using an SPR-based screening system, and the potential active ingredients were collected, enriched, and identified as paeoniflorin and paeonol. Next, the affinity constants of paeoniflorin and paeonol were determined as 4.9 and 11.8 µM, respectively. Then, SPR-based competition assays and molecular docking were performed to show that the two compounds could compete with tumor necrosis factor-α (TNF-α) while binding to the subdomain 1 site of TNF-R1. Finally, in biological assays, the two compounds suppressed cytotoxicity and apoptosis induced by TNF-α in the L929 cell line. These findings prove that SPR technology is a useful tool for determining the active ingredients of TCM at the molecular level and can be used in various aspects of drug development. The SPR-based integrated strategy is reliable and feasible in TCM studies and will shed light on the elucidation of the pharmacological mechanism of TCM and facilitate its modernization.