Metabolomics of the anti-inflammatory effect of Pueraria lobata and Pueraria lobata var. Thomsonii in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi and Pueraria lobata var. Thomsonii (Benth.) Maesen are essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata and P. thomsonii are widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata and P. thomsonii have pharmacological effects in the clinic, such as antipyretic, analgesic, anti-inflammatory and antioxidant effects. These plants are commonly used in the treatment of inflammatory diseases and other related diseases. However, the potential mechanisms of the anti-inflammatory effects of P. lobata and P. thomsonii have not been elucidated. AIM OF THE STUDY: This study aimed to confirm the anti-inflammatory effects of P. lobata and P. thomsonii on inflammatory model diseases and to investigate the mechanism of their anti-inflammatory effects from the perspective of plasma metabolomics. MATERIALS AND METHODS: First, P. lobata and P. thomsonii were identified by high‒performance liquid chromatography (HPLC). Second, we established the following three inflammation models: an acute inflammation model of auricular swelling in mice induced by xylene, an acute inflammation model of foot swelling in rats induced by carrageenan gum, and a chronic inflammation model of cotton ball granuloma in rats. Then we examined the weight and swelling rate of auricular swelling in mice; the residence time, contact area, and mean contact pressure in rats on the gait meter; and the weight of granulomas in rats and the content of IL-1ß and TNF-α in plasma to investigate the anti-inflammatory pharmacodynamics of P. lobata and P. thomsonii. Third, we used LC‒MS‒based plasma metabolomics techniques to obtain potential biomarkers of P. lobata and P. thomsonii related to inflammation. Then, the potential biomarkers were enriched by MetaboAnalyst and KEGG metabolomics analysis tools to obtain metabolic pathways related to inflammation. Finally, we tested the indicators of COX-2, 5-LOX, GSH, GSSG and γ⁃GCL in rat plasma from the granuloma model by enzyme-linked immunosorbent assays (ELISAs) to verify the inflammation-related metabolic pathway. RESULTS: The experimental results showed that P. lobata and P. thomsonii could reduce the swollen weight and swelling rate of the auricle in mice, and could increase the residence time, contact area and mean contact pressure in rats on the gait meter. Moreover, P. lobata and P. thomsonii could inhibit the growth of granulomas and reduce the content of IL-1ß and TNF-α in plasma in rats. The above results preliminarily verified that P. lobata and P. thomsonii have different anti-inflammatory effects. We identified eighteen plasma biomarkers associated with P. lobata and sixteen plasma biomarkers related to P. thomsonii in regulating inflammation by a plasma metabolomics analysis. The following two major metabolic pathways were further screened and enriched: arachidonic acid metabolism and glutathione metabolism. Then we noted that P. lobata and P. thomsonii could reduce the COX-2, 5-LOX and GSSG levels and increase the GSH, GSH/GSSG and γ⁃GCL levels based on the ELISA results, which demonstrated that P. lobata and P. thomsonii affect the anti-inflammatory mechanism through arachidonic acid metabolism and glutathione metabolism. CONCLUSIONS: The results of this study further elucidate the anti-inflammatory mechanism of action of P. lobata and P. thomsonii, providing a scientific basis for developing new drugs for the treatment of inflammation-related diseases and laying a foundation for the development of herbal resources, such as P. lobata and P. thomsonii.

Characterization of Neoantigen Load Subgroups in Gynecologic and Breast Cancers.

OBJECTIVE: Although gynecologic and breast (Pan-Gyn) cancers share a variety of similar characteristics, their response to immunotherapy is different. Immune checkpoint inhibitor therapy is not effective in all patients, while neoantigen load (NAL) may be a predictive biomarker. However, the selection of a NAL cutoff point and its predictive effect remain to be elucidated. METHODS: We divided 812 Pan-Gyn cancer samples from The Cancer Genome Atlas into three groups based on 60 and 80% of their load percentile. We then correlated the identified NAL subgroups with gene expression, somatic mutation, DNA methylation, and clinicopathological information. We also characterized each subgroup by distinct immune cell enrichment, PD-1 signaling, and cytolytic activity. Finally, we predicted the response of each subgroup to chemotherapy and immunotherapy. RESULTS: Across Pan-Gyn cancers, we identified three distinct NAL subgroups. These subgroups showed differences in biological function, genetic information, clinical variables, and immune infiltration. Eighty percent was identified as a meaningful cutoff point for NAL. In all patients, a higher NAL (top 20%) was associated with better overall survival as well as high immune infiltration and low intra-tumor heterogeneity. Furthermore, an interesting lncRNA named AC092580.4 was found, which was associated with two significantly different immune genes (CXCL9 and CXCL13). CONCLUSIONS: Our novel findings provide further insights into the NAL of Pan-Gyn cancers and may open up novel opportunities for their exploitation toward personalized treatment with immunotherapy.

Cardioprotective Effects of Latifolin Against Doxorubicin-Induced Cardiotoxicity by Macrophage Polarization in Mice.

Latifolin, one of the major flavonoids extracted from lignum dalbergiae odoriferae, has been documented to protect the heart from acute myocardial ischemia induced by pituitrin and isoproterenol in rats and has also been found to inhibit inflammation. In this study, we aimed to investigate whether latifolin could protect the heart from doxorubicin (DOX)-induced cardiotoxicity and elucidate its underlying mechanisms. Male mice were treated with an intraperitoneal dose of DOX (20 mg/kg) plus oral latifolin at a dose of 50 or 100 mg/kg for 12 days. After exposure, we assessed cardiac function, myocardial injury, and macrophage polarization in excised cardiac tissue. Our results demonstrated that latifolin prevented DOX-induced cardiac dysfunction and produced macrophage polarization in mice challenged with latifolin. In cultured peritoneal macrophages, latifolin significantly reduced inflammatory cytokines (P < 0.05). Furthermore, latifolin remarkably decreased the percentage of macrophage M1/M2 polarization (P < 0.05). The results from the present study highlight the benefits of treatment with latifolin in DOX-induced cardiotoxicity, and the mechanism involved in mediating the polarization phenotype change of M1/M2 macrophages.

A Pre-operative Nomogram for Prediction of Lymph Node Metastasis in Bladder Urothelial Carcinoma.

The status of lymph node (LN) metastases plays a decisive role in the selection of surgical procedures and post-operative treatment. Several histopathologic features, known as predictors of LN metastasis, are commonly available post-operatively. Medical imaging improved pre-operative diagnosis, but the results are not fully satisfactory due to substantial false positives. Thus, a reliable and robust method for pre-operative assessment of LN status is urgently required. We developed a prediction model in a training set from the TCGA-BLCA cohort including 196 bladder urothelial carcinoma samples with confirmed LN metastasis status. Least absolute shrinkage and selection operator (LASSO) regression was harnessed for dimension reduction, feature selection, and LNM signature building. Multivariable logistic regression was used to develop the prognostic model, incorporating the LNM signature, and a genomic mutation of MLL2, and was presented with a LNM nomogram. The performance of the nomogram was assessed with respect to its calibration, discrimination, and clinical usefulness. Internal validation was evaluated by the testing set from the TCGA cohort and independent validation was assessed by two independent cohorts. The LNM signature, which consisted of 48 selected features, was significantly associated with LN status (p < 0.005 for both the training and testing sets of the TCGA cohort). Predictors contained in the individualized prediction nomogram included the LNM signature and MLL2 mutation status. The model demonstrated good discrimination, with an area under the curve (AUC) of 98.7% (85.3% for testing set) and good calibration with p = 0.973 (0.485 for testing set) in the Hosmer-Lemeshow goodness of fit test. Decision curve analysis demonstrated that the LNM nomogram was clinically useful. This study presents a pre-operative nomogram incorporating a LNM signature and a genomic mutation, which can be conveniently utilized to facilitate pre-operative individualized prediction of LN metastasis in patients with bladder urothelial carcinoma.

Immune Signature-Based Subtypes of Cervical Squamous Cell Carcinoma Tightly Associated with Human Papillomavirus Type 16 Expression, Molecular Features, and Clinical Outcome.

Substantial heterogeneity exists within cervical cancer that is generally infected by human papillomavirus (HPV). However, the most common histological subtype of cervical cancer, cervical squamous cell carcinoma (CSCC), is poorly characterized regarding the association between its heterogeneity and HPV oncoprotein expression. We filtered out 138 CSCC samples with infection of HPV16 only as the first step; then we compressed HPV16 E6/E7 expression as HPVpca and correlated HPVpca with the immunological profiling of CSCC based on supervised clustering to discover subtypes and to characterize the differences between subgroups in terms of the HPVpca level, pathway activity, epigenetic dysregulation, somatic mutation frequencies, and likelihood of responding to chemo/immunotherapies. Supervised clustering of immune signatures revealed two HPV16 subtypes (namely, HPV16-IMM and HPV16-KRT) that correlated with HPVpca and clinical outcomes. HPV16-KRT is characterized by elevated expression of genes in keratinization, biological oxidation, and Wnt signaling, whereas HPV16-IMM has a strong immune response and mesenchymal features. HPV16-IMM exhibited much more epigenetic silencing and significant mutation at FBXW7, while MUC4 and PIK3CA were mutated frequently for HPV16-KRT. We also imputed that HPV16-IMM is much more sensitive to chemo/immunotherapy than is HPV16-KRT. Our characterization tightly links the expression of HPV16 E6/E7 with biological and clinical outcomes of CSCC, providing valuable molecular-level information that points to decoding heterogeneity. Together, these results shed light on stratifications of CSCC infected by HPV16 and shall help to guide personalized management and treatment of patients.