Objective To clarify the anti-inflammatory quality markers(Q-markers)of Salvia plebeia and determine their contents,so as to provide a reference for the quality control of Salvia plebeia.Methods The main components of Salvia plebeia were characterized by high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS).According to the fragmentation information of the secondary mass spectrometry and the literature data,the analysis and identification were carried out.Further,the active components with high oral bioavailability(OB)and in line with the five principles of drug-like(DL)were screened from the identified chemical components through the SwissADME platform.The SwissTargetPrediction database was used to find and predict the component targets of Salvia plebeia.Disease targets were screened through online Mendelian Inheritance in Man(OMIM),GeneCards database,etc.String11.5 database and Cytoscape3.7.2 software were used to construct the PPI network and screen core targets.Gene Ontology(GO)annotation and KEGG pathway enrichment analysis were performed using DAVID6.8,and the enriched pathways were verified by experiments to clarify their mechanism of action.Reverse traceability was used to analyze the potential pharmacodynamic material basis of key pathways,experimental verification was carried out,and high-performance liquid chromatography(HPLC)content determination method was established.Results 36 main components were identified,including flavonoids and terpenoids.Further screening of 190 active ingredients and disease intersection targets;through network topology screening,18 core targets were obtained;enrichment analysis showed that the primary pathways involved in the anti-inflammatory effect of Salvia plebeia include:NF-κB signaling pathway,PI3K-Akt signaling pathway and TNF signaling pathway.Four related components including homoplantaginin,hispidulin,luteolin and isorhamnetin were obtained by reverse traceability of the NF-κB signaling pathway.Molecular docking demonstrated excellent docking activities of the 4 components to PTGS2 with the binding energies of-9.5,-9.7,-9.4,and-9.4 kcal·mol-1,respectively.According to the measurability of quality markers,it was determined that homoplantaginin and hispidulin could be used as anti-inflammatory quality markers of Salvia plebeia.Western Blot results showed that homoplantaginin and hispidulin could significantly reduce the expression of COX-2 and NF-κB p-p65(P<0.05,P<0.01).Conclusion The anti-inflammatory quality markers of Salvia plebeia are homoplantaginin and hispidulin,which can exert anti-inflammatory effects through the NF-κB pathway.

Metabolic-associated fatty liver disease (MAFLD), which is previously known as non-alcoholic fatty liver disease (NAFLD), represents a major health concern worldwide with limited therapy. Here, we provide evidence that ferroptosis, a novel form of regulated cell death characterized by iron-driven lipid peroxidation, was comprehensively activated in liver tissues from MAFLD patients. The canonical-GPX4 (cGPX4), which is the most important negative controller of ferroptosis, is downregulated at protein but not mRNA level. Interestingly, a non-canonical GPX4 transcript-variant is induced (inducible-GPX4, iGPX4) in MAFLD condition. The high fat-fructose/sucrose diet (HFFD) and methionine/choline-deficient diet (MCD)-induced MAFLD pathologies, including hepatocellular ballooning, steatohepatitis and fibrosis, were attenuated and aggravated, respectively, in cGPX4-and iGPX4-knockin mice. cGPX4 and iGPX4 isoforms also displayed opposing effects on oxidative stress and ferroptosis in hepatocytes. Knockdown of iGPX4 by siRNA alleviated lipid stress, ferroptosis and cell injury. Mechanistically, the triggered iGPX4 interacts with cGPX4 to facilitate the transformation of cGPX4 from enzymatic-active monomer to enzymatic-inactive oligomers upon lipid stress, and thus promotes ferroptosis. Co-immunoprecipitation and nano LC-MS/MS analyses confirmed the interaction between iGPX4 and cGPX4. Our results reveal a detrimental role of non-canonical GPX4 isoform in ferroptosis, and indicate selectively targeting iGPX4 may be a promising therapeutic strategy for MAFLD.

Objective: To understand the consistency of ALK Ventana-D5F3 immunohistochemistry (IHC) interpretation in Chinese lung adenocarcinoma among histopathologists from different hospitals, and to recommend solution for the problems found during the interpretation of ALK IHC in real world, with the aim of the precise selection of patients who can benefit from ALK targeted therapy. Methods: This was a multicenter and retrospective study. A total of 109 lung adenocarcinoma cases with ALK Ventana-D5F3 IHC staining were collected from 31 lung cancer centers in RATICAL research group from January to June in 2018. All cases were scanned into digital imaging with Ventana iSCANcoreo Digital Slide Scanning System and scored by 31 histopathologists from different centers according to ALK binary (positive or negative) interpretation based on its manufacturer′s protocol. The cases with high inconsistency rate were further analyzed using FISH/RT-PCR/NGS. Results: There were 49 ALK positive cases and 60 ALK negative cases, confirmed by re-evaluation by the specialist panel. Two cases (No. 2302 and No.2701) scored as positive by local hospitals were rescored as negative, and were confirmed to be negative by RT-PCR/FISH/NGS. The false interpretation rate of these two cases was 58.1% (18/31) and 48.4% (15/31), respectively. Six out of 31 (19.4%) pathologists got 100% accuracy. The minimum consistency between every two pathologists was 75.8%.At least one pathologist gave negative judgement (false negative) or positive judgement (false positive) in the 49 positive or 60 negative cases, accounted for 26.5% (13/49), 41.7% (25/60), respectively, with at least one uncertainty interpretation accounted for 31.2% (34/109). Conclusion There are certain heterogeneities and misclassifications in the real world interpretation of ALK-D5F3 IHC test, which need to be guided by the oncoming expert consensus based on the real world data.