ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， network pharmacology， molecular docking and cell experiments， the active ingredients， possible targets and molecular mechanisms of Baoxin granules（BXG） regulating ferroptosis in the treatment of heart failure（HF） were explored. MethodsBXG intestinal absorption fluid was prepared by everted gut sac and the chemical composition contained therein were identified by UPLC-Q-TOF-MS. According to the obtained components， the potential targets of BXG were predicted， and the HF-related targets and related genes of ferroptosis were retrieved at the same time， and the intersecting targets were obtained by Venn diagram. In addition， the protein-protein interaction（PPI） network and the component-target network were constructed， and the core components and core targets were obtained by topological analysis. Then Gene Ontology（GO） function and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis were performed on the core targets， and molecular docking validation of the key targets and main components was carried out by AutoDockTools 1.5.7. H9c2 cells were used to establish a oxygen-glucose deprivation model， and the protective effect of BXG on cells was investigated by detecting cell viability， cell survival rate and reactive oxygen species（ROS） level. The protein expression levels of signal transducer and activator of transcription 3（STAT3）， phosphorylation（p）-STAT3 and glutathione peroxidase 4（GPX4） were detected by Western blot to clarify the regulatory effect of BXG on ferroptosis. ResultsA total of 61 chemical components in BXG intestinal absorption fluid were identified， and network pharmacology obtained 27 potential targets of BXG for the treatment of HF， as well as 139 signaling pathways. BXG may act on core targets such as STAT3， tumor protein p53（TP53）， epidermal growth factor receptor（EGFR）， JUN and prostaglandin-endoperoxide synthase 2（PTGS2） through core components such as glabrolide and limonin， which in turn intervene in lipid and atherosclerosis， phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt）， endocrine resistance and other signaling pathways to exert therapeutic effects on HF. Molecular docking showed that the docking results of multiple groups of targets and compounds were good. In vitro cell experiments showed that compared with the blank group， the cell viability and survival rate of the model group were significantly decreased， the level of ROS was significantly increased（P<0.01）， the expression levels of STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 proteins were significantly decreased（P<0.05， P<0.01）. Compared with the model group， the cell viability and survival rate of the BXG group were significantly increased， the ROS level was significantly decreased（P<0.01）， the STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 protein levels were significantly increased（P<0.05， P<0.01）. ConclusionBXG may inhibit the occurrence of ferroptosis by up-regulating the expression of STAT3 and GPX4， thus exerting a therapeutic effect on HF， and flavonoids may be the key components of this role.

ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， network pharmacology， molecular docking and cell experiments， the active ingredients， possible targets and molecular mechanisms of Baoxin granules（BXG） regulating ferroptosis in the treatment of heart failure（HF） were explored. MethodsBXG intestinal absorption fluid was prepared by everted gut sac and the chemical composition contained therein were identified by UPLC-Q-TOF-MS. According to the obtained components， the potential targets of BXG were predicted， and the HF-related targets and related genes of ferroptosis were retrieved at the same time， and the intersecting targets were obtained by Venn diagram. In addition， the protein-protein interaction（PPI） network and the component-target network were constructed， and the core components and core targets were obtained by topological analysis. Then Gene Ontology（GO） function and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analysis were performed on the core targets， and molecular docking validation of the key targets and main components was carried out by AutoDockTools 1.5.7. H9c2 cells were used to establish a oxygen-glucose deprivation model， and the protective effect of BXG on cells was investigated by detecting cell viability， cell survival rate and reactive oxygen species（ROS） level. The protein expression levels of signal transducer and activator of transcription 3（STAT3）， phosphorylation（p）-STAT3 and glutathione peroxidase 4（GPX4） were detected by Western blot to clarify the regulatory effect of BXG on ferroptosis. ResultsA total of 61 chemical components in BXG intestinal absorption fluid were identified， and network pharmacology obtained 27 potential targets of BXG for the treatment of HF， as well as 139 signaling pathways. BXG may act on core targets such as STAT3， tumor protein p53（TP53）， epidermal growth factor receptor（EGFR）， JUN and prostaglandin-endoperoxide synthase 2（PTGS2） through core components such as glabrolide and limonin， which in turn intervene in lipid and atherosclerosis， phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt）， endocrine resistance and other signaling pathways to exert therapeutic effects on HF. Molecular docking showed that the docking results of multiple groups of targets and compounds were good. In vitro cell experiments showed that compared with the blank group， the cell viability and survival rate of the model group were significantly decreased， the level of ROS was significantly increased（P<0.01）， the expression levels of STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 proteins were significantly decreased（P<0.05， P<0.01）. Compared with the model group， the cell viability and survival rate of the BXG group were significantly increased， the ROS level was significantly decreased（P<0.01）， the STAT3， p-STAT3， p-STAT3/STAT3 and GPX4 protein levels were significantly increased（P<0.05， P<0.01）. ConclusionBXG may inhibit the occurrence of ferroptosis by up-regulating the expression of STAT3 and GPX4， thus exerting a therapeutic effect on HF， and flavonoids may be the key components of this role.

Generalized anxiety disorder (GAD) belongs to the category of emotional diseases in TCM, and its occurrence is closely related to liver depression and qi stagnation, and liver depression and fire syndrome is one of the TCM syndromes of GAD. With the deepening of modern medical research on GAD, microglia have been found to play an important role in the development of liver depression and fire syndrome of GAD. Excessive activation of microglia and transformation to M1 type would cause inflammatory mediator secretion up-regulation and destruction of neurons, etc. When using liver-soothing and heat-clearing medicines such as Bupleuri Radix, Gardeniae Fructus, Dangzhi Xiaoyao San, Yuejiu Pills and other Chinese materia medica or compounds, activation of microglia would be suppressed, and the inflammatory mediators mediated by microglia would also be suppressed, and the neurons could be protected. Therefore, this article discussed the modern biological connotation of the liver depression and fire syndrome of GAD from the perspective of microglia, and summarized the effects of TCM modulation of microglia on the liver depression and fire syndrome of GAD, and concluded that microglia could mediate the development of liver depression and fire syndrome of GAD.

Objective: To investigate the regulatory relationship between long non-coding RNA LINC01578 and c-Myc, and to explore the effect of LINC01578 on the metabolic process of oral squamous cell carcinoma(OSCC). Methods: After c-Myc was knocked down in OSCC cell line CAL27, LINC01578, a long non-coding RNA that is positively regulated by c-Myc, was identified by high-throughput sequencing technology. qRT-PCR was employed to measure the expression levels of c-Myc and LINC01578 in OSCC tissues and adjacent normal tissues. Following overexpression or knockdown of c-Myc in CAL27 and HN6 cells, qRT-PCR was conducted to validate the consistency with sequencing results. The binding of c-Myc to the LINC01578 promoter was confirmed using a dual luciferase reporter assay. Seahorse, ATP production and lactate production assays were utilized to examine the impact of c-Myc on glucose metabolism in OSCC via LINC01578. Colony formation assays assessed the proliferative capacity of OSCC cell lines. Results: qRT-PCR analysis revealed significantly higher expression levels of c-Myc LINC01578 in OSCC tissues compared to adjacent tissues( P < 0. 05 ) , confirming that c⁃Myc positively regulates LINC01578 expression. Consistent with sequencing data , c⁃Myc overexpression markedly upregulated LINC01578 (P < 0. 001) , while c⁃Myc knockdown led to a significant decrease in LINC01578 levels(P < 0. 000 1) . Dual lu ciferase reporter gene assays demonstrated that c⁃Myc directly targets and transcriptionally enhanced LINC01578 ex⁃ pression(P < 0. 001) . Seahorse experiments indicated that c⁃Myc promoted glucose metabolism in OSCC through LINC01578 regulation(P < 0. 05) . Colony formation assays showed that LINC01578 overexpression enhanced OS⁃ CC cell proliferation , whereas LINC01578 knockdown inhibited it. Conclusion c⁃Myc upregulates LINC01578 expression in OSCC cells , thereby modulating glycolysis and promoting cell proliferation.

Objective: To explore the role of leukemia inhibitory factor (LIF) in dental pulp inflammation . Methods: Human dental pulp stem cells (hDPSCs) were cultured in vitro as the target cells , the inflammatory response was induced by lipopolysaccharide (LPS) , and high-throughput sequencing was used to detect relevant highly ex- pressed genes in the inflammatory state . The expression of LIF under graded concentrations of LPS stimulation was detected by real-time fluorescence quantitative PCR (qPCR) . The expression levels of interleukin-6 (IL-6) , inter- leukin-1β(IL-1β) , and tumor necrosis factor-α(TNF-α) were detected after LIF knockdown and overexpression in hDPSCs by qPCR . Normal and inflammatory pulp tissues were collected , and the expression of LIF in both tis- sues was detected by qPCR and immunofluorescence (IF) . Results: The expression level of LIF increased in hu- man dental pulp cells after LPS stimulation . The expression level of LIF was subsequently elevated in inflammatory pulp induced by graded concentrations of LPS . The expression of IL-6 , IL-1β, and TNF-αwas significantly down- regulated in hDPSCs after LIF knockdown in response to LPS stimulation , while LIF overexpression upregulated the expression of these cytokines . qPCR and IF assays showed high expression of LIF in inflamed pulp tissue . Conclusion LIF is involved in dental pulp inflammation and promotes the development of pulpitis .

Objective To explore the action mechanism of long non-coding RNA(lncRNAs)lncRNA KCTD13-DT in oral squamous cell carcinoma(OSCC)and its potential interaction with transcription factor c-Myc,providing a potential diagnostic and therapeutic target for patients with OSCC.Methods The expression of lncRNA KCTD13-DT in OSCC and paracancerous tissues was detected by qRT-PCR.The effects of c-Myc overexpression and knock-down on human tongue squamous carcinoma cells HN6 and CAL27 were detected by qRT-PCR.Fluorescence in si-tu hybridization(FISH)assessed the localization of lncRNA KCTD13-DT in cells.A dual luciferase reporter gene was used to analyze the role of c-Myc in target binding to the promoter region of lncRNA KCTD13-DT.Stable cell lines with knockdown or overexpression of lncRNA KCTD13-DT were constructed in human OSCC cell lines HN6 and CAL27 by lentiviral infection,and the knockdown and overexpression efficiencies of lncRNA KCTD13-DT were detected by qRT-PCR.Cell proliferation changes were detected by growth curve assay,CCK-8 assay,colony forma-tion assay,and cell migration was detected by scratch assay and Transwell.Results lncRNA KCTD13-DT expres-sion level was reduced in OSCC tissues and OSCC cells(HN6,CAL27),and Western blot verified that after knoc-king down and overexpression of c-Myc in HN6 and CAL27,the qRT-PCR experiments showed that c-Myc nega-tively regulated lncRNA KCTD13-DT,and overexpression of c-Myc significantly down-regulated lncRNA KCTD13-DT;knockdown of c-Myc significantly up-regulated lncRNA KCTD13-DT levels.Dual luciferase reporter gene showed that c-Myc could target lncRNA KCTD13-DT,and c-Myc could be involved in regulating and repressing the transcriptional activity of lncRNA KCTD13-DT.FISH showed that lncRNA KCTD13-DT mainly existed in the nu-cleus.Growth curve assay,CCK-8 assay,cell scratch assay,Transwell,and colony formation assay showed that knockdown of lncRNA KCTD13-DT promoted the growth and proliferation of OSCC cells,and overexpression of ln-cRNA KCTD13-DT significantly inhibited the proliferation and migration of OSCC cells.Conclusion lncRNA KCTD13-DT is negatively regulated by c-Myc.Knockdown of lncRNA KCTD13-DT promotes cell proliferation,while overexpression of it inhibits cell growth.

Post-stroke depression (PSD) is a serious and common complication of stroke, which seriously affects the rehabilitation of stroke patients. To date, the pathogenesis of PSD is unclear and effective treatments remain unavailable. Here, we established a mouse model of PSD through photothrombosis-induced focal ischemia. By using a combination of brain imaging, transcriptome sequencing, and bioinformatics analysis, we found that the hippocampus of PSD mice had a significantly lower metabolic level than other brain regions. RNA sequencing revealed a significant reduction of miR34b-3p, which was expressed in hippocampal neurons and inhibited the translation of eukaryotic translation initiation factor 4E (eIF4E). Furthermore, silencing eIF4E inactivated microglia, inhibited neuroinflammation, and abolished the depression-like behaviors in PSD mice. Together, our data demonstrated that insufficient miR34b-3p after stroke cannot inhibit eIF4E translation, which causes PSD by the activation of microglia in the hippocampus. Therefore, miR34b-3p and eIF4E may serve as potential therapeutic targets for the treatment of PSD.
Animals ; Mice ; Depression ; Eukaryotic Initiation Factor-4E/metabolism* ; MicroRNAs/metabolism* ; Neurons/metabolism* ; Stroke/metabolism*

It has been demonstrated that APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) is involved in the regulation of several growth-related signaling pathways and thus closely associated with the development and progression of some cancers. Diallyl trisulfide (DAT), a garlic-derived bioactive compound, exerts selective cytotoxicity to various human cancer cells through interfering with pro-survival signaling pathways. However, whether and how DAT affects survival of human hepatocellular carcinoma (HCC) cells remain unclear. Herein, we tested the hypothesis of the involvement of APPL1 in DAT-induced cytotoxicity in HCC HepG2 cells. We found that Lys 63 (K63)-linked polyubiquitination of APPL1 was significantly decreased whereas phosphorylation of APPL1 at serine residues remained unchanged in DAT-treated HepG2 cells. Compared with wild-type APPL1, overexpression of APPL1 K63R mutant dramatically increased cell apoptosis and mitigated cell survival, along with a reduction of phosphorylation of STAT3, Akt, and Erk1/2. In addition, DAT administration markedly reduced protein levels of intracellular TNF receptor-associated factor 6 (TRAF6). Genetic inhibition of TRAF6 decreased K63-linked polyubiquitination of APPL1. Moreover, the cytotoxicity impacts of DAT on HepG2 cells were greatly attenuated by overexpression of wild-type APPL1. Taken together, these results suggest that APPL1 polyubiquitination probably mediates the inhibitory effects of DAT on survival of HepG2 cells by modulating STAT3, Akt, and Erk1/2 pathways.

Objective:To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures (Dowling-Degos disease, DDD) .Methods:HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats (CRISPR) -CRISPR-associated protein 9 (Cas9) technology (experimental group) , and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn) separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 (Pmel17) in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results:Sanger sequencing showed a heterozygous mutation (c.1delA) at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group (0.60 ± 0.05) than in the control group (1.00 ± 0.00, t = 32.38, P = 0.001) . Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% ( t = -3.48, P = 0.025) in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion:The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.

Objective:To explore the selection of surgical methods for different sites of symptomatic Rathke's cleft cyst (RCC) and the clinical efficacies of these patients.Methods:Forty-seven patients with symptomatic RCC, admitted to our hospital from January 2016 to December 2019, were chosen in our study; 21 patients with intrasellar symptomatic RCC accepted surgery via unilateral nasal approach at the right side, 19 patients with intra-suprasellar symptomatic RCC accepted surgery via bilateral nasal approach, 3 patients with suprasellar symptomatic RCC accepted endonasal transsphenoidal surgery under endoscope, and 4 patients with suprasellar symptomatic RCC accepted craniotomy via pterion approach. The clinical efficacies and complications of patients accepted different surgical methods were compared. All patients were followed up for 3-36 months to observe the recurrence.Results:The postoperative symptoms of the patients were effectively improved, including headache relief ratio of 27/31, vision loss improvement ratio of 5/5, high prolactin relief ratio of 11/13, pituitary function improvement ratio of 9/18. Complications occurred in 6 patients, presenting as diabetes insipidus. Four patients recurred during follow-up.Conclusion:Intrasellar and intra-suprasellar symptomatic RCC accepted surgery via endoscopic transnasal transsphenoidal approach are safe and effective; selection of surgical methods for suprasellar symptomatic RCC should be determined according to the sizes and growth directions of cysts.