Molecular mechanism of Chang Shen Hua volatile oil modulating brain cAMP-PKA-CREB pathway to improve depression-like behavior in rats.

BACKGROUND: Depression is a common and complex mental illness that manifests as persistent episodes of sadness, loss of interest, and decreased energy, which might lead to self-harm and suicide in severe cases. Reportedly, depression affects 3.8 % of the world's population and has been listed as one of the major global public health concerns. In recent years, aromatherapy has been widely used as an alternative and complementary therapy in the prevention and treatment of depression; people can relieve anxiety and depression by sniffing plant aromatic essential oils. Acorus tatarinowii and Panax ginseng essential oils in Chang Shen Hua volatile oil (CSHVO) are derived from Acorus tatarinowii and Panax ginseng, respectively, the main medicines in the famous Chinese medicine prescription Kai Xin San (KXS), Then, these oils are combined with the essential oil of Albizia julibrissin flower to form a new Chinese medicine inhalation preparation, CSHVO. KXS has been widely used in the treatment of depression; however, whether CSHVO can ameliorate depression-like behavior, its pharmacological effects, and the underlying mechanisms of action are yet to be elucidated. STUDY DESIGN AND METHODS: A rat model of chronic and unpredictable mild stimulation (CUMS) combined with orphan rearing was treated with CSHVO for 4 weeks. Using behavioral tests (sucrose preference, force swimming, tail suspension, and open field), the depression-like degree was evaluated. Concurrently, brain homogenate and serum biochemistry were analyzed to assess the changes in the neurotransmitters and inflammatory and neurotrophic factors. Furthermore, tissue samples were collected for histological and protein analyses. In addition, network pharmacology and molecular docking analyses of the major active compounds, potential therapeutic targets, and intervention pathways predicted a role of CSHVO in depression relief. Subsequently, these predictions were confirmed by in vitro experiments using a corticosterone (CORT)-induced PC12 cell damage model. RESULTS: CSHVO inhalation can effectively improve the weight and depression-like behavior of depressed rats and regulate the expression of inflammatory factors and neurotransmitters. Hematoxylin-eosin, Nissl, and immunofluorescence staining indicated that compared to the model group, the pathological damage to the brain tissues of rats in the CSHVO groups was improved. The network pharmacological analysis revealed that 144 CSHVO active compounds mediate 71 targets relevant to depression treatment, most of which are rich in the cAMP signaling and inflammatory cytokine pathways. Protein-protein interaction analysis showed that TNF, IL6, and AKT are the core anti-depressive targets of CSHVO. Molecular docking analysis showed an adequate binding between the active ingredients and the key targets. In vitro experiments showed that compared to the model group, the survival rate of PC12 cells induced by CSHVO intervention was increased, the apoptosis rate was decreased, and the expression of inflammatory cytokines in the cell supernatant was improved. Western blot analysis and immunofluorescence staining confirmed that CSHVO regulates PC12 cells in the CORT model through the cAMP-PKA-CREB signaling pathway, and pretreatment with PKA blocker H89 eliminates the protective effect of CSHVO on CORT-induced PC12 cells. CONCLUSIONS: CSHVO improves CORT-induced injury in the PC12 cell model and CUMS combined with orphan rearing-induced depression model in rats. The antidepressant mechanism of CSHVO is associated with the modulation of the cAMP-PKA-CREB signaling pathway.

Vitamin D inhibits ferroptosis and mitigates the kidney injury of prediabetic mice by activating the Klotho/p53 signaling pathway.

Diabetic nephropathy (DN) is a serious public health problem worldwide, and ferroptosis is deeply involved in the pathogenesis of DN. Prediabetes is a critical period in the prevention and control of diabetes and its complications, in which kidney injury occurs. This study aimed to explore whether ferroptosis would induce kidney injury in prediabetic mice, and whether vitamin D (VD) supplementation is capable of preventing kidney injury by inhibiting ferroptosis, while discussing the potential mechanisms. High-fat diet (HFD) fed KKAy mice and high glucose (HG) treated HK-2 cells were used as experimental subjects in the current study. Our results revealed that serious injury and ferroptosis take place in the kidney tissue of prediabetic mice; furthermore, VD intervention significantly improved the kidney structure and function in prediabetic mice and inhibited ferroptosis, showing ameliorated iron deposition, enhanced antioxidant capability, reduced reactive oxygen species (ROS) and lipid peroxidation accumulation. Meanwhile, VD up-regulated Klotho, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression, and down-regulated p53, transferrin receptor 1 (TFR1) and Acyl-Coenzyme A synthetase long-chain family member 4 (ACSL4) expression. Moreover, we demonstrated that HG-induced ferroptosis is antagonized by treatment of VD and knockdown of Klotho attenuates the protective effect of VD on ferroptosis in vitro. In conclusion, ferroptosis occurs in the kidney of prediabetic mice and VD owns a protective effect on prediabetic kidney injury, possibly by via the Klotho/p53 pathway, thus inhibiting hyperglycemia-induced ferroptosis.

Hypoxia-regulated secretion of IL-12 enhances antitumor activity and safety of CD19 CAR-T cells in the treatment of DLBCL.

CD19-targeted chimeric antigen receptor-modified T (CD19 CAR-T) cell therapy has been demonstrated as one of the most promising therapeutic strategies for treating B cell malignancies. However, it has shown limited treatment efficacy for diffuse large B cell lymphoma (DLBCL). This is, in part, due to the tumor heterogeneity and the hostile tumor microenvironment. Human interleukin-12 (IL-12), as a potent antitumor cytokine, has delivered encouraging outcomes in preclinical studies of DLBCL. However, potentially lethal toxicity associated with systemic administration precludes its clinical application. Here, an armed CD19 CAR expressing hypoxia-regulated IL-12 was developed (CAR19/hIL12ODD). In this vector, IL-12 secretion was restricted to hypoxic microenvironments within the tumor site by fusion of IL-12 with the oxygen degradation domain (ODD) of HIF1α. In vitro, CAR19/hIL12ODD-T cells could only secrete bioactive IL-12 under hypoxic conditions, accompanied by enhanced proliferation, robust IFN-γ secretion, increased abundance of CD4+, and central memory T cell phenotype. In vivo, adoptive transfer of CAR19/hIL12ODD-T cells significantly enhanced regression of large, established DLBCL xenografts in a novel immunodeficient Syrian hamster model. Notably, this targeted and controlled IL-12 treatment was without toxicity in this model. Taken together, our results suggest that armed CD19 CARs with hypoxia-controlled IL-12 (CAR19/hIL12ODD) might be a promising and safer approach for treating DLBCL.

Transcriptomic analysis the mechanisms of anti-osteoporosis of desert-living Cistanche herb in ovariectomized rats of postmenopausal osteoporosis.

Desert-living Cistanche herb (DC), as a traditional Chinese medicine for tonifying kidney yang, is often used to treat postmenopausal osteoporosis (PMOP). Total phenylethanoid glycosides are instruction ingredients for discrimination and assay according to the China pharmacopoeia for DC. This research aimed to reveal the anti-osteoporosis mechanism of total phenylethanoid glycosides of DC (PGC) by transcriptomic analysis of ovariectomized rats. Serum levels of BGP were evaluated by ELISA, the bone weight was measured, and transmission electron microscopy was used to examine the ultrastructure of osteoblasts in rats. In addition, micro-CT was used to detect the bone volume (Tb.BS/BV), bone mineral density (Tb.BMD), and bone mineral content (Tb.BMC) in trabecular bone, and the ratio of cortical bone area to total area (Ct.ar/Tt.ar), and the level of bone mineral content (Ct.BMC) in cortical bone. Differential expressed genes (DEGs) after PGC treatment were analyzed by transcriptomics. Then, a bioinformatics analysis of DEGs was carried out through GO enrichment, KEGG enrichment, and selection of the nucleus gene through the protein-protein interaction network. Through qRT-PCR analysis, the DEGs were verified. The analysis results indicated that PGC increased the secretion of osteogenic markers, and ultrastructural characterization of osteoblasts and bone morphology were improved in ovariectomized rats. A total of 269 genes were differentially expressed, including 201 genes that were downregulated and 68 genes that were upregulated between the model group and the PGC group. Bioinformation analysis results prompt the conclusion that PGC could promote the bone metabolism by muscle cell development, myofibril assembly, etc. In addition, our study also found that PGC has a good effect on osteoporosis complicated with cardiomyopathy, and it also provided evidence for the correlation between sarcopenia and osteoporosis.

A novel microenvironment regulated system CAR-T (MRS.CAR-T) for immunotherapeutic treatment of esophageal squamous carcinoma.

Chimeric antigen receptor T cell immunotherapy has achieved promising therapeutic effects in the treatment of hematological malignancies. However, there are still many obstacles, including on-target off-tumor antigen expression, that prevent successful application to solid tumors. We designed a tumor microenvironment (TME) regulated system chimeric antigen receptor T (MRS.CAR-T) which can only be auto-activated in the solid TME. B7-H3 was selected as the target antigen for esophageal carcinoma. An element comprising a human serum albumin (HSA) binding peptide and a matrix metalloproteases (MMPs) cleavage site was inserted between the 5' terminal signal peptide and single chain fragment variable (scFv) of the CAR skeleton. Upon administration, HSA bound the binding peptide in MRS.B7-H3.CAR-T effectively and promoted proliferation and differentiation into memory cells. MRS.B7-H3.CAR-T was not cytotoxic in normal tissues expressing B7-H3 as the antigen recognition site in the scFv was cloaked by HSA. The anti-tumor function of MRS.B7-H3.CAR-T was recovered once the cleavage site was cleaved by MMPs in the TME. The anti-tumor efficacy associated with MRS.B7-H3.CAR-T cells was improved compared to classic B7-H3.CAR-T cells in vitro and less IFN-γ was released, suggesting a treatment that may induce less extent of cytokine release syndrome-mediated toxicity. In vivo, MRS.B7-H3.CAR-T cells had strong anti-tumor activity and were safe. MRS.CAR-T represents a novel strategy to improve the efficacy and safety of CAR-T therapy in solid tumors.

Identification of optimal reference genes in golden Syrian hamster with ethanol- and palmitoleic acid-induced acute pancreatitis using quantitative real-time polymerase chain reaction.

BACKGROUND: Acute pancreatitis (AP) is a severe disorder that leads to high morbidity and mortality. Appropriate reference genes are important for gene analysis in AP. This study sought to study the expression stability of several reference genes in the golden Syrian hamster, a model of AP. METHODS: AP was induced in golden Syrian hamster by intraperitoneal injection of ethanol (1.35 g/kg) and palmitoleic acid (2 mg/kg). The expression of candidate genes, including Actb, Gapdh, Eef2, Ywhaz, Rps18, Hprt1, Tubb, Rpl13a, Nono, and B2m, in hamster pancreas at different time points (1, 3, 6, 9, and 24 h) posttreatment was analyzed using quantitative polymerase chain reaction. The expression stability of these genes was calculated using BestKeeper, Comprehensive Delta CT, NormFinder, and geNorm algorithms and RefFinder software. RESULTS: Our results show that the expression of these reference genes fluctuated during AP, of which Ywhaz and Gapdh were the most stable genes, whereas Tubb, Eef2, and Actb were the least stable genes. Furthermore, these genes were used to normalize the expression of TNF-α messenger ribonucleic acid in inflamed pancreas. CONCLUSIONS: In conclusion, Ywhaz and Gapdh were suitable reference genes for gene expression analysis in AP induced in Syrian hamster.

Red blood cell membrane-coated functionalized Au nanocage as a biomimetic platform for improved MicroRNA delivery in hepatocellular carcinoma.

Dysregulation of microRNAs (miRNAs) expression is closely related to cancers and managing miRNA expression holds great promise for cancer therapy. However, their wide clinical application has been hampered by their poor stability, short half-life and non-specific biodistribution in vivo. Herein, a novel biomimetic platform designated as RHAuNCs-miRNA for improved miRNA delivery was prepared through wrapping miRNA-loaded functionalized Au nanocages (AuNCs) with red blood cell (RBC) membrane. RHAuNCs-miRNA not only successfully loaded miRNAs but also effectively protected them from enzymatic degradation. With good stability, RHAuNCs-miRNA had the characteristics of photothermal conversion and sustained release. Cellular uptake of RHAuNCs-miRNA by SMMC-7721 cells was in a time-dependent manner via clathrin- and caveolin-mediated endocytosis. The uptake of RHAuNCs-miRNAs was affected by cell types and improved by mild near infrared (NIR) laser irradiation. More importantly, RHAuNCs-miRNA exhibited a prolonged circulation time without the occurrence of accelerated blood clearance (ABC) in vivo, resulting in efficient delivery to tumor tissues. This study may demonstrate the great potential of RHAuNCs-miRNA for improved miRNAs delivery.

Lesion-aware generative adversarial networks for color fundus image to fundus fluorescein angiography translation.

BACKGROUND AND OBJECTIVE: Fundus fluorescein angiography (FFA) is widely used in clinical ophthalmic diagnosis and treatment with the requirement of adverse fluorescent dyes injection. Recently, many deep Convolutional Neural Network(CNN)-based methods have been proposed to estimate FFA from color fundus (CF) images to eliminate the use of adverse fluorescent dyes. However, the robustness of these methods is affected by pathological changes. METHOD: In this work, we present a CNN-based approach, lesion-aware generative adversarial networks (LA-GAN), to enhance the visual effect of lesion characteristics in the generated FFA images. First, we lead the generator notice lesion information by joint learning with lesion region segmentation. A new hierarchical correlation multi-task framework for high-resolution images is designed. Second, to enhance the visual contrast between normal regions and lesion regions, a newly designed region-level adversarial loss is used rather than the image-level adversarial loss. The code is publicly available at: https://github.com/nicetomeetu21/LA-GAN. RESULTS: The effectiveness of LA-Net has been verified in data with branch retinal vein occlusion. The proposed model reported as measures of generation performance a mean structural similarity (SSIM) of 0.536, mean learned perceptual image patch similarity (LPIPS) 0.312, outperforming other FFA generation and general image generation methods. Further, due to the proposed multi-task learning framework, the lesion-region segmentation performance was further reported as the mean Dice increased from 0.714 to 0.797 and the mean accuracy increased from 0.873 to 0.905, outperforming general single-task image segmentation methods. CONCLUSIONS: The results show that the visual effect of lesion characteristics can be improved by employing the region-level adversarial loss and the hierarchical correlation multi-task framework respectively. Based on the results of comparison with the state-of-the-art methods, LA-GAN is not only effective for CF-to-FFA translation, but also effective for lesion-region segmentation. Thus, it may be used for various image translation and lesion segmentation tasks in future research.

CHCHD2 p.Thr61Ile knock-in mice exhibit motor defects and neuropathological features of Parkinson's disease.

The p.Thr61Ile (p.T61I) mutation in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) was deemed a causative factor in Parkinson's disease (PD). However, the pathomechanism of the CHCHD2 p.T61I mutation in PD remains unclear. Few existing mouse models of CHCHD2-related PD completely reproduce the features of PD, and no transgenic or knock-in (KI) mouse models of CHCHD2 mutations have been reported. In the present study, we generated a novel CHCHD2 p.T61I KI mouse model, which exhibited accelerated mortality, progressive motor deficits, and dopaminergic (DA) neurons loss with age, accompanied by the accumulation and aggregation of α-synuclein and p-α-synuclein in the brains of the mutant mice. The mitochondria of mouse brains and induced pluripotent stem cells (iPSCs)-derived DA neurons carrying the CHCHD2 p.T61I mutation exhibited aberrant morphology and impaired function. Mechanistically, proteomic and RNA sequencing analysis revealed that p.T61I mutation induced mitochondrial dysfunction in aged mice likely through repressed insulin-degrading enzyme (IDE) expression, resulting in the degeneration of the nervous system. Overall, this CHCHD2 p.T61I KI mouse model recapitulated the crucial clinical and neuropathological aspects of patients with PD and provided a novel tool for understanding the pathogenic mechanism and therapeutic interventions of CHCHD2-related PD.

Clinical features of Danon disease and insights gained from LAMP-2 deficiency models.

Danon disease (DD) is an X-linked multisystem disorder with clinical features characterized by the triad of hypertrophic cardiomyopathy, skeletal muscle weakness, and mental retardation. Cardiac involvement can be fatal in the absence of an effective treatment option such as heart transplantation. Molecular studies have proved that LAMP-2 protein deficiency, mainly LAMP-2B isoform, resulting from LAMP2 gene mutation, is the culprit for DD. Autophagy impairment due to LAMP-2 deficiency mediated the accumulation of abnormal autophagic vacuoles in cells. While it is not ideal for mimicking DD phenotypes in humans, the emergence of LAMP-2-deficient animal models and induced pluripotent stem cells from DD patients provided powerful tools for exploring DD mechanism. In both in vitro and in vivo studies, much evidence has demonstrated that mitochondria dysfunction and fragmentation can result in DD pathology. Fundamental research contributes to the therapeutic transformation. By targeting the molecular core, several potential therapies have demonstrated promising results in partial phenotypes improvement. Among them, gene therapies anticipate inaugurate a class of symptom control and prevention drugs as their in vivo effects are promising, and one clinical trial is currently underway.

Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system.

BACKGROUND: SHARPIN (SHANK-associated RH domain interactor) is a component of the linear ubiquitination complex that regulates the NF-κB signaling pathway. To better understand the function of SHARPIN, we sought to establish a novel genetically engineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system. METHODS: A single-guide ribonucleic acid targeting exon 1 of SHARPIN gene was designed and constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatal mutants were identified by genotyping. SHARPIN protein expression was detected using Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymic weights were measured, and organ coefficients were calculated. Histopathological examination of the spleen, liver, lung, small intestine, and esophagus was performed independently by a pathologist. The expression of lymphocytic markers and cytokines was evaluated using reverse transcriptase-quantitative polymerase chain reaction. RESULTS: All the offspring harbored germline-transmitted SHARPIN mutations. Compared with wild-type hamsters, SHARPIN protein was undetectable in SHARPIN-/- hamsters. Spleen enlargement and splenic coefficient elevation were spotted in SHARPIN-/- hamsters, with the descent of MLNs and thymuses. Further, eosinophil infiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagi were obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless, the expression of CCR3, CCL11, Il4, and Il13 was upregulated in the esophagi. The expression of NF-κB and phosphorylation of NF-κB and IκB protein significantly diminished in SHARPIN-/- animals. CONCLUSIONS: A novel SHARPIN KO hamster was successfully established using the CRISPR/Cas9 system. Abnormal development of secondary lymphoid organs and eosinophil infiltration in multiple organs reveal its potential in delineating SHARPIN function and chronic inflammation.

[Mechanism of Puerariae Lobatae Radix against lung cancer by inhibiting histone demethylase LSD1].

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.

Deep Learning Models for Segmenting Non-perfusion Area of Color Fundus Photographs in Patients With Branch Retinal Vein Occlusion.

Purpose: To develop artificial intelligence (AI)-based deep learning (DL) models for automatically detecting the ischemia type and the non-perfusion area (NPA) from color fundus photographs (CFPs) of patients with branch retinal vein occlusion (BRVO). Methods: This was a retrospective analysis of 274 CFPs from patients diagnosed with BRVO. All DL models were trained using a deep convolutional neural network (CNN) based on 45 degree CFPs covering the fovea and the optic disk. We first trained a DL algorithm to identify BRVO patients with or without the necessity of retinal photocoagulation from 219 CFPs and validated the algorithm on 55 CFPs. Next, we trained another DL algorithm to segment NPA from 104 CFPs and validated it on 29 CFPs, in which the NPA was manually delineated by 3 experienced ophthalmologists according to fundus fluorescein angiography. Both DL models have been cross-validated 5-fold. The recall, precision, accuracy, and area under the curve (AUC) were used to evaluate the DL models in comparison with three types of independent ophthalmologists of different seniority. Results: In the first DL model, the recall, precision, accuracy, and area under the curve (AUC) were 0.75 ± 0.08, 0.80 ± 0.07, 0.79 ± 0.02, and 0.82 ± 0.03, respectively, for predicting the necessity of laser photocoagulation for BRVO CFPs. The second DL model was able to segment NPA in CFPs of BRVO with an AUC of 0.96 ± 0.02. The recall, precision, and accuracy for segmenting NPA was 0.74 ± 0.05, 0.87 ± 0.02, and 0.89 ± 0.02, respectively. The performance of the second DL model was nearly comparable with the senior doctors and significantly better than the residents. Conclusion: These results indicate that the DL models can directly identify and segment retinal NPA from the CFPs of patients with BRVO, which can further guide laser photocoagulation. Further research is needed to identify NPA of the peripheral retina in BRVO, or other diseases, such as diabetic retinopathy.

Deoxythymidylate Kinase as a Promising Marker for Predicting Prognosis and Immune Cell Infiltration of Pan-cancer.

Background: Deoxythymidylate kinase (DTYMK) serves as a pyrimidine metabolic rate-limiting enzyme that catalyzes deoxythymidine monophosphate (dTMP) to generate deoxythymidine diphosphate (dTDP). It remains unclear whether DTYMK expression has the potential to predict outcome and immune cell infiltration in cancers. Methods: DTYMK expression profile was analyzed using Oncomine, TIMER, GEPIA and UALCAN databases. The influence of DTYMK on immune infiltration was examined using TIMER and TISIDB databases. DTYMK interactive gene hub and co-expressing genes were obtained and analyzed by STRING and Linkedomics, respectively. The relationship between DTYMK expression and patient prognosis was validated using GEPIA, Kaplan-Meier plotter, and PrognoScan databases. The functions of DTYMK in cancer cells were also biologically validated in vitro. Results: DTYMK expression was elevated in tumor tissues compared with their control counterparts. DTYMK expression varied in different stages and discriminatorily distributed in different immune and molecular subtypes. Higher expression of DTYMK predicted worse outcome in several cancer types such as liver hepatocellular carcinoma (LIHC) and lung adenocarcinoma (LUAD). High DTYMK expression was positively or negatively correlated with immune cell infiltration, including B cell, CD8+ cell, CD4+ T cell, macrophage, neutrophil and dendritic cell, depending on the type of cancers. Additionally, DTYMK co-expressing genes participated in pyrimidine metabolism as well as in T helper cell differentiation in LIHC and LUAD. In vitro, knockdown of DTYMK suppressed cell migration of liver and lung cancer cells. Conclusion: DTYMK might be taken as an useful prognostic and immunological marker in cancers and further investigation is warrented.

The two-faced role of ATF2 on cisplatin response in gastric cancer depends on p53 context.

BACKGROUND: Activating transcription factor-2 (ATF2) is a member of the basic leucine zipper family of DNA-binding proteins, which exhibits both oncogenic and tumor suppression activity in different tumors. However, the molecular mechanism of its dual function in cancer chemotherapy especially in gastric cancer has still not been elucidated. METHODS: The protein expression and location of ATF2 in gastric cancer tissues was detected with immunohistochemistry assay, and the clinical significance was analyzed using TCGA and GEO database. The activation and impact of ATF2 in cisplatin treated cells were evaluated with western blot, incucyte live cell analysis, clone formation and tumor xenografts assays. Interaction between ATF2 and p53 was confirmed with immunoprecipitation and GST-pull down. Potential molecular mechanism of ATF2 in different p53 status cells was analyzed with RNA sequencing and real-time quantitative PCR. RESULTS: ATF2 mainly located in the nucleus of cancer cells, higher ATF2 level was associated with poor five-year survival of gastric patients, especially in those undergone chemotherapy treatment. Cisplatin treatment significantly activated ATF2 in p53 mutant cells. ATF2 could interact with the trans-activation domain of p53 and enhance cisplatin sensitivity in p53 wild type cell lines, while promoted cell survival in mutant p53 cancer cells by affecting ERK1/2 pathway. CONCLUSIONS: This study confirmed the effect of ATF2 on cisplatin sensitivity was associated with the functional status of p53 in gastric cancer cells. Integrated analysis of ATF2 expression and P53 status could be used to evaluate the chemotherapy sensitivity and prognosis of gastric cancer patients.

MiRNA-363-3p/DUSP10/JNK axis mediates chemoresistance by enhancing DNA damage repair in diffuse large B-cell lymphoma.

Anthracycline-based chemotherapy resistance represents a major challenge in diffuse large B-cell lymphoma (DLBCL). MiRNA and gene expression profiles (n = 47) were determined to uncover potential chemoresistance mechanisms and therapeutic approaches. An independent correlation between high expression of miRNA-363-3p and chemoresistance was observed and validated in a larger cohort (n = 106). MiRNA-363-3p was shown to reduce doxorubicin-induced apoptosis and tumor shrinkage in in vitro and in vivo experiments by ectopic expression and CRISPR/Cas9-mediated knockout in DLBCL cell lines. DNA methylation was found to participate in transcriptional regulation of miRNA-363-3p. Further investigation revealed that dual specificity phosphatase 10 (DUSP10) is a target of miRNA-363-3p and its suppression promotes the phosphorylation of c-Jun N-terminal kinase (JNK). The miRNA-363-3p/DUSP10/JNK axis was predominantly associated with negative regulation of homologous recombination (HR) and DNA repair pathways. Ectopic expression of miRNA-363-3p more effectively repaired doxorubicin-induced double-strand break (DSB) while enhancing non-homologous end joining repair and reducing HR repair. Targeting JNK and poly (ADP-ribose) polymerase 1 significantly inhibited doxorubicin-induced DSB repair, increased doxorubicin-induced cell apoptosis and tumor shrinkage, and improved the survival of tumor-bearing mice. In conclusion, the miRNA-363-3p/DUSP10/JNK axis is a novel chemoresistance mechanism in DLBCL that may be reversed by targeted therapy.

[Effect of fresh Phragmitis Rhizoma on airway inflammation in chronic bronchitis based on TGF-ß signaling pathway].

This study aims to explore the mechanism of fresh Phragmitis Rhizoma against chronic bronchitis airway inflammation. The SD rats of SPF grade were divided into control group, model group, Guilongkechuanning group(GLKCN, 1.125 g·kg~(-1)), high-dose fresh Phragmitis Rhizoma group(LG-HD, 15 g·kg~(-1)), and low-dose fresh Phragmitis Rhizoma group(LG-LD, 7.5 g·kg~(-1)). The chronic bronchitis models of rats in other groups except the control group were induced by the modified smoking method. From the 15 th day of modeling, the rats were given corresponding agents by gavage for 20 consecutive days. After the last administration, the rats were sacrificed for sample collection. Enzyme-linked immunosorbent assay(ELISA) was employed to detect serum transforming growth factor-ß(TGF-ß) and interleukin-6(IL-6) levels. The protein expression of TGF-ß, IL-1ß and IL-6 in lung tissue was detected by immunohistochemical method. Masson staining was performed to detect collagen fibers and muscle fibers in lung tissue, and HE staining to detect the pathological changes of lung tissue. Human bronchial epithelial(16 HBE) cells were cultured in vitro, and CCK-8(cell counting kit-8) method was used to detect the cytotoxicity of cigarette smoke extract(CSE) and fresh Phragmitis Rhizoma. After the exposure of 16 HBE cells to 3.5% CSE and appropriate concentration(800, 400 µg·mL~(-1)) of fresh Phragmitis Rhizoma for 24 h, quantitative real-time PCR was conducted to determine the mRNA levels of TGF-ß and IL-1ß, and Western blot was employed to determine the protein levels of TGF-ß and IL-6 in the cells. The rat model of chronic bronchitis induced by smoking was successfully established. Fresh Phragmitis Rhizoma reduced serum TGF-ß and IL-6 levels, down-regulated the protein levels of TGF-ß, IL-1ß, and IL-6 in lung tissue, and alleviated pathological changes and fibrotic lesions in lung tissue. Moreover, it down-regulated the CSE-induced protein expression of TGF-ß and IL-6 as well as the mRNA level of TGF-ß in 16 HBE cells. These results indicated that fresh Phragmitis Rhizoma could prevent airway inflammation from chronic bronchitis and promote cell repair by inhibiting the TGF-ß signaling pathway.

Sinomenine Relieves Airway Remodeling By Inhibiting Epithelial-Mesenchymal Transition Through Downregulating TGF-ß1 and Smad3 Expression In Vitro and In Vivo.

Airway remodeling is associated with dysregulation of epithelial-mesenchymal transition (EMT) in patients with asthma. Sinomenine (Sin) is an effective, biologically active alkaloid that has been reported to suppress airway remodeling in mice with asthma. However, the molecular mechanisms behind this effect remain unclear. We aimed to explore the potential relationship between Sin and EMT in respiratory epithelial cells in vitro and in vivo. First, 16HBE cells were exposed to 100 µg/mL LPS and treated with 200 µg/mL Sin. Cell proliferation, migration, and wound healing assays were performed to evaluate EMT, and EMT-related markers were detected using Western blotting. Mice with OVA-induced asthma were administered 35 mg/kg or 75 mg/kg Sin. Airway inflammation and remodeling detection experiments were performed, and EMT-related factors and proteins in the TGF-ß1 pathway were detected using IHC and Western blotting. We found that Sin suppressed cell migration but not proliferation in LPS-exposed 16HBE cells. Sin also inhibited MMP7, MMP9, and vimentin expression in 16HBE cells and respiratory epithelial cells from mice with asthma. Furthermore, it decreased OVA-specific IgE and IL-4 levels in serum, relieved airway remodeling, attenuated subepithelial collagen deposition, and downregulating TGF-ß1and Smad3 expression in mice with asthma. Our results suggest that Sin suppresses EMT by inhibiting IL-4 and downregulating TGF-ß1 and Smad3 expression.

Cancer spectrum in TP53-deficient golden Syrian hamsters: A new model for Li-Fraumeni syndrome.

BACKGROUND: The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. Humans who inherit mutant TP53 alleles develop a wide range of early onset cancers, a disorder called Li-Fraumeni Syndrome (LFS). Trp53-deficient mice recapitulate most but not all of the cancer phenotypes observed in TP53-deficient human cancers, indicating that new animal models may complement current mouse models and better inform on human disease development. MATERIALS AND METHODS: The recent application of CRISPR/Cas9 genetic engineering technology has permitted the emergence of golden Syrian hamsters as genetic models for wide range of diseases, including cancer. Here, the first cancer phenotype of TP53 knockout golden Syrian hamsters is described. RESULTS: Hamsters that are homozygous for TP53 mutations become moribund on average ~ 139 days of age, while hamsters that are heterozygous become moribund at ~ 286 days. TP53 homozygous knockout hamsters develop a wide range of cancers, often synchronous and metastatic to multiple tissues, including lymphomas, several sarcomas, especially hemangiosarcomas, myeloid leukemias and several carcinomas. TP53 heterozygous mutants develop a more restricted tumor spectrum, primarily lymphomas. CONCLUSIONS: Overall, hamsters may provide insights into how TP53 deficiency leads to cancer in humans and can become a new model to test novel therapies.

Total flavonoids of Taraxacum mongolicum inhibit non-small cell lung cancer by regulating immune function.

ETHNOPHARMACOLOGICAL RELEVANCE: Taraxacum mongolicum Hand.-Mazz. has been used in lung cancer treatment in Chinese medicine. However, its specific mechanism of action has not yet been reported, and developing pharmaceutical anti-cancer resources is important. Here, we aimed to elucidate the anti-tumor effects of dandelion in vitro and in vivo and assess its effects on immune function in lung cancer patients. AIM OF THE STUDY: In the present study, we mainly observed the therapeutic effects of total flavonoids from Taraxacum mongolicum Hand.-Mazz. (TFTM) on non-small cell lung cancer and its influence on the body's immune function. MATERIALS AND METHODS: In vitro experiments on A549 and H1299 cells were performed using the CCK8 method; the proliferation and migration of cells were observed to investigate the wound healing effects of TFTM, and flow cytometry was used to detect the apoptotic rate of TFTM on lung cancer cells. In vivo experiments were preformed to establish a non-small cell lung cancer mouse model using subcutaneously transplanted Lewis cells, and the body weight and tumor growth of the mice were recorded. Hematoxylin and eosin staining was performed for tumor tissue to assess pathological changes. The thymus, spleen, and lungs were isolated for to calculate organ index. The CD4+, CD8+, and CD4+/CD8+ levels were detected in mouse spleen using flow cytometry, and IL-2, IL-3, IFN-γ, and TNF-α levels were determined in serum using enzyme-linked immunosorbent assay. Expressions of IL-2, IL-3, IFN-γ, and TNF-α were detected using quantitative real-time PCR in tumor tissues, and Ki67 expression was observed by immunofluorescence. RESULTS: At 24 h, TFTM (100 and 200 µg/mL) had the best inhibitory effect on the proliferation of A549 and H1299 cells. The cell migration rate significantly reduced (P < 0.01), and the tumor inhibition rate increased (P < 0.01) and promoted apoptosis (P < 0.01). The mouse thymus index significantly increased (P < 0.05) and mouse spleen index reduced (P < 0.05). The CD4+, CD8+, and CD4+/CD8+ levels in Lewis lung cancer mouse model increased, as did the levels of IL-2, IL-3, IFN-γ, and TNF-α in the serum and tumor of mice; Ki67 expression in tumor tissues significantly reduced (P < 0.01). CONCLUSION: TFTM has an inhibitory effect on lung cancer. The mechanism may be that it improves the host's protective immune response by having a milder tumor growth inhibitory effect than cyclophosphamide.