Autophagy regulation combined with stem cell therapy for treatment of spinal cord injury

Stem cells are a group of cells with unique self-renewal and differentiation abilities that have great prospects in the repair of spinal cord injury. However, stem cell renewal and differentiation require strict control of protein turnover in the stem cells to achieve cell remodeling. As a highly conserved “gatekeeper” of cell homeostasis, autophagy can regulate cell remodeling by precisely controlling protein turnover in cells. Recently, it has been found that the expression of autophagy markers changes in animal models of spinal cord injury. Therefore, understanding whether autophagy can affect the fate of stem cells and promote the repair of spinal cord injury is of considerable clinical value. This review expounds the importance of autophagy homeostasis control for the repair of spinal cord injury from three aspects—pathophysiology of spinal cord injury, autophagy and stem cell function, and autophagy and stem cell function in spinal cord injury—and proposes the synergistic therapeutic effect of autophagy and stem cells in spinal cord injury.

Predictive value of co-expression patterns of immune checkpoint molecules for clinical outcomes of hematological malignancies

Co-expression of immune checkpoint(IC)molecules can exacerbate T cell exhaustion in patients with hematological malignancies(HMs)and contribute to the immune escape of tumor cells,which is related to poor clinical outcome.It is worth establishing and optimizing an ideal prediction model based on the co-expression patterns of IC molecules to evaluate the immune status of HM patients and predict their clinical outcome.In this perspective,we summarize the co-expression patterns of IC molecules and their importance as biomarkers that predict the prognosis of patients with different HMs,providing new insights for designing dual IC blockades(ICBs).

Outlook of PINK1/Parkin signaling in molecular etiology of Parkinson's disease,with insights into Pink1 knockout models

Parkinson’s disease(PD)relates to defective mitochondrial quality control in the dopaminergic motor network.Genetic studies have revealed that PINK1 and Parkin mutations are indicative of a heightened propensity to PD onset,pinpointing mitophagy and inflammation as the culprit pathways involved in neuronal loss in the substantia nigra(SNpc).In a reciprocal manner,LRRK2 functions in the regulation of basal flux and inflammatory responses responsible for PINK1/Parkin-dependent mitophagy activation.Pharmacological intervention in these diseasemodifying pathways may facilitate the development of novel PD therapeutics,despite the current lack of an established drug evaluation model.As such,we reviewed the feasibility of employing the versatile global Pink1knockout(KO)rat model as a self-sufficient,spontaneous PD model for investigating both disease etiology and drug pharmacology.These rats retain clinical features encompassing basal mitophagic flux changes with PD progression.We demonstrate the versatility of this PD rat model based on the incorporation of additional experimental insults to recapitulate the proinflammatory responses observed in PD patients.

Genome-wide identification and spatiotemporal expression profiling of zinc finger SWIM domain-containing protein family genes

The biological function of the novel zinc-finger SWIM domain-containing protein family(ZSWIM)during embryonic development remains elusive.Here,we conducted a genome-wide analysis to explore the evolutionary processes of the ZSWIM gene family members in mice,Xenopus tropicalis,zebrafish,and humans.We identified nine putative ZSWIM genes in the human and mouse genome,eight in the Xenopus genome,and five in the zebrafish genome.Based on multiple sequence alignment,three members,ZSWIM5,ZSWIM6,and ZSWIM8,demonstrated the highest homology across all four species.Using available RNA sequencing(RNAseq)data,ZSWIM genes were found to be widely expressed across different tissues,with distinct tissuespecific properties.To identify the functions of the ZSWIM protein family during embryogenesis,we examined temporal and spatial expression patterns of zswim family genes in Xenopus embryos.Quantitative real-time polymerase chain reaction(qRT-PCR)revealed that each member had a distinct expression profile.Whole-mount in situ hybridization showed that both zswim1 and zswim3 were maternally expressed genes;zswim5 and zswim6were expressed throughout embryogenesis and displayed dynamic expression in the brain,eyes,somite,and bronchial arch at the late tailbud stages;zswim7 was detected in the eye area;zswim8 showed a dynamic expression pattern during the tailbud stages,with expression detected in the brain,eyes,and somite;zswim9 was faintly expressed throughout embryonic development.This study provides a foundation for future research to delineate the functions of ZSWIM gene members.

Decoding hereditary spastic paraplegia pathogenicity through transcriptomic profiling

Hereditary spastic paraplegia(HSP)is a group of genetic motor neuron diseases resulting from length-dependent axonal degeneration of the corticospinal upper motor neurons.Due to the advancement of next-generation sequencing,more than 70 novel HSP disease-causing genes have been identified in the past decade.Despite this,our understanding of HSP physiopathology and the development of efficient management and treatment strategies remain poor.One major challenge in studying HSP pathogenicity is selective neuronal vulnerability,characterized by the manifestation of clinical symptoms that are restricted to specific neuronal populations,despite the presence of germline disease-causing variants in every cell of the patient.Furthermore,disease genes may exhibit ubiquitous expression patterns and involve a myriad of different pathways to cause motor neuron degeneration.In the current review,we explore the correlation between transcriptomic data and clinical manifestations,as well as the importance of interspecies models by comparing tissue-specific transcriptomic profiles of humans and mice,expression patterns of different genes in the brain during development,and single-cell transcriptomic data from related tissues.Furthermore,we discuss the potential of emerging single-cell RNA sequencing technologies to resolve unanswered questions related to HSP pathogenicity.

Facile and rapid fabrication of a novel 3D-printable,visible light-crosslinkable and bioactive polythiourethane for large-to-massive rotator cuff tendon repair

Facile and rapid 3D fabrication of strong,bioactive materials can address challenges that impede repair of large-to-massive rotator cuff tears including personalized grafts,limited mechanical support,and inadequate tissue regeneration.Herein,we developed a facile and rapid methodology that generates visible light-crosslinkable polythiourethane(PHT)pre-polymer resin(~30 min at room temperature),yielding 3D-printable scaffolds with tendon-like mechanical attributes capable of delivering tenogenic bioactive factors.Ex vivo characterization confirmed successful fabrication,robust human supraspinatus tendon(SST)-like tensile properties(strength:23 MPa,modulus:459 MPa,at least 10,000 physiological loading cycles without failure),excellent suture retention(8.62-fold lower than acellular dermal matrix(ADM)-based clinical graft),slow degradation,and controlled release of fibroblast growth factor-2(FGF-2)and transforming growth factor-β3(TGF-β3).In vitro studies showed cytocompatibility and growth factor-mediated tenogenic-like differentiation of mesenchymal stem cells.In vivo studies demonstrated biocompatibility(3-week mouse subcutaneous implantation)and ability of growth factor-containing scaffolds to notably regenerate at least 1-cm of tendon with native-like biomechanical attributes as uninjured shoulder(8-week,large-to-massive 1-cm gap rabbit rotator cuff injury).This study demonstrates use of a 3D-printable,strong,and bioactive material to provide mechanical support and pro-regenerative cues for challenging injuries such as large-to-massive rotator cuff tears.

Engineering an extracellular matrix-functionalized,load-bearing tendon substitute for effective repair of large-to-massive tendon defects

A significant clinical challenge in large-to-massive rotator cuff tendon injuries is the need for sustaining high mechanical demands despite limited tissue regeneration,which often results in clinical repair failure with high retear rates and long-term functional deficiencies.To address this,an innovative tendon substitute named“BioTenoForce”is engineered,which uses(i)tendon extracellular matrix(tECM)’s rich biocomplexity for tendon-specific regeneration and(ii)a mechanically robust,slow degradation polyurethane elastomer to mimic native tendon’s physical attributes for sustaining long-term shoulder movement.Comprehensive assessments revealed outstanding performance of BioTenoForce,characterized by robust core-shell interfacial bonding,human rotator cuff tendon-like mechanical properties,excellent suture retention,biocompatibility,and tendon differentiation of human adipose-derived stem cells.Importantly,BioTenoForce,when used as an interpositional tendon substitute,demonstrated successful integration with regenerative tissue,exhibiting remarkable efficacy in repairing large-to-massive tendon injuries in two animal models.Noteworthy outcomes include durable repair and sustained functionality with no observed breakage/rupture,accelerated recovery of rat gait performance,and>1 cm rabbit tendon regeneration with native tendon-like biomechanical attributes.The regenerated tissues showed tendon-like,wavy,aligned matrix structure,which starkly contrasts with the typical disorganized scar tissue observed after tendon injury,and was strongly correlated with tissue stiffness.Our simple yet versatile approach offers a dual-pronged,broadly applicable strategy that overcomes the limitations of poor regeneration and stringent biomechanical requirements,particularly essential for substantial defects in tendon and other load-bearing tissues.

In vivo genome editing thrives with diversified CRISPR technologies

Prokaryotic type II adaptive immune systems have been developed into the versatile CRISPR technology, which has been widely applied in site- specific genome editing and has revolutionized biomedical research due to its superior efficiency and flexibility. Recent studies have greatly diversified CRISPR technologies by coupling it with various DNA repair mechanisms and targeting strategies. These new advances have significantly expanded the generation of genetically modified animal models, either by including species in which targeted genetic modification could not be achieved previously, or through introducing complex genetic modifications that take multiple steps and cost years to achieve using traditional methods. Herein, we review the recent developments and applications of CRISPR-based technology in generating various animal models, and discuss the everlasting impact of this new progress on biomedical research.

Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs

A comprehensive understanding of the cellular heterogeneity and molecular mechanisms underlying the development,homeostasis,and disease of human intervertebral disks(IVDs)remains challenging.Here,the transcriptomic landscape of 108108 IVD cells was mapped using single-cell RNA sequencing of three main compartments from young and adult healthy IVDs,including the nucleus pulposus(NP),annulus fibrosus,and cartilage endplate(CEP).The chondrocyte subclusters were classified based on their potential regulatory,homeostatic,and effector functions in extracellular matrix(ECM)homeostasis.Notably,in the NP,a PROCR+resident progenitor population showed enriched colony-forming unit-fibroblast(CFU-F)activity and trilineage differentiation capacity.Finally,intercellular crosstalk based on signaling network analysis uncovered that the PDGF and TGF-βcascades are important cues in the NP microenvironment.In conclusion,a single-cell transcriptomic atlas that resolves spatially regulated cellular heterogeneity together with the critical signaling that underlies homeostasis will help to establish new therapeutic strategies for IVD degeneration in the clinic.

Higher PD-1 expression concurrent with exhausted CD8+ T cells in patients with de novo acute myeloid leukemia

Objective： To investigate the association between the T cell inhibitory receptor programmed death 1（PD-1）and T cell exhaustion status in T cells from patients with de novo acute myeloid leukemia（AML） and AML in complete remission（CR）.Methods：Surface expression of PD-1 and the exhaustion and immunosenescence markers CD244 and CD57 on CD3＋,CD4＋ and CD8＋ T cells from peripheral blood samples from 20 newly diagnosed,untreated AML patients and 10 cases with AML in CR was analyzed by flow cytometry.Twenty-three healthy individuals served as control.Results：A significantly higher percentage of PD-1＋ cells were found for CD3＋ T cells in the de novo AML group compared with healthy controls.In addition,an increased level of PD-1＋ CD8＋ T cells,but not PD-1＋ CD4＋,was found for CD3＋ T cells in the de novo AML and AML-CR samples.A higher percentage of CD244＋ CD4＋,CD244＋ CD8＋,CD57＋ CD4＋ and CD57＋ CD8＋ T cells was found in CD3＋ T cells in samples from those with de novo AML compared with those from healthy controls.Strong increased PD-1＋ CD244＋ and PD-1＋ CD57＋ coexpression was found for CD4＋ and CD8＋ T cells in the de novo AML group compared with healthy controls.Conclusions：We characterized the major T cell defects,including co-expression of PD-1 and CD244,CD57-exhausted T cells in patients with de novo AML,and found a particular influence on CD8＋ T cells,suggesting a poor anti-leukemia immune response in these patients.

Characteristics of neural growth and cryopreservation of the dorsal root ganglion using three-dimensional collagen hydrogel culture versus conventional culture

In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion(DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates(T-DRG) versus three-dimensional collagen hydrogels(C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis-and myelination-related genes were highly expressed in C-DRG, while epithelial–mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2′-deoxyuridine(EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B(S100 B) and lower α-smooth muscle actin(α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China(approval No. 2020-IRB16), on March 15, 2020.

Developmental expression of three prmt genes in Xenopus

DEAR EDITOR Protein arginine methyltransferases (PRMTs)are involved in many cellular processes via the arginine methylation of histone or non-histone proteins.We examined the expression patterns of prmt4,prmt7,and prmt9 during embryogenesis in Xenopus using whole-mount in situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-PCR).Xenopus prmt4 and prmt7 were expressed in the neural crest,brain,and spinal cord,and also detected in the eye,branchial arches,and heart at the tailbud stage.Specific print9 signals were not detected in Xenopus embryos until the late tailbud stage when weak expression was observed in the branchial arches.Quantitative RT-PCR indicated that the expression of prmt4 and prmt7 was up-regulated during the neurula stage,whereas prmt9 maintained its low expression until the late tailbud stage,consistent with the whole-mount in situ hybridization results.Thus,the developmental expression patterns of these three print genes in Xenopus embryos provide a basis for further functional study of such genes.

The gene expression patterns of BMPR2,EP300,TGFβ2,and TNFAIP3 in B-Lymphoma cells

Objective: The results of a previous study showed that a clear dysregulation was evident in the global gene expression of the BCL11A-suppressed B-lymphoma cells. In this study, the bone morphogenetic protein receptor, type II(BMPR2), E1 A binding protein p300(EP300), transforming growth factor-β2(TGFβ2), and tumor necrosis factor, and alpha-induced protein 3(TNFAIP3) gene expression patterns in B-cell malignancies were studied. Methods: The relative expression levels of BMPR2, EP300, TGFβ2, and TNFAIP3 mRNA in B-lymphoma cell lines, myeloid cell lines, as well as in cells from healthy volunteers, were determined by real-time quantitative reverse transcriptpolymerase chain reaction(qRT-PCR) with SYBR Green Dye. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was used as reference. Results: The expression level of TGFβ2 mRNA in B-lymphoma cell lines was significantly higher than those in the cells from the healthy control(P<0.05). However, the expression level of TNFAIP3 mRNA in B-malignant cells was significantly lower than that of the healthy control(P<0.05). The expression levels of BMPR2 and EP300 mRNA showed no significant difference between B-malignant cell lines and the healthy group(P>0.05). In B-lymphoma cell lines, correlation analyses revealed that the expression of BMPR2 and TNFAIP3(r=0.882, P=0.04) had significant positive relation. The expression levels of BMPR2, EP300, and TNFAIP3 mRNA in cell lines from myeloid leukemia were significantly lower than those in the cells from the healthy control(P<0.05). The expression levels of TGFβ2 mRNA showed no significant difference between myeloid leukemia cell lines and the healthy control or B-malignant cell lines(P>0.05). The expression levels of BMPR2, EP300, and TNFAIP3 mRNA in B-lymphoma cells were significantly higher than those of the myeloid leukemia cells(P<0.05).Conclusion: Different expression patterns of BMPR2, EP300, TGFβ2, and TNFAIP3 genes in B-lymphoma cells exist.

Insulin exerts direct, IGF-1 independent actions in growth plate chondrocytes

Insufficient insulin production or action in diabetic states is associated with growth retardation and impaired bone healing, while the underling mechanisms are unknown. In this study, we sought to define the role of insulin signaling in the growth plate. Insulin treatment of embryonic metatarsal bones from wild-type mice increased chondrocyte proliferation. Mice lacking insulin receptor （IR） selectively in chondrocytes （CartIR-/-） had no discernable differences in total femoral length compared to control littermates. However, CartIR-/- mice exhibited an increase in chondrocyte numbers in the growth plate than that of the controls. Chondrocytes lacking IR had elevated insulin-like growth factor （IGF）-IR mRNA and protein levels. Subsequently, IGF-1 induced phosphorylafion of Akt and ERK was enhanced, while this action was eliminated when the cells were treated with IGF-1R inhibitor Picropodophyllin. Deletion of the IR impaired chondrogenic differentiation, and the effect could not be restored by treatment of insulin, but partially rescued by IGF-1 treatment. Intriguingly, the size of hypertrophic chondrocytes was smaller in CartIR-/- mice when compared with that of the control littermates, which was associated with upregnlation of tuberous sclerosis complex 2 （TSC2）. These results suggest that deletion of the IR in chondrocytes sensitizes IGF-1R signaling and action, IR and IGF-1R coordinate to regulate the proliferation, differentiation and hypertrophy of growth plate chondrocytes.

Chloride channel involved in the regulation of curcumin-induced apoptosis of human breast cancer cells

Objective: To investigate the role of ClC-3 chloride channel in the proliferation of breast cancer cell line Mcf-7 treated with curcumin and its specific mechanism. Methods: MTT assay was used to detect the effect of chloride channel blocker(DIDS) and curcumin on Mcf-7 and human normal cell viability. Patch-clamp technique was used to determine the current density before and after drug treatment. Apoptosis assay by flow cytometry was performed for further examination of cell apoptosis. Results: Curcumin had toxicity on Mcf-7 and HUVEC cells and DIDS reduced the survival rate of Mcf-7 cells by inhibiting proliferation. Curcumin could activate the chloride ion current on MCF-7 cell membrane, which would be inhibited by DIDS.Finally, curcumin in low concentration combined with DIDS could significantly promote the MCF-7 cells apoptosis. Conclusions: Our results suggest that ClC-3 protein is involved in the regulation of curcumin induced proliferation inhibiting in breast cancer cells through inducing cell apoptosis. ClC-3 may be a potential target of tumor therapy.

Extensive exploration of T cell heterogeneity in cancers by single cell sequencing

Human T cells are a highly heterogeneous population and can recognize a wide variety of antigens by their T cell receptors(TCRs). Tumor cells display a large repertoire of antigens that serve as potential targets for recognition,thus making T cells in the tumor micro-environment more complicated. Making a connection between TCRs and the transcriptional information of individual T cells will be interesting for investigating clonal expansion within T cell populations under pathologic conditions. Advances in single cell RNA-sequencing(scRNA-seq) have allowed for comprehensive analysis of T cells. In this review, we briefly describe the research progress on tumor microenvironment T cells using single cell RNA sequencing, and then discuss how scRNA-seq can be used to resolve immune system heterogeneity in health and disease. Finally, we point out future directions in this field and potential for immunotherapy.

Characterization of three-dimensional multipotent adipose-derived stem cell spheroids

Human adipose stem cells(hADSCs)are reliable sources for cell therapy.However,the clinical applications are limited by the decrease in activity during in vitro culture.We used a knockout serum replacement(KSR)medium,Eppendorf(EP)tube culture,and a simulated microgravity(SMG)culture system to establish hADSC spheroids.We found that hADSCs aggregated and formed spheroids in the KSR culture medium.The EP tube culture method revealed many biological cell characteristics,such as good cell viabilities,rough surfaces,polar growth,fusion phenomenon,and injectability.The findings show its advantages for hADSCs spherical cultures.When cultured in SMG,hADSC spheroids produced large-scale spheroids.Additionally,confocal examination and viability assays revealed that SMG-cultured hADSC spheroids had higher cell viabilities and looser spherical structures,relative to those cultured in EP tubes.hADSC spheroids in static EP tube culture had tighter structures and more dead cells with rough and irregular surfaces,while hADSC spheroids in dynamic SMG condition exhibited looser structures and better cell viabilities with flat and regular surfaces.Therefore,the KSR media promotes spherical formation by hADSCs,which showed polar growth,fusion,and injectability in vitro.The dynamic SMG culture enhances the formation of a looser structure and better cell viabilities for hADSC spheroids.

Expression pattern of BIM,BCL-6,and c-MYC in adult B-cell acute lymphoblastic leukemia

Objective We aimed to evaluate the expression pattern of the genes BIM, BCL-6, and c-MYC in adult patients at initial diagnosis of B-cell acute lymphoblastic leukemia(B-ALL).Methods Relative m RNA levels of BIM, BCL-6, and c-MYC in peripheral blood mononuclear cells(PBMCs) from B-ALL patients were determined by quantitative reverse-transcription polymerase chain reaction(q RT-PCR) using SYBR Green dye. PBMCs from healthy volunteers served as a control. GAPDH was used as a reference gene.Results Relative expression of BIM, BCL-6, and c-MYC m RNA in B-ALL patients was significantly lower than in healthy controls(P < 0.05). Furthermore, this result was observed for both newly diagnosed B-ALL patients and those incomplete remission(CR)(P < 0.05). There were no statistically significant differences in the expression levels of BIM, BCL-6, and c-MYC between these B-ALL patient groups(P > 0.05). Spearman's rank correlation analyses revealed the expression level of BIM to be positively correlated with that of BCL-6 in B-ALL patients.Conclusion Expression of the genes BIM, BCL-6, and c-MYC is decreased in adult B-ALL patients. Moreover, the expression pattern of these genes may be similar in such patients at initial diagnosis and following CR. The expression characteristics of BIM, BCL-6, and c-MYC may constitute useful markers for the diagnosis of adult B-ALL.

Hsa_circ_0002137 stabled by LIN28B promotes osteosarcoma cell growth through the hsa-miR-1246/BCL2 axis

Circular RNAs(circRNAs)are a novel class of non-coding RNA that have recently shown to have huge capabilities in the regulation of gene expression at the posttranscriptional level.Growing evidence has indicated that circRNAs could serve as competing endogenous RNAs(ceRNAs)to sponge microRNAs(miRNAs)and suppress functions of targeted miRNAs.Osteosarcoma(OS)is the most common malignant primary bone cancer.Hsa_circ_0002137 is upregulated in OS.However,the role of hsa_circ_0002137 in OS remains unclear.Using miRNA pull-down assay,we showed that cir_0002137 sponged hsa-miR-1246,and BCL2 apoptosis regulator(BCL2)mRNA was a potential target of hsa-miR-1246 in human osteosarcoma(HOS)cells.Further,we found that hsa_cir_0002137 could enhance the expression of BCL2 hsa-miR-1246 and promote HOS cell growth through sponging hsa-miR-1246.Moreover,RNA binding protein immunoprecip itation(RIP)assay revealed that lin-28 homolog B(LIN28B)protein associated with hsa_circ_0002137,and LIN28B could increase hsa_circ_0002137 stability and thus accelerate OS cell growth.Our work was the first to study the functions of hsa_circ_0002137,has-miR-1246 and LIN28B in OS,and these results may provide novel therapeutic targets for OS treatment.

Psychological and Emotional Responses during Different Stages of the COVID-19 Pandemic Based on a Survey of a Mental Health Hotline

Background:The coronavirus(COVID-19)outbreak in 2019 triggered psychological and emotional responses.This research investigates the psychological status and emotional problems of those who sought psychological assistance during the epidemic period by calling a mental health hotline.Methods:This study aims to combine qualitative and quantitative research.Descriptive analysis was used for undertaking qualitative research.We ana-lyzed the data from group 1(n=706),in which the people used the mental health hotline from 25 January 2020 to 23 June 2020.A self-designed questionnaire was developed in accordance with the classification and sum-marized items from group 1’s psychological problems and emotional status.To implement the quantitative research,we conducted a cross-sectional descriptive survey and used the self-compiled scale and HADS to inves-tigate group 2(n=553)from May 2020 to June 2020.Results:Descriptive statistics and comparative analysis revealed that:①Visitors mainly reported behavior,emotional,family relationship problems and sleep disorders.②Anxiety,comorbidities,sleep disorders and coping problems were the most frequently reported problems.③There were significant differences in the number of visitors experiencing various problems or exhibiting harmful behaviors(sorrow,worry,fear,depression,sleep disorders,self-harm or suicide,and coping problems,anxiety,hypochondria,and comorbidity)in the four stages of the epidemic.④More than a quarter of participants still suffered from anxiety or depression in the later stages of the epidemic.Conclusion:Different problems manifested at different stages of the epidemic,and psychological interventions and assistance should be tailored to reflect this.