Tooth pulpitis is a prevalent oral disorder. Understanding the underlying mechanisms of pulpitis and developing effective treatment strategies hold great significance. Ferroptosis has recently emerged as a new form of cell death, but the role of ferroptosis in pulpitis remains largely unknown. In our study, single-cell RNA sequencing (scRNA-seq) was used to identify cellular heterogeneity between 3 pulpitis tissue and 3 healthy pulp tissue, and explored ferroptosis occurrence in pulpitis tissue and inflamed dental pulp cells (DPCs). In scRNA-seq, 40 231 cells (Pulpitis: 17 814; Healthy pulp: 22 417) were captured, and visualized into 12 distinct cell clusters. Differentially expressed ferroptosis-related genes (DE-FRGs) were almost presented in each cluster in pulpitis vs healthy pulp. ROS and Fe²⁺ levels significantly rose, and immunohistochemistry showed low expression of GPX4 and high expression of PTGS2 in pulpitis. In LPS-stimulated DPCs, thymosin α1 increased the expression of GPX4 and FTL, and decreased expression of TNF-α, IL-1β, IL-6, and Fe²⁺ levels. In rat pulpitis models, both prothymosin α (PTMA, precursor of thymosin α1) gelatin sponge placed at the hole of pulp (LPS-P(gs)) and PTMA injection in pulp (LPS-P(i)) significantly reduced infiltration of inflammatory cells and expression of PTGS2, and increased the expression of GPX4. In RNA sequencing, the expression of DE-FRGs were reversed when thymosin α1 were added in LPS-stimulated DPCs. Collectively, single-cell atlas reveals cellular heterogeneity between pulpitis and healthy pulp, and ferroptosis occurrence in pulpitis. Thymosin α1 may reduce ferroptosis in DPCs to alleviate pulpitis and thus potentially has the ability to treat pulpitis.
Ferroptosis/drug effects* ; Dental Pulp/drug effects* ; Animals ; Pulpitis/pathology* ; Rats ; Thymalfasin/pharmacology* ; Humans ; Male ; Thymosin/pharmacology* ; Disease Models, Animal ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the role and mechanism of thymosin beta 4 (Tβ4) in oxidative stress injury of spinal cord-derived neural stem/progenitor cells (NSPCs) induced by hydrogen peroxide (H₂O₂). METHODS: NSPCs were isolated from Sprague-Dawley (SD) adult male rats, and divided into control group (untreated NSPCs cells), H₂O₂ group (NSPCs cells damaged by 500 μM H₂O₂), Tβ4 -3 groups (NSPCs were treated with 1, 2.5, 5 μg/ml Tβ4 on the basis of H₂O₂ treatment) and TAK-242 group [NSPCs were treated with 5 μg/ml Tβ4 and Toll-like receptor 4(TLR4) inhibitor TAK-242 on the basis of H₂O₂ treatment]. NSPCs were transfected with lentivirus vector of myeloid differentiation factor 88(MyD88) to construct MyD88-overexpressing cell lines, which were treated with H₂O₂ and Tβ4. The expression of Tβ4, TLR4, MyD88 were detected by qRT-PCR and Western blot. Cell viability was detected by MTT assay and lactate dehydrogenase(LDH) assay kit. Ca2+ concentration was detected by Fluo-3/AM probe method. The apoptosis of NSPCs was detected by flow cytometry and Caspase-3 and Caspase-9 kits;reactive oxygen species (ROS), superoxi dedismu-tase dismutase(SOD) activity and glutathione (GSH) content were detected by corresponding kits. Interleukin(IL)-6 and IL-1β were detected by enzyme-linked immunosorbent assay. RESULTS: The expression of Tβ4 was decreased in H₂O₂ injured NSPCs(P<0.05). Compared with H₂O₂ group, the cell viability and Ca2+ concentration was significantly increased, release of LDH and apoptosis were significantly decreased, production of ROS and pro-inflammatory cytokines were significantly decreased, and the expression levels of TLR4 and MyD88 protein were significantly decreased in Tβ4-3 groups and TAK-242 group (P<0.05). After overexpression of MyD88, cell viability, SOD activity and GSH content of NSPCs decreased, LDH release and apoptosis increased significantly (P<0.05), while after treatment with Tβ4, cell viability, SOD activity and GSH content increased, LDH release and apoptosis decreased (P<0.05). CONCLUSION Tβ4 attenuates H₂O₂-induced NSPCs oxidative stress, apoptosis and inflammation in NSPCs via inhibiting TLR4 and MyD88 pathways.
Animals ; Apoptosis ; Calcium/pharmacology* ; Cell Survival ; Hydrogen Peroxide/pharmacology* ; Male ; Myeloid Differentiation Factor 88/pharmacology* ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/pharmacology* ; Spinal Cord Injuries/drug therapy* ; Stem Cells ; Superoxide Dismutase/pharmacology* ; Thymosin/metabolism* ; Toll-Like Receptor 4/metabolism*

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
Animals ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Nanog Homeobox Protein ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Patch-Clamp Techniques ; Primary Cell Culture ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Thymosin ; pharmacology

OBJECTIVETo study the regulatory effect of thymosin α1 (Tα1) on immunosuppression of bone marrow mesenchymal stem cells (MSCs) from children with aplastic anemia (AA) through Toll-like receptor 9(TLR9)and indoleamine 2,3-dioxygenase (IDO) signaling pathway.

METHODBone marrow T cell subsets from children with AA and normal individuals were measured by using flow cytometry. Expressions of TLR9/IDO mRNA of MSCs cocultured with Tα1 were determined by reverse-transcription PCR (RT-PCR). Inhibition of PHA-activated T cell proliferation and activation by MSCs cocultured with Tα1 was detected by using MTT assay and flow cytometry.

RESULTCD4(+)/CD8(+) ratio (0.64 ± 0.02) in children with AA was significantly lower than that in normal individuals (1.42 ± 0.05); but CD8(+)/CD38(+) ratio (0.92 ± 0.04) was significantly higher than that in normal individuals (0.65 ± 0.05). AA MSCs obviously expressed TLR9, but not IDO; AA MSCs treated with Tα1 downregulated TLR9 expression but upregulated IDO expression in concentration- and time-dependent manners. The inhibition of AA MSCs on T cell proliferation (21.38% ± 12.34%) was lower than that in normal individuals (62.72% ± 17.79%, P < 0.05), while AA MSCs treated with Tα1 for 18 h exhibited a stronger inhibition (42.83% ± 16.54%, P < 0.05).

CONCLUSIONThe immunosuppression mediated by MSCs could be improved by Tα1 through upregulation of IDO expression via TLR9-dependent signaling pathway. This research provides a new idea for targeted immunomodulatory therapy with bone marrow MSCs from children with AA.

Adolescent ; Anemia, Aplastic ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; Case-Control Studies ; Child ; Female ; Humans ; Immune Tolerance ; Immunosuppression ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Male ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Signal Transduction ; immunology ; Thymosin ; analogs & derivatives ; pharmacology ; Toll-Like Receptor 9 ; metabolism

The purpose of this study was to explore the regulatory effect of thymosin α1 (Tα1) on expression of TOLL-like receptor 9 (TLR9)/indoleamine2, 3-dioxygenase (ido) mRNA in bone marrow mesenchymal stem cells (MSC) from children with aplastic anemia (AA). Culture system of bone marrow MSC from AA children and normal children in vitro was established, and the effects of Tα1 on expressions of tlr9 mRNA and ido mRNA of MSC from AA children and normal children were determined by RT-PCR. The results showed that the bone marrow MSC from normal children did not express tlr9 and ido mRNA. Bone marrow MSC from children with AA obviously expressed tlr9 mRNA , but did not express ido mRNA; AA children's MSC treated with Tα1 for 18 hours markedly down-regulated tlr9 mRNA expression, but up-regulated ido mRNA expression in the concentration- and time-dependent ways. It is concluded that Tα1 can up-regulate the expression of ido mRNA in bone marrow MSC from children with AA.
Adolescent ; Anemia, Aplastic ; metabolism ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; Child ; Female ; Gene Expression Regulation ; HL-60 Cells ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Male ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; RNA, Messenger ; genetics ; Thymosin ; analogs & derivatives ; pharmacology ; Toll-Like Receptor 9 ; metabolism

Adolescent ; Adult ; Case-Control Studies ; Dendritic Cells ; drug effects ; Female ; Hepatitis B, Chronic ; Humans ; Male ; Thymosin ; pharmacology ; Young Adult

OBJECTIVETo investigate the effect of Thymosin and growth hormone(GH) on inflammatory response in burn rats or burn rats with sepsis.

METHODSSixty-four SD rats were randomly divided into normal control group (NC, without treatment), sepsis group (S, with injection of LPS), sepsis + Thymosin group (ST, with successive injection of Thymosin and LPS), sepsis + GH group [SGH, with successive injection of recombinant human GH (rhGH) and LPS], burn group, burn + sepsis group (BS, with injection of LPS after burn), burn + sepsis + Thymosin group (BST, with successive injection of Thymosin and LPS after burn), burn + sepsis + GH (BSGH, with successive injection of rhGH and LPS after burn), with 8 rats in each group. Specimens of spleen tissues were harvested to determine HLA-DR in lymphocyte and evaluate inflammatory cell infiltration (score). Specimens of peripheral blood were collected to determine Toll-like receptor 4 (TLR4) level in monocyte and serum level of TNF-alpha, IL-4, IL-6, IL-10.

RESULTSCompared with those in NC group, serum level of IL-10 in S group decreased obviously, while other indices increased obviously (P < 0.01). The levels of HLA-DR and TLR4 and serum level of TNF-alpha were similar between SGH and ST groups (P > 0.05). Compared with those in SGH group [(2.87 +/- 0.04) score, and IL-6 (0.0083 +/- 0.0018) microg/mg, IL-4 (0.0102 +/- 0.0021) microg/mg, IL-10 (0.0310 +/- 0.0027) microg/mg, respectively], degree of inflammatory cell infiltration (1.50 +/- 0.76) score and serum levels of IL-6, IL-4, IL-10 of rats in ST group decreased obviously (0.0064 +/- 0.0012, 0.0058 +/- 0.0024, 0.0230 +/- 0.0021 microg/mg, respectively, P < 0.01). The levels of HLA-DR, TLR4 and inflammatory cell infiltration degree of spleen in B group were respectively higher than those in NC group and lower than those in BS group. Compared with those in NC group, serum levels of TNF-alpha, IL-6 in B group increased significantly, while IL-4, IL-10 showed an opposite tendency. There was no obvious difference between BST and BSGH groups in serum levels of HLA-DR and IL-6 (P > 0.05). Compared with those in BST group, inflammatory cell infiltration degree in spleen and the levels of TLR, TNF-alpha obviously decreased (P < 0.01), while IL-4 and IL-10 levels increased in BSGH group (P < 0.01).

CONCLUSIONSInhibitive effects between Thymosin and GH on extensive inflammatory reaction were similar with or without trauma, and GH has better effect as compared with Thymosin when with trauma.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Burns ; immunology ; Human Growth Hormone ; pharmacology ; Inflammation ; immunology ; Male ; Rats ; Rats, Sprague-Dawley ; Sepsis ; immunology ; Thymosin ; pharmacology

This study aimed to obtain recombinant fusion protein of thymosin alphal(TM-alpha1) and consensus IFNalpha (IFNalpha-con) which have bath TM-alpha1 and IFNalpha-con activities. The DNA sequence for the fusion protein was cloned into expression vector of pET-22b (+) and expressed in BL21 (DE3)-Codon plus-RP-X. The expressed product (TM-alpha1-IFN-con) was soluble, and amounted to more than 20% in total proteins of E. coli. By precipitation of (NH4)2SO4, hydrophobic interaction chromatography (HIC, Phenyl Sepharose 6 Fast Flow), anion-exchange chromatography (Q Sepharose Fast Flow), cation-exchange chromatography (SP Sepharose Fast Flow) and gel filtration (Sephadex G-75), it was purified to more than 96% purity. The activity of fusion protein for antivirus was tested by cytopathic-effect inhibition assay and activity for promoting lymphocyte proliferation was tested by cell proliferative assay. The activity for antivirus was higher than commercial IFNalpha1b and IFNalpha2a and activity for promoting lymphocyte proliferation was similar to commercial TM-alpha1. The fusion protein had better effect for anti-HBV in vitro, its effect was stronger than combination of IFNalpha and TM-alpha1 and cell toxicity was less than combination of IFNalpha and TM-alpha1. The above results show that it has effect bath antivirus of IFNalpha and promoting lymphocyte proliferation of the soluble fusion protein expressed in E. coli.
Amino Acid Sequence ; Animals ; Antiviral Agents ; pharmacology ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Humans ; Interferon Type I ; biosynthesis ; genetics ; Interferon-alpha ; Mice ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Recombinant Proteins ; Thymosin ; analogs & derivatives ; biosynthesis ; genetics

BACKGROUNDTo evaluate the efficacy of alpha-2b interferon alone or combined with alpha1-thymosin in treatment of patients with chronic hepatitis B (CHB) resistant to lamivudine.

METHODSSixty six patients with CHB resistant to lamivudine were enrolled and randomized into treatment group A, treatment group B and control group. In the treatment group A 26 cases, after giving interferon-alpha alone for 1 month, lamivudine was withdrawn and continuously treated with interferon-alpha for 5 months. In the treatment group B 10 cases, after treatment with interferon-alpha and thymosin-alpha1 for 1 month, lamivudine was withdrawn and continuously treated with interferon-alpha and thymosin-alpha1 for 5 months. In control group (30 cases), after lamivudine was directly withdrawn, no anti-virus drug was given. Hepatic function tests and serum virological index were detected at regular intervals in all patients.

RESULTSNormalization rate of hepatic function, HBV DNA seroconversion rate and HBeAg/HBeAb seroconversion rate in two treatment groups were significantly higher than those in control group.

CONCLUSIONThe study suggested that interferon-alpha alone or combined with thymosin-alpha1 in treatment of patients with chronic hepatitis B resistant to lamivudine showed a beneficial effect.

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; genetics ; Drug Resistance, Viral ; Drug Therapy, Combination ; Female ; Hepatitis B Antibodies ; blood ; Hepatitis B e Antigens ; immunology ; Hepatitis B virus ; drug effects ; genetics ; immunology ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; therapeutic use ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; Recombinant Proteins ; Thymosin ; therapeutic use ; Treatment Outcome

OBJECTIVETo investigate the anti-HBV effect of fusion protein thymosin alpha1-interferon alpha (TA1-IFN) in vitro and to compare its effect with a combination of interferon alpha and thymosin alpha1.

METHODSAfter 2.2.15 cells were seeded for 24 hours, drugs of five serial concentrations (8000, 4000, 2000, 1000, 500 U/ml) were added to the wells, then the medium was changed every three days. After 2.2.15 cells were treated with drugs for 6 days, the medium was collected. The inhibitory rates on HBsAg and HBeAg were determined using Abbot kit, and the cytotoxicity of different drugs by means of MTT colorimetric assays was also observed.

RESULTSThe inhibitory rate of fusion protein on HBsAg, HBeAg was dose-dependent and reached the maximum at 8000 U/ml concentration. In the meantime, the inhibitory rates of fusion protein on HBsAg and HBeAg were 72.2% +/- 0.8% and 60.4% +/- 1.1% respectively, and the cell survival rate was 85.2% +/- 2.0%; In the corresponding concentration, the inhibitory rates of combination thymosin alpha 1 and interferon alpha on HBsAg and HBeAg were 40.0% +/- 0.7%, 34.5% +/- 3.2% respectively. The results showed significant statistical differences between them; cell survival rate 70.0% +/- 1.9%, and the difference of the results was also significant. Cytotoxicity of fusion protein was weaker than a combination of thymosin alpha 1 and interferon alpha.

CONCLUSIONFusion protein TA1-IFN exerted stronger anti-HBV effects in vitro. Its anti-HBV effects in vitro were stronger than the combination of thymosin alpha and interferon alpha, and its cytotoxicity was weaker than the combination of thymosin alpha and interferon alpha. Our studies provided important evidence for clinical research on TA1-IFN, and also brought new hope for hepatitis B therapy.

Antiviral Agents ; pharmacology ; Hepatitis B virus ; drug effects ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Thymosin ; biosynthesis ; genetics ; pharmacology