Deficiency of two-pore segment channel 2 contributes to systemic lupus erythematosus via regulation of apoptosis and cell cycle

Systemic lupus erythematosus (SLE) is a complex autoimmune disease, and the mechanism of SLE is yet to be fully elucidated. The aim of this study was to explore the role of two-pore segment channel 2 (TPCN2) in SLE pathogenesis.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression of TPCN2 in SLE. We performed a loss-of-function assay by lentiviral construct in Jurkat and THP-1 cell.
Knockdown of TPCN2 were confirmed at the RNA level by qRT-PCR and protein level by Bio Med Frontiers Western blotting. Cell Count Kit-8 and flow cytometry were used to analyze the cell proliferation, apoptosis, and cell cycle of TPCN2-deficient cells. In addition, gene expression profile of TPCN2-deficient cells was analyzed by RNA sequencing (RNA-seq).
TPCN2 knockdown with short hairpin RNA (shRNA)-mediated lentiviruses inhibited cell proliferation, and induced apoptosis and cell-cycle arrest of G2/M phase in both Jurkat and THP-1 cells.
We analyzed the transcriptome of knockdown-TPCN2-Jurkat cells, and screened the differential genes, which were enriched for the G2/M checkpoint, complement, and interleukin-6-Janus kinase-signal transducer and activator of transcription pathways, as well as changes in levels of forkhead box O, phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin, and T cell receptor pathways; moreover, TPCN2 significantly influenced cellular processes and biological regulation.

Effect of Ultraviolet B Irradiation on Melanin Content Accompanied by the Activation of p62/GATA4-Mediated Premature Senescence in HaCaT Cells

  • To explore the effect and mechanism of ultraviolet B (UVB) on melanin synthesis and premature senescence in human immortalized keratinocytes (HaCaT) cells. 
  • HaCaT cells were irradiated with 0, 20, 50, 80, 100, 150, and 200 mJ/cm2 of UVB. NaOH method was used for melanin content assay, cellular tyrosinase (TYR) activity was determined by 3,4-Dihydroxy-L-phenylalanine (L-DOPA) oxidation to dopachrome, premature senescence was analyzed by senescence-associated beta-galactosidase (SA-β-gal) staining kit, and the levels of p21, p16, p62, and GATA4 proteins were detected by Western blotting.
  • Premature senescence was inhibited by the inhibitors of ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad3-related (ATR), and the p53 signaling pathway was activated by Nutlin-3.
  • The mRNA levels of senescence-associated secretory phenotype (SASP) factors including tumor necrosis factor alpha (TNF-α), Learn More vascular endothelial growth factor A (VEGF-A), and interleukin-8 (IL-8) were measured by real-time quantitative polymerase chain reaction in HaCaT cells after 80 mJ/cm2 of UVB irradiation.
  • The melanin level increased significantly with the elevation of irradiation dose (F = 28.19, 43.82, 143.60, P < .05), reaching the peak at the dose of 80 mJ/cm2.
  • The tyrosinase activity increased significantly (F = 84.50, P < .05), the percentage of premature senescence increased (F = 16.31, P < .05), the levels of p62 decreased, and the level of GATA4 increased obviously with the increase of UVB dose after irradiation.
  • The UVB-induced promotion of GATA4 level was significantly inhibited by being treated with ATM or ATR inhibitor. However, this did not occur in the Nutlin-3-treated group. The mRNA and protein expression of TNF-α increased significantly at 72 h at 80 mJ/cm2 of UVB irradiation.
Melanin contents increased first and decreased afterward with the increase of UVB irradiation. The decrease of p62-mediated selective autophagy was accompanied by the accumulation of GATA4 after different doses of UVB irradiation. Activation of this p62/GATA4 pathway depends on the ATM and ATR but is independent of p53, and the SASP factor was activated in HaCaT cells at 80 mJ/cm2 of UVB irradiation.

Ponicidin attenuates streptozotocin-induced diabetic nephropathy in rats via modulating hyperlipidemia, oxidative stress, and inflammatory markers

The present research work was proposed to discover the beneficial roles of ponicidin against the streptozotocin (STZ)-induced diabetic nephropathy (DN) in rats via modulating the oxidative stress and inflammation.
The DN was initiated to the Wistar rats via administering 45 mg/kg of STZ and then diabetic animals were supplemented with 50 mg/kg of ponicidin and 150 mg/kg of metformin (standard drug) for 8 weeks.
The body weight and food intake of animals were checked every week.
The glucose, insulin, and homeostasis model assessment- insulin resistance (HOMA-IR) levels in the serum were assessed using kits.
The levels of reactive oxygen species (ROS) accumulation, oxidative stress and antioxidant markers, and pro-inflammatory cytokines were examined using assay kits.
The levels of lipid profiles and renal function markers were investigated using respective kits. The renal tissues were analyzed microscopically to detect the histological alterations.
The ponicidin treatment effectively decreased the body weight, food intake, HOMA-IR, and HbAlc levels in the DN animals.
The levels of ROS and MDA were decreased and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities were improved by the ponicidin. The ponicidin also reduced the blood urea nitrogen (BUN), creatinine, lactate dehydrogenase (LDH), and kidney injury molecule (KIM-1) levels.
The levels of low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), free fatty acid (FFA), and total cholesterol (TC) were decreased and the high-density lipoprotein (HDL) level was improved by the ponicidin treatment to the DN rats.
The tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), nuclear factor-kappa B (NF-κB), and IL-6 levels were appreciably attenuated by the ponicidin. The ponicidin also ameliorated the DM-provoked histological alterations in the renal tissues.
In conclusion, this study work evidenced that ponicidin has the therapeutic action in ameliorating the development of DN via averting oxidative stress, inflammation, and renal injury. It could be a promising therapeutic agent to treat DN in the future.

Network Pharmacology Combined with Molecular Docking and Experimental Verification Reveals the Bioactive Components and Potential Targets of Danlong Dingchuan Decoction against Asthma

Danlong Dingchuan Decoction has a definite effect in the clinical treatment of asthma. This study aimed to explore the material and molecular biological basis of Danlong Dingchuan Decoction in treating asthma through network pharmacology combined with animal experiments.
 First, the chemical constituents of Danlong Dingchuan Decoction were screened from the Traditional Chinese Medicine Systematic Pharmacology Analysis Platform (TCMSP) and the Traditional Chinese Medicine and Chemical Composition Database.
Literature reports on asthma targets were obtained from the Online Mendelian Inheritance in Man (OMIM), Therapeutic Targets Database (TTD), and other databases.
Then, the protein-protein interaction network was constructed according to the matching results of Danlong Dingchuan Decoction and asthma targets.
Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID).
Finally, the interaction between the active compounds of Danlong Dingchuan Decoction and key targets was simulated using molecular docking. In animal experiments, ovalbumin was used to induce asthma in mice.
After treating the mice by oral gavage administration of Danlong Dingchuan Decoction, the expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected in the lung tissue of the mice by enzyme-linked immunosorbent assay kit, whereas TLR4 mRNA expression was detected by quantitative reverse transcription-polymerase chain reaction.
A total of 247 active compounds and 155 potential targets were obtained.
Enrichment analysis showed that quercetin, xanthine, lysine, kaempferol, ß-sitosterol, and four other active compounds were the main components of Danlong Dingchuan Decoction; IL-6, TNF, CXCL8, VEGFA, MAPK3, IL-10, PTGS2, IL-1β, IL-4, and TLR4 were the potential targets for therapy.
KEGG analysis showed that the cAMP signaling pathway, cGMP-PKG signaling pathway, NF-κB signaling pathway, and PI3K-Akt signaling pathway might play an important role in treating asthma.
Molecular docking analysis showed that quercetin combined well with TNF, CXCL8, and TLR4. Animal experiments showed that Danlong Dingchuan Decoction effectively reduced the expression levels of TNF-α, IL-4, TGF-ββ in the lung tissue of asthmatic mice and inhibited TLR4 mRNA expression.

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 Danlong Dingchuan Decoction may act on key targets (such as IL-6, TNF, CXCL8, VEGFA, and MAPK3) with key active ingredients (such as quercetin, xanthine, lysine, kaempferol, and ß-sitosterol) to reduce the expression levels of IL-4, IL-6, IL-8, and other Th2 cytokines.
This may be the mechanism by which Danlong Dingchuan Decoction reduces airway inflammation and treats asthma mediated by Th2 cytokines.

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