A Low-Producing Haplotype of Interleukin-6 Disrupting CTCF Binding Is Protective against Severe COVID-19.

Interleukin6 (IL-6) is a key driver of hyperinflammation in COVID-19, and its level strongly correlates with disease progression. To investigate whether variability in COVID-19 severity partially results from differential IL-6 expression, functional single-nucleotide polymorphisms (SNPs) of IL-6 were determined in Chinese COVID-19 patients with mild or severe illness. An Asian-common IL-6 haplotype defined by promoter SNP rs1800796 and intronic SNPs rs1524107 and rs2066992 correlated with COVID-19 severity. Homozygote carriers of C-T-T variant haplotype were at lower risk of developing severe symptoms (odds ratio, 0.256; 95% confidence interval, 0.088 to 0.739; P = 0.007). This protective haplotype was associated with lower levels of IL-6 and its antisense long noncoding RNA IL-6-AS1 by cis-expression quantitative trait loci analysis. The differences in expression resulted from the disturbance of stimulus-dependent bidirectional transcription of the IL-6/IL-6-AS1 locus by the polymorphisms. The protective rs2066992-T allele disrupted a conserved CTCF-binding locus at the enhancer elements of IL-6-AS1, which transcribed antisense to IL-6 and induces IL-6 expression in inflammatory responses. As a result, carriers of the protective allele had significantly reduced IL-6-AS1 expression and attenuated IL-6 induction in response to acute inflammatory stimuli and viral infection. Intriguingly, this low-producing variant that is endemic to present-day Asia was found in early humans who had inhabited mainland Asia since ∼40,000 years ago but not in other ancient humans, such as Neanderthals and Denisovans. The present study suggests that an individual's IL-6 genotype underlies COVID-19 outcome and may be used to guide IL-6 blockade therapy in Asian patients. IMPORTANCE Overproduction of cytokine interleukin-6 (IL-6) is a hallmark of severe COVID-19 and is believed to play a critical role in exacerbating the excessive inflammatory response. Polymorphisms in IL-6 account for the variability of IL-6 expression and disparities in infectious diseases, but its contribution to the clinical presentation of COVID-19 has not been reported. Here, we investigated IL-6 polymorphisms in severe and mild cases of COVID-19 in a Chinese population. The variant haplotype C-T-T, represented by rs1800796, rs1524107, and rs2066992 at the IL-6 locus, was reduced in patients with severe illness; in contrast, carriers of the wild-type haplotype G-C-G had higher risk of severe illness. Mechanistically, the protective variant haplotype lost CTCF binding at the IL-6 intron and responded poorly to inflammatory stimuli, which may protect the carriers from hyperinflammation in response to acute SARS-CoV-2 infection. These results point out the possibility that IL-6 genotypes underlie the differential viral virulence during the outbreak of COVID-19. The risk loci we identified may serve as a genetic marker to screen high-risk COVID-19 patients.

[Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on silent information regulator-1/nuclear factor κB signaling pathway in gastric antrum of diabetic gastroparesis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on silent information regulator-1 (SIRT1)/nuclear factor kappa B (NF-κB) signal pathway and inflammatory factor expression in gastric antrum tissue of diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. METHODS: Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+moxibustion groups (n=12 per group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension. EA (10 Hz /50 Hz, 2 mA) or moxibustion (3 cones) or EA+moxibustion was applied to "Zhongwan"(CV12), bilateral "Neiguan"(PC6) and bilateral "Sanyinjiao"(SP6) of the related group for 20 min, once a day for 3 weeks. Blood glucose, gastric emptying rate and intestinal propulsion rate were measured. The levels of serum interleukin (IL)-6, IL-8, IL-10 and tumor necrosis factor-α(TNF-α) were detected by ELISA; the phosphorylation level of the phosphorylated inhibitor of nuclear factor κBα inhibitor (pIκ-Bα), the protein and mRNA expression of NF-κB p65 and SIRT1 in the gastric antrum tissue were detected by Western blot and real-time quantifitative PCR, respectively. RESULTS: (1) Compared with the normal group, the levels of blood glucose, serum IL-6, IL-8, TNF-α, and gastric pIκ-Bα and NF-κB p65 protein and mRNA expressions were significantly increased (P<0.01), and the gastric emptying rate, intestinal propulsion rate, serum IL-10 level, and SIRT1 protein and mRNA expressions were considerably decreased in the model group (P<0.01). (2) In contrast to the model group, the blood glucose in the EA, moxibustion and EA+moxibustion groups, serum IL-6, IL-8 and TNF-α levels in the 4 treatment groups, as well as NF-κB p65 protein expression in the medication and EA+moxibustion groups, and NF-κB p65 mRNA expression and pIκ-Bα protein and mRNA expression in the 4 treatment groups were significantly decreased (P<0.01, P<0.05); while the gastric emptying rate and intestinal propulsive rate and IL-10 content in the 4 treatment groups, and SIRT1 protein and mRNA expression in the medication and EA+moxibustion groups were obviously increased (P<0.05, P<0.01). (3) The effects of EA+moxibustion were significantly superior to those of simple EA and moxibustion in increasing gastric emptying rate, IL-10, SIRT1 protein expression (P<0.05, P<0.01), and in lowering IL-8 and TNF-α contents, pIκ-Bα protein and mRNA expression and NF-κB p65 mRNA expression (P<0.05, P<0.01). No significant differences were found among the 4 intervention groups in promoting the intestinal propulsive rate and among the EA, moxibustion and EA+moxibustion groups in lowering blood glucose (P>0.05). CONCLUSION: EA combined with Zhuang-medicine-thread moxibustion can effectively reduce the level of serum inflammatory factors and regulate SIRT1/NF-κB signal pathway in DGP rats, which may contribute to its function in improving gastrointestinal movement.

lncRNA NEAT1 aggravates sepsis-induced lung injury by regulating the miR-27a/PTEN axis.

Sepsis is an acute inflammatory reaction and a cause of acute respiratory distress syndrome (ARDS). In the present study, we explored the roles and underlying mechanism of the lncRNA Nuclear enriched abundant transcript 1 (NEAT1) in ARDS. The expression levels of genes, proteins and pro-inflammatory cytokines in patients with ARDS, LPS-stimulated cells and septic mouse models were quantified using qPCR, western blotting and ELISA assays, respectively. The molecular targeting relationship was validated by conducting a dual-luciferase reporter assay. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. The cell cycle phase was determined by flow cytometry assay. The expression levels of NEAT1 and pro-inflammatory cytokines were higher in patients with ARDS and septic models than in controls. Knockdown of NEAT1 significantly increased cell proliferation and cycle progression and prolonged mouse survival in vitro and in vivo. Mechanistically, miR-27a was identified as a downstream target of NEAT1 and directly inhibited PTEN expression. Further rescue experiments revealed that inhibition of miR-27a impeded the promoting effects of NEAT1 silence on cell proliferation and cycle progression, whereas inhibition of PTEN markedly weakened the inhibitory effects of NEAT1 overexpression on cell proliferation and cycle progression. Altogether, our study revealed that NEAT1 plays a promoting role in the progression of ARDS via the NEAT1/miR-27a/PTEN regulatory network, providing new insight into the pathologic mechanism behind ARDS.

The KBTBD6/7-DRD2 axis regulates pituitary adenoma sensitivity to dopamine agonist treatment.

Pituitary adenoma (PA) is one of the most common intracranial tumors, and approximately 40% of all PAs are prolactinomas. Dopamine agonists (DAs), such as cabergoline (CAB), have been successfully used in the treatment of prolactinomas. The expression of dopamine type 2 receptor (DRD2) determines the therapeutic effect of DAs, but the molecular mechanisms of DRD2 regulation are not fully understood. In this study, we first demonstrated that DRD2 underwent proteasome-mediated degradation. We further employed the yeast two-hybrid system and identified kelch repeat and BTB (POZ) domain containing 7 (KBTBD7), a substrate adaptor for the CUL3-RING ubiquitin (Ub) ligase complex, as a DRD2-interacting protein. KBTBD6/7 directly interacted with, and ubiquitinated DRD2 at five ubiquitination sites (K221, K226, K241, K251, and K258). CAB, a high-affinity DRD2 agonist, induced DRD2 internalization, and cytoplasmic DRD2 was degraded via ubiquitination under the control of KBTBD6/7, the activity of which attenuated CAB-mediated inhibition of the AKT/mTOR pathway. KBTBD7 knockout (KO) mice were generated using the CRISPR-Cas9 technique, in which the static level of DRD2 protein was elevated in the pituitary gland, thalamus, and heart, compared to that of WT mice. Consistently, the expression of KBTBD6/7 was negatively correlated with that of DRD2 in human pituitary tumors. Moreover, KBTBD7 was highly expressed in dopamine-resistant prolactinomas, but at low levels in dopamine-sensitive prolactinomas. Knockdown of KBTBD6/7 sensitized MMQ cells and primary pituitary tumor cells to CAB treatment. Conversely, KBTBD7 overexpression increased CAB resistance of estrogen-induced in situ rat prolactinoma model. Together, our findings have uncovered the novel mechanism of DRD2 protein degradation and shown that the KBTBD6/7-DRD2 axis regulates PA sensitivity to DA treatment. KBTBD6/7 may thus become a promising therapeutic target for pituitary tumors.

Immunotherapeutic effects of allogeneic mesenchymal stem cells on systemic lupus erythematosus.

Mesenchymal stem cells have been applied to treat graft versus host disease as they have immunosuppressive ability and can overcome the major histocompatibility complex-histocompatibility barrier. The potential of allogeneic mesenchymal stem cells in treating systemic lupus erythematosus (SLE) was investigated in this study. MRL/lpr mice which can develop acquired SLE-like phenotypes were selected as an animal model. Mesenchymal stem cells obtained from green fluorescent protein-transgenic ICR mice were infused into MRL/lpr mice at either the early or late stage of disease. The dosage was 1 × 106/mice per infusion. Mice were stratified into six groups including negative controls and those receiving one, two, three, four or five doses at 2-weekly intervals. The phenotypes were monitored regularly. After treatment, the spleen CD3+CD4-CD8- T and CD19+ B cells of two-dose mesenchymal stem cell-treated mice were significantly lower than those of the phosphate-buffered saline control. In terms of reducing the severity of SLE such as hair loss, skin ulcers, proteinuria and anti-dsDNA level, mesenchymal stem cells given at the early stage responded better and mice receiving two doses of mesenchymal stem cells performed better than those receiving either a lower dose (one dose) or higher doses (three, four or five doses). In conclusion, early treatment and an optimal dose of mesenchymal stem cells can effectively suppress the murine SLE model.

Osteogenic Ability Detection of Human Bone Morphogenetic Protein-2 Gene-activated Nano Bone Putty by Reusable Double-Cavity Bone Harvest Chamber.

OBJECTIVE: To explore the feasibility of implanting a self-designed reusable double-cavity bone harvest chamber into Guizhou mini-pigs for observation of the osteogenic effect of human bone morphogenetic protein-2 (hBMP-2) gene-activated nano bone putty on bone in growth. METHODS: Eight healthy 12-month-old female Guizhou mini-pigs were used for the present experiment. In the first operation, empty double-cavity bone harvest chambers (n = 8) were implanted into the femoral metaphysis of the animals as a blank control group. In the second operation, the femoral metaphyses were implanted with the chambers filled by the nano bone putty+hBMP-2 plasmid in one cavity and nothing in the other cavity, respectively (experiment group, n = 8). The time interval between every operation was 3 months. The cavity materials were retrieved and replaced for assessment by gross observation, histological examination, and bone morphology metrology analysis to compare osteogenesis ability and alkaline phosphatase. RESULTS: Three months after surgery, the nano bone putty+hBMP-2 plasmid in one cavity of the chambers had hard gray and white tissues inside, while the cavities pre-installed with nothing were filled with soft brown tissues. Light microscopy showed new generated bone tissue around the filled material, but only fibrous tissues in the empty cavities. Osteogenesis ability and alkaline phosphatase of the nano bone putty+hBMP-2 plasmid group were significantly higher than those of the blank control group (P < 0.05). CONCLUSION: The reusable double-cavity bone harvest chamber can be used to observe the osteogenic potential of the hBMP-2 gene-activated nano bone putty.

[Plasma from patients with systemic lupus erythematosus inhibits suppressive activity of mesenchymal stem cells against lupus B lymphocytes].

OBJECTIVE: To investigate whether plasma from patients with systemic lupus erythematosus (SLE) inhibits the suppressive effects of mesenchymal stem cells (MSCs) on lupus B lymphocytes. METHODS: MSCs isolated and expanded from the bone marrow of healthy donors were co-cultured with B cells purified from the peripheral blood of SLE patients in the presence of fetal bovine serum or pooled plasma from SLE patients, and the proliferation and maturation of the B lymphocytes were analyzed. RESULTS: s Co-culture with normal MSCs obviously inhibited the proliferation of lupus B cells and suppressed the maturation of B lymphocytes, which showed lowered expressions of CD27 and CD38. The pooled plasma from SLE patients significantly inhibited the suppressive effects of normal MSCs on B cell proliferation and maturation. CONCLUSION: Plasma from SLE patients negatively modulates the effects of normal MSCs in suppressing lupus B cell proliferation and maturation to affect the therapeutic effect of MSC transplantation for treatment of SLE. Double filtration plasmapheresis may therefore prove beneficial to enhance the therapeutic effects of MSC transplantation for SLE.

Exploration of the regulatory effect of miR-21 on breast cancer cell line proliferation and invasion as well as the downstream target genes.

OBJECTIVE: To study the regulatory effects of miR-21 on breast cancer cell line proliferation and invasion as well as the downstream target genes. METHODS: Breast cancer cell lines MCF-7 were cultured and transfected with miR-21 mimics and the corresponding negative control mimics (NC mimics), and then MTS kits were used to detect cell viability. Transwell experiment was used to detect cell invasion ability, and fluorescence quantitative PCR was used to detect the expression of proliferation and invasion-related genes in cells. RESULTS: 24 h after transfection of miR-21 mimics and NC mimics, cell OD value and the number of invasive cells of miR-21 group were significantly higher than those of NC group, and mRNA contents of PDCD-4, FasL, PTEN, RhoB, Maspin, TIMP3 and RECK in cells were significantly lower than those of NC group. CONCLUSION: miR-21 can promote the proliferation and invasion of breast cancer cell lines, and its downstream target genes include PDCD-4, FasL, PTEN, RhoB, Maspin, TIMP3 and RECK.