Effect of combination antiviral therapy on hematological profiles in 151 adults hospitalized with severe coronavirus disease 2019.

OBJECTIVES: The current diagnosis and medicines approach in coronavirus disease 2019 (COVID-19) does not reflect the heterogeneous characteristics of this disease. This study aims to find a new antiviral combination regimen by investigating the frequency of clinically relevant and objectively identified comorbidities, and the clustering of these clinical syndromes and varying results of treatment with antiviral drugs in patients hospitalized with severe COVID-19. METHODS: This study recruited 151 severe COVID-19 infection cases diagnosed in our hospital examination and illustrated the clinical potential during a consecutive 25-day medication period. Potential differences in disease severity and clinical characteristics, hematological profile, and current pharmacologic treatments (single agent, double or triple combinations, and the combined antiviral drugs plus Lianhua Qingwen) among comorbidity clusters were explored. RESULTS: Although disease severity was comparable among three clusters, it was markedly different in terms of laboratory test status. Coagulable abnormality was mainly present in cluster 1 and cluster 2. Other indicators were normal, except for a significant increase of neutrophils presented in cluster 2. Patients showed the most complicated haematological results in cluster 3, including severe coagulation abnormalities, leukocytosis, neutrophilic granulocytosis, and lymphopenia. Our results for the first time suggest that a quadruple combination therapy (Ribavirin, Lopinavir/ritonavir, Umifenovir, and Lianhua Qingwen) can be considered as a preferred treatment approach to severe COVID-19 patients. After treatment, abnormal coagulation and leukocyte had markedly improved with a better prognosis. CONCLUSION: This study expands the understanding of the co-occurrence of combination therapy in patients with COVID-19, which provides the probability of developing novel combined therapy. Furthermore, explore clinical trials of variable antivirus treatments based on subgroup analyses or on using subgroups in the selection criteria would be the next step.

(2R,3R)Dihydromyricetin inhibits osteoclastogenesis and bone loss through scavenging LPS-induced oxidative stress and NF-κB and MAPKs pathways activating.

Osteolysis is a serious complication of several chronic inflammatory diseases and is closely associated with a local chronic inflammatory reaction with a variety of causes. However, similarities exist in the mechanisms of their pathological processes. Inflammatory factors and oxidative stress-induced nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways play a center role in bone erosion. Dihydromyricetin (DMY) is a natural compound with anti-inflammatory and antioxidative effect, which are commonly used in chronic pharyngitis and alcohol use disorders. In the current study, we identified that DMY attenuated lipopolysaccharide (LPS)-induced oxidative stress through inhibiting the production of reactive oxygen species (ROS) and nitric oxide (NO), downregulated COX-2 and iNOS, and promoted the activity of the antioxidative system by activating superoxide dismutase (SOD) and Nrf2/HO-1 pathway. To further investigate the underlying mechanism, we found that DMY inhibits osteoclast (OC) differentiation and bone resorption activity through blocking the RANKL-induced activation of the NF-κB and MAPKs signaling pathways and then downregulated c-Fos and NFATc1, which is essential for OC differentiation. Furthermore, DMY inhibited LPS-induced osteolysis in vivo. Collectively, these results indicate that DMY might be a promising prophylactic antiosteoclastic/resorptive agent in preventing or treating bone lysis diseases.

Inhibitory effects of high glucose/insulin environment on osteoclast formation and resorption in vitro.

Patients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 µmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 µmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.

Rosiglitazone inhibits bone regeneration and causes significant accumulation of fat at sites of new bone formation.

Thiazolidinediones (TZDs), peroxisome proliferator-activated receptor gamma activators, and insulin sensitizers represent drugs used to treat hyperglycemia in diabetic patients. Type 2 diabetes mellitus (T2DM) is associated with a twofold increase in fracture risk, and TZDs use increases this risk by an additional twofold. In this study, we analyzed the effect of systemic administration of the TZD rosiglitazone on new bone formation in two in vivo models of bone repair, a model of drilled bone defect regeneration (BDR) and distraction osteogenesis (DO) and a model of extended bone formation. Rosiglitazone significantly inhibited new endosteal bone formation in both models. This effect was correlated with a significant accumulation of fat cells, specifically at sites of bone regeneration. The diminished bone regeneration in the DO model in rosiglitazone-treated animals was associated with a significant decrease in cell proliferation measured by the number of cells expressing proliferating cell nuclear antigen and neovascularization measured by both the number of vascular sinusoids and the number of cells producing proangiogenic vascular endothelial growth factor at the DO site. In summary, rosiglitazone decreased new bone formation in both BDR and DO models of bone repair by mechanisms which include both intrinsic changes in mesenchymal stem cell proliferation and differentiation and changes in the local environment supporting angiogenesis and new bone formation. These studies suggest that bone regeneration may be significantly compromised in T2DM patients on TZD therapy.

Gender-associated difference following COVID-19 virus infection: Implications for thymosin alpha-1 therapy.

Gender influences clinical presentations, duration and severity of symptoms, and therapy outcome in coronavirus disease 2019 (COVID-19) infection. Whether the immune response to Tα1 treatment for SARS-CoV-2 differs between the sexes, and whether this difference explains the male susceptibility to COVID-19, is unclear. This study aimed to investigate the efficiency and safety of Tα1 treatment and provide a basis for practically identifying gender differences characteristics and features of COVID-19. One hundred twenty-seven patients had COVID-19 symptoms and tested COVID19-positive (female 42.52%) in Wuhan union hospital were enrolled for medication. They were randomly divided into groups Control and Tα1 intervention. Seventy-eight patients received a subcutaneous injection of 1.6 mg Tα1, based on supportive treatment for 15 days. The control group included untreated 49 COVID19 patients closely matched for gender and age and received regular supportive treatment. In this retrospective analysis, we found that COVID-19-infected males reported more symptoms than COVID-19-infected females. A high degree of gender differences-related variability was observed in CRP and PCT levels and the cell counts of many lymphocyte subpopulations in the COVID-19 patients after Tα1 intervention. Levels of CRP and IL-6 were higher in Tα1-treated male group than Tα1-treated female group, while the level of PCT was significantly lower in Tα1-treated male group. Gender differences may be a factor in sustaining COVID-19 immunity responded to Tα1, male and female show statistically significant differences in relevance to cytokine production associated with the development of a more significant number of symptoms. This leaves the question of identifying gender-specific risk factors to explain these differences.

Multitarget and promising role of dihydromyricetin in the treatment of metabolic diseases.

Dihydromyricetin (DMY or DHM), also known as ampelopsin, is the main natural flavonol compound extracted from the plant Ampelopsis grossedentata (Hand. -Mazz) W.T. Wang. In recent years, accumulating studies have been conducted to explore the extensive biological functions of DMY, including antitumor, anti-inflammation, organ-protective, and metabolic regulation effects. DMY acts as a potential preventive or therapeutic agent in treating multiple diseases, such as diabetes mellitus, atherosclerosis, nonalcoholic fatty liver disease and osteoporosis. This review article summarizes the preventive and therapeutic potential of DMY in multiple metabolic diseases and the main signaling pathways in which DMY participates to offer a comprehensive understanding and guidance for future studies.

Flavonoids as inducers of white adipose tissue browning and thermogenesis: signalling pathways and molecular triggers.

BACKGROUND: Flavonoids are a class of plant and fungus secondary metabolites and are the most common group of polyphenolic compounds in the human diet. In recent studies, flavonoids have been shown to induce browning of white adipocytes, increase energy consumption, inhibit high-fat diet (HFD)-induced obesity and improve metabolic status. Promoting the activity of brown adipose tissue (BAT) and inducing white adipose tissue (WAT) browning are promising means to increase energy expenditure and improve glucose and lipid metabolism. This review summarizes recent advances in the knowledge of flavonoid compounds and their metabolites. METHODS: We searched the following databases for all research related to flavonoids and WAT browning published through March 2019: PubMed, MEDLINE, EMBASE, and the Web of Science. All included studies are summarized and listed in Table 1. RESULT: We summarized the effects of flavonoids on fat metabolism and the specific underlying mechanisms in sub-categories. Flavonoids activated the sympathetic nervous system (SNS), promoted the release of adrenaline and thyroid hormones to increase thermogenesis and induced WAT browning through the AMPK-PGC-1α/Sirt1 and PPAR signalling pathways. Flavonoids may also promote brown preadipocyte differentiation, inhibit apoptosis and produce inflammatory factors in BAT. CONCLUSION: Flavonoids induced WAT browning and activated BAT to increase energy consumption and non-shivering thermogenesis, thus inhibiting weight gain and preventing metabolic diseases.

[Immortalization of rat epiphysis cartilage cells induced by simian virus 40 large T antigen gene transfection].

OBJECTIVE: To establish immortalized epiphysis cartilage cell strains in order to provide a stable cell resource for cell substitution and gene therapies of growth retardation. METHODS: Plasmid pEGFP-IRES2-SV40LTag containing simian virus 40 large T antigen gene was transfected into primarily cultured epiphysis cartilage cells of the newborn rat using the lipofectin transfection method. Colonies were isolated by G418 selection and cultured to immortalized cell strains. Fibroblast growth factor receptor-3 (FGFR-3), anti-collagen type II and type X antibodies were used to identify cultured cells and to investigate the capability of differentiation of the transfected cells. SV40LTag expression in expanded cell strains was identified by RT-PCR, Southern blot and immunocytochemistry method. RESULTS: Anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive epiphysis cartilage cells with the capability of stable proliferation. mRNA and protein of SV40LTag were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains and named as immortalized epiphysis cartilage cells. The immortalized cells were elliptic or triangular, with two or three short axons. The immortalized epiphysis cartilage cell strains had stable biological characters. CONCLUSIONS: SV40LTag gene transfection can immortalize epiphysis cartilage cells. The establishment of FGFR-3 positive immortalized epiphysis cartilage cell strains may provide a stable cell resource for cell substitution and gene therapies of growth retardation.

Marrow Adipose Tissue: Skeletal Location, Sexual Dimorphism, and Response to Sex Steroid Deficiency.

Marrow adipose tissue (MAT) is unique with respect to origin, metabolism, and function. MAT is characterized with high heterogeneity which correlates with skeletal location and bone metabolism. This fat depot is also highly sensitive to various hormonal, environmental, and pharmacologic cues to which it responds with changes in volume and/or metabolic phenotype. We have demonstrated previously that MAT has characteristics of both white (WAT) and brown (BAT)-like or beige adipose tissue, and that beige phenotype is attenuated with aging and in diabetes. Here, we extended our analysis by comparing MAT phenotype in different locations within a tibia bone of mature C57BL/6 mice and with respect to the presence of sex steroids in males and females. We report that MAT juxtaposed to trabecular bone of proximal tibia (pMAT) is characterized by elevated expression of beige fat markers including Ucp1, HoxC9, Prdm16, Tbx1, and Dio2, when compared with MAT located in distal tibia (dMAT). There is also a difference in tissue organization with adipocytes in proximal tibia being dispersed between trabeculae, while adipocytes in distal tibia being densely packed. Higher trabecular bone mass (BV/TV) in males correlates with lower pMAT volume and higher expression of beige markers in the same location, when compared with females. However, there is no sexual divergence in the volume and transcriptional profile of dMAT. A removal of ovaries in females resulted in decreased cortical bone mass and increased volume of both pMAT and dMAT, as well as volume of gonadal WAT (gWAT). Increase in pMAT volume was associated with marked increase in Fabp4 and Adiponectin expression and relative decrease in beige fat gene markers. A removal of testes in males resulted in cortical and trabecular bone loss and the tendency to increased volume of both pMAT and dMAT, despite a loss of gWAT. Orchiectomy did not affect the expression of white and beige adipocyte gene markers. In conclusion, expression profile of beige adipocyte gene markers correlates with skeletal location of active bone remodeling and higher BV/TV, however bone loss resulted from sex steroid deficiency is not proportional to MAT expansion at the same skeletal location.

Pioglitazone affects the OPG/RANKL/RANK system and increase osteoclastogenesis.

Thiazolidinediones are traditional anti­diabetic therapeutic agents that have been associated with bone loss and increased fracture risk. However, the underlying mechanisms of this side effect require further elucidation. The present study aimed to investigate the effect of pioglitazone (PIO), a thiazolidinedione, on osteoblastogenesis, osteoclastogenesis and the osteoprotegerin (OPG) / receptor activator of nuclear factor­κB ligand (RANKL) / RANK system. The MC3T3­E1 murine pre­osteoblastic cell line was treated with PIO and processed for reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis of OPG, RANKL, peroxisome proliferator­activated receptor Î³ (PPARγ), Runt­related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN), and western blotting analysis of OPG and RANKL. The culture medium was collected for ELISA analysis of OPG and RANKL. Murine bone marrow monocytes (BMMCs) were treated with PIO in the presence of RANKL and macrophage­colony stimulating factor and subjected to tartrate­resistant acid phosphatase (TRAP) staining and activity measurement, and RT­qPCR analysis of cathepsin K, TRAP and RANK. Co­culture of MC3T3­E1 and BMMCs was performed in the presence of PIO, and TRAP staining was also conducted. PIO inhibited the osteoblastic differentiation of MC3T3­E1 cells, and promoted the osteoclastic differentiation of BMMCs with or without co­culturing with MC3T3­E1 cells. ELISA analysis indicated increased RANKL and decreased OPG expression levels in the medium of MC3T3­E1 cells treated with PIO. PIO upregulated expression of RANKL and PPARγ and downregulated expression of OPG, RUNX2, ALP and OCN in MC3T3­E1 cells, while expression levels of RANK in BMMCs remained unchanged. These results suggest that PIO suppresses osteoblastogenesis and enhances osteoclastogenesis. In addition, PIO may also promote osteoclastogenesis by affecting the OPG­RANKL­RANK system.

Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro.

Zoledronic acid (ZA), which is one of the most potent and efficacious bisphosphonates, has been commonly used in clinical practice for the treatment of various bone disorders. The extensive use of ZA has been associated with increasing occurrence of jaw complications, now known as bisphosphonate­associated osteonecrosis of the jaw (BRONJ). However, the mechanism underlying BRONJ remains to be fully elucidated. The aim of the present study was to investigate the effects of different concentrations of ZA on the MC3T3­E1 murine preosteoblast cell line cells and examine the possible pathogenesis of BRONJ. In the present study, the effect of ZA on the viability, apoptosis, differentiation and maturation of MC3T3­E1 cells, as well as its relevant molecular mechanism, were examined The results of a Cell Counting Kit 8 assay, a flow cytometric Annexin­V/propidium iodide assay and western blot analysis demonstrated that ZA exhibited a significant inhibition of cell viability and induction of apoptosis at concentrations >10 µM. Subsequently, the effect of ZA on cell differentiation at concentrations <1 µM were investigated. In this condition, ZA inhibited bone nodule formation and decreased the activity of alkaline phosphatase. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses indicated that ZA downregulated the expression levels of the marker genes and proteins associated with osteogenic differentiation. Further investigation revealed that the suppression of differentiation by ZA was associated with decreased expression of bone morphogenetic protein­2 (BMP­2) and downregulation of the phosphorylation levels in the downstream extracellular signal­regulated kinase 1/2 and p38 pathways. These adverse effects of ZA were observed to be concentration­dependent. The results from the present study suggested that ZA at higher concentrations induces cytotoxicity towards osteoblasts, and ZA at lower concentrations suppresses osteoblast differentiation by downregulation of BMP-2. These results assist in further understanding the mechanisms of BRONJ.

[Regulation of differentiation and proliferation of epiphysis stem cells by Notch1 signaling system].

OBJECTIVE: To investigate the regulation of differentiation and proliferation of epiphysis stem cells by Notch1 signaling system. METHODS: Costocostal cartilage was taken from a SD rat. Epiphysis stem cells were isolated and cultured. Recombinant human nuclear factor-kappaB (rhNF-kappaB), an activator of the Notch signaling system, and gamma-secretase inhibitor (MW167), an inhibitor of the Notch signaling system, were added into the culture medium respectively. The cells cultured in the medium added with phosphate-buffered saline were used as control group. Then the cultured cells were collected. The expression of the homologous Notch receptors and homologous Notch ligands was detected by RT-PCR. Immunohistochemistry was used to detect the levels of collagen II, collagen X, and proliferating cell nuclear antigen (PCNA). MTT method was used to calculate the growth curve. The cell phase was examined by flow cytometry. The level of alkaline phosphatase (AP) was measured. Western blotting was used to detect the protein expression of collagen II, collagen X, and stathmin, a signaling protein of proliferation. RESULTS: Only 2 the expression of the receptor Notch1 and the ligand Jagged1 was found. The expression of PCNA was stronger in the rhNF-kappaB group than in the other 2 groups. rhNF-kappaB remarkably promoted the expression of collagen II and inhibited the expression of collagen X and MW167 remarkably promoted the expression of collagen X and did not remarkably influence the expression of collagen II. MTT method showed that rhNF-kappaB significantly promote the proliferation of the cells (P = 0.027), and MW167 did not significantly promote the cell proliferation (P > 0.05). The percentage of cells at S phase of the rhNF-kappaB group was 26.54%, significantly higher than those of the MW167 group and control group (8.22% and 6.15%). AP was significantly expressed in the MW167 group, and less expressed in the other groups. Western blotting showed a significantly increased expression of collagen X protein and decreased expression of collagen II protein and stathmin. CONCLUSION: When the Notch signaling system is activated the epiphysis stem cells proliferate, and when the Notch signaling system is suppressed the epiphysis stem cells differentiate.

Protective effects of ciliary neurotrophic factor on denervated skeletal muscle.

To study the effects of ciliary neurotrophic factor (CNTF) on denervated skeletal muscle atrophy and to find a new approach to ameliorate atrophy of denervated muscle, a model was established by cutting the right sciatic nerve in 36 Wistar mice, with the left side serving as control. Then they were divided into two groups randomly. CNTF (1 U/ml) 0.1 ml was injected into the right tibial muscle every day in experimental group, and saline was used into another group for comparison. The muscle wet weight, muscle total protein, Ca2+, physiological response and morphology were analyzed on the 7th, 14th and 28th day after operation. Our results showed that compared to control group, there was a significant increase in muscle wet weight, total protein, Ca2+, muscle fiber cross-section area in CNTF group (P < 0.05). CNTF could ameliorate the decrease of tetanic tension (PO), post-tetanic twitch potentiation (PTP), and the prolonged muscle relaxation time (RT) caused by denervation (P < 0.05). The motor end-plate areas 7 days and 14 days after denervation was similar (P > 0.05), but significantly larger 28 days after the denervation (P < 0.05). Our results suggest that CNTF exerts myotrophic effects by attenuating the morphological and functional changes associated with denervation of rat muscles and has protective effects on denervated muscle and motor end plate.

Decreased osteoclastogenesis, osteoblastogenesis and low bone mass in a mouse model of type 2 diabetes.

The effect of type 2 diabetes mellitus (T2DM) on bone is controversial. Therefore, the present study investigated whether T2DM causes osteoporosis and explored the underlying mechanisms involved in this process. The effects of T2DM on bone physiology were analyzed in a mouse model of T2DM; KK/Upj­Ay/J (KK­Ay) mice develop diabetes after 8 weeks and exhibit stable diabetes symptoms and signs after 10 weeks when fed a KK­Ay mouse maintenance fodder. Diabetic mice exhibited hyperglycemia, hyperinsulinemia and increased body and fat pad weight in comparison with C57BL/6 non-diabetic mice. Furthermore, diabetic mice demonstrated low bone weight and bone mineral density in the femur, tibia and fifth lumbar vertebra. Using von Kossa and tartrate-resistant acid phosphatase (TRAP) staining, alkaline phosphatase and TRAP activity analyses and gene profiling it was demonstrated that osteoblastogenesis and osteoclastogenesis were impaired in diabetic mice. To evaluate the bone biomechanics, the ultimate load of the bone was analyzed. It was found that the ultimate load of the tibia in diabetic mice was lower than that in the controls. The results from the present study suggest that bone metabolism is impaired in T2DM, resulting in decreased osteoblastogenesis, osteoclastogenesis and bone mass.

Purification and characterization of a novel antithrombotic peptide from Scolopendra subspinipes mutilans.

ETHNOPHARMACOLOGICAL RELEVANCE: The centipede has been prescribed for the treatment of cardiovascular diseases in Korea, China and other Far Eastern Asian countries for several hundred years. MATERIALS AND METHODS: A novel antithrombotic peptide was isolated from Scolopendra subspinipes mutilans using a combination of ultrafiltration, Sephadex G-50 column, Source 15Q anion exchange column and RP-HPLC C18 column. RESULTS: The molecular mass of the purified peptide is 346Da measured by Electrospray Ionization Mass Spectrometry (ESI-MS). The primary structure of the peptide is Ser-Gln-Leu (SQL) determined by Edman degradation. SQL potently prolonged the activated partial thromboplastin time (aPTT), and inhibited platelet aggregation. CONCLUSIONS: These results help to clarify the mechanism of the antithrombotic activity of the centipede for effective treatment of cardiovascular and cerebrovascular diseases.