METTL14 knockdown inhibits the pyroptosis in the sepsis-induced acute lung injury through regulating the m6A modification of NLRP3.

Background: Sepsis has become one of the main factors inducing the development of acute lung injury (ALI) in clinical practice. Currently, inhibiting the activation of NLRP3 mediated pyroptosis is the target of multiple drugs in the treatment of sepsis induced ALI. This study aimed to explore the effects of METTL14 on the pyroptosis in the sepsis induced ALI progression.Methods: LPS-stimulated A549 cells and cecal ligation and puncture (CLP)-treated mice were used to establish the ALI model in vitro and in vivo. Then, the cell viability was measured by CCK-8 assay. ELISA kits were used to determine the IL-18 and IL-1ß contents. Pyroptosis rate was tested by flow cytometry. M6A dot blot was conducted to analyze the global m6A levels and MeRIP assay was performed to detect the m6A levels of NLRP3. The relationship between METTL14 and NLRP3 was confirmed by RIP and dual-luciferase report assays.Results: The global m6A levels were significantly increased in the LPS-stimulated A549 cells and CLP-treated mice. METTL14 knockdown decreased the cell viability, IL-18 and IL-1ß contents, and pyroptosis rate of the LPS-stimulated A549 cells. Furthermore, the increase of pyroptosis-related proteins in LPS-stimulated A549 cells was significantly decreased after METTL14 knockdown. Additionally, METTL14 knockdown decreased the m6A and mRNA levels of NLRP3, and NLRP3 overexpression reversed the effects of METTL14 knockdown on the pyroptosis in the LPS-stimulated A549 cells. In CLP-treated mice, METTL14 knockdown relieved the injury and decreased the IL-18 and IL-1ß contents in the lung tissues, serum and bronchoalveolar lavage fluid.Conclusion: This study demonstrated that METTL14 knockdown inhibited the pyroptosis in the sepsis-induced ALI progression through decreasing the NLRP3 levels dependent on m6A methylation modification.

Combination of optimized tissue engineering bone implantation with heel-strike like mechanical loading to repair segmental bone defect in New Zealand rabbits.

In this study, effects of combining optimized tissue engineering bone (TEB) implantation with heel-strike like mechanical loading to repair segmental bone defect in New Zealand rabbits were investigated. Physiological characteristics of bone marrow mesenchymal stem cells (BMMSCs), compact bone cells (CBCs), and bone marrow and compact bone coculture cells (BMMSC-CBCs) were compared to select the optimal seed cells for optimized TEB construction. Rabbits with segmental bone defects were treated in different ways (cancellous bone scaffold for group A, cancellous bone scaffold and mechanical loading for group B, optimized TEB for group C, optimized TEB and mechanical loading for group D, n = 4), and the bone repair were compared. BMMSC-CBCs showed better proliferation capacity than CBCs (p < 0.01) and stronger osteogenic differentiation ability than BMMSCs (p < 0.05). Heel-strike like mechanical loading improved proliferation and osteogenic differentiation ability and expression levels of TGFß1 as well as BMP2 of seed cells in vitro (p < 0.05). At week 12 post-operation, group D showed the best bone repair, followed by groups B and C, while group A finished last (p < 0.05). During week 4 to 12 post-operation, group D peaked in terms of expression levels of TGFß1, BMP2, and OCN, followed by groups B and C, while group A finished last (p < 0.05). Thus, BMMSC-CBCs showed good proliferation and osteogenic differentiation ability, and they were thought to be better as seed cells than BMMSCs and CBCs. The optimized TEB implantation combined with heel-strike like mechanical loading had a synergistic effect on bone defect healing, and enhanced expression of TGFß1 and BMP2 played an important role in this process.

Combination of Heel-strike like Mechanical Loading with Deproteinized Cancellous Bone Scaffold Implantation to Repair Segmental Bone Defects in Rabbits.

Under physiological conditions bone defects often occur at mechanical load bearing sites and bone substitutes used for regeneration should be similarly subjected to mechanical loading stress. In this study, we investigated whether a novel heel-strike like mechanical loading method can be used as a complementary therapy to promote bone regeneration following bone substitute grafting. To test this, three groups of rabbits with segmental bone defects in the tibia were implanted with bovine deproteinized cancellous bone scaffold (DCBS), with one group also receiving heel-strike like mechanical loading generated by a rap stress stimulator. From weeks 4-12 post-operation X-ray and micro-CT scanning showed that rabbits receiving combination therapy had significantly more callus at the bone defect. Moreover, bone defects in the combination group were completely replaced with new bone at week 12, while the DCBS implantation alone group healed only partially and rabbits receiving neither DCBS nor mechanical loading developed only small calluses throughout the observation period. Analysis of micro-CT scanning results demonstrated that new bone density in the combination group was significantly higher than the DCBS only group at weeks 4 and 12 (p<0.05). H&E staining results also indicated a significantly higher percentage of new bone in the bone defect area and a lower percentage of residual scaffold in the combination group compared to the DCBS only group (p<0.05). Thus, this heel-strike like mechanical loading method appears to accelerate bone regeneration following substitute implantation by restoring a local mechanical loading environment in segmental bone defects.

The baicalein amorphous solid dispersion to enhance the dissolution and bioavailability and effects on growth performance, meat quality, antioxidant capacity and intestinal flora in Taihang chickens.

Baicalein (BAI) is a natural flavonoid with antioxidant, antitumor and antibacterial properties. However, the bioavailability of BAI was limited due to low solubility. This study aims to improve the solubility of BAI through the amorphous solid dispersion (ASD) and evaluate changes in its pharmacokinetics and pharmacodynamics in Taihang chickens. Polyethylene caprolactam-polyvinyl acetate-polyethylene glycol grafted copolymer (Soluplus) was chosen as the carrier, and ASD was prepared by rotary evaporation and was characterized by powder X-ray diffractions (PXRD), differential scanning calorimetry (DSC) and fourier transform infrared spectroscopy (FT-IR). In vitro dissolution assays were used to screen the optimal ratio of drug to carrier, in vivo pharmacokinetic assays were conducted to investigate the promoting effect on the absorption. In addition, the effects of ASD on the growth performance, meat quality, antioxidant capacity and intestinal flora were investigated. ASD (1:9 and 2:8) did not exhibit crystal diffraction peaks of BAI in PXRD or endothermic peaks in DSC, indicating the successful preparation of ASD. The results of in vitro dissolution assay showed that the cumulative dissolution rate of ASD (2:8) within 600 min was 52.67%, which was 7.84-fold higher than BAI. The pharmacokinetic results showed that the peak concentration (Cmax) and the area under the drug-time curve (AUC0∼24) of ASD (2:8) was (5.20 ± 0.82) µg/mL and (17.03 ± 0.67) µg·h/mL, which was 1.91 and 2.64-fold higher than BAI, respectively. Dietary supplementation of BAI and ASD could increase average daily gain (ADG), while decrease feed conversion ratio (FCR), but there was no significant difference (P > 0.05). The drip loss of BAIASD group was lower than BAI group (P < 0.05). In addition, the antioxidant capacity of Taihang chickens were enhanced, the diversity and the abundance of beneficial bacteria was improved. Results of BAI upon the dietary supplementation tested in Taihang chickens, after preparation of ASD, indicating a superior enhancement effect in growth performance, meat quality, antioxidant capacity and intestinal flora due to an improved solubility and optimized bioavailability.

Tongfu Lifei Decoction Attenuated Sepsis-Related Intestinal Mucosal Injury Through Regulating Th17/Treg Balance and Modulating Gut Microbiota.

Intestinal damage and secondary bacterial translocation are caused by the inflammatory response induced by sepsis. Tongfu Lifei (TLF) decoction has a protective effect on sepsis-related gastrointestinal function injury. However, the relation between gut microbiota, immune barrier, and sepsis under the treatment of TLF have not been well clarified yet. Here, rats were subjected to cecal ligation and puncture (CLP) to create a sepsis model. Subsequently, the TLF decoction was given to CLP rats by gavage, fecal microbiota transplantation (FMT), and antibiotic were used as positive control. TLF suppressed the inflammatory response and improved the pathological changes in the intestines of CLP rats. Besides, TLF promoted the balance of the percentage of the Th17 and Treg cells. Intestinal barrier function was also improved by TLF through enhancing ZO-1, and Occludin and Claudin 1 expression, preventing the secondary translocation of other gut microbiota. TLF dramatically boosted the gut microbiota's alpha- and beta-diversity in CLP rats. Moreover, it increased the relative abundance of anti-inflammatory gut microbiota and changed the progress of the glucose metabolism. In short, TLF regulated the gut microbiota to balance the ratio of Th17/Treg cells, reducing the inflammation in serum and intestinal mucosal injury in rats.

Solid Dispersions of Genistein via Solvent Rotary Evaporation for Improving Solubility, Bioavailability, and Amelioration Effect in HFD-Induced Obesity Mice.

Genistein (GEN) is an active pharmaceutical ingredient that presents the challenges of poor water solubility and low oral bioavailability. To tackle these challenges, a GEN solid dispersion was prepared by solvent rotary evaporation using polyvinylpyrrolidone K30 (PVP K30) as a carrier. The optimal formulation was determined by drug loading efficiency and in vitro release. The physical state of the solid dispersion was characterized by DSC, XRD, SEM and FT-IR. And the results of the in vitro release study indicate that the drug release of SD (1:7) increased 482-fold that of pure GEN at 60 min. Following oral administration to rats, the Cmax and AUC0-24 of SD (1:7) was increased 6.86- and 2.06-fold to that of pure GEN. The adipose fat index and body weight of the SD (1:7) group were significantly lower than those of the GEN group (p < 0.05). Meanwhile, the levels of TC and TG in the serum were significantly decreased in the SD (1:7) group compared with the GEN group (p < 0.05). All experiments revealed that solid dispersion could be a promising formulation approach to improve the dissolution rate, oral bioavailability, and effect on the reduction of lipid accumulation in high-fat diet-induced obesity mice.

[Effect of stromal cell-derived factor 1α/cysteine X cysteine receptor 4 signaling pathway on axial stress stimulation promoting bone regeneration].

OBJECTIVE: To observe the change of stromal cell-derived factor 1α/cysteine X cysteine receptor 4 (SDF-1α/CXCR4) signaling pathway during the process of axial stress stimulation promoting bone regeneration, and to further explore its mechanism. METHODS: A total of 72 male New Zealand white rabbits were selected to prepare the single cortical bone defect in diameter of 8 mm at the proximal end of the right tibia that repaired with deproteinized cancellous bone. All models were randomly divided into 3 groups ( n=24). Group A was treated with intraperitoneally injection of PBS; Group B was treated with stress stimulation and intraperitoneally injection of PBS; Group C was treated with stress stimulation and intraperitoneally injection of AMD3100 solution. The X-ray films were taken and Lane-Sandhu scores of bone healing were scored at 2, 4, 8, and 12 weeks after operation, while specimens were harvested for HE staining, immunohistochemical staining of vascular endothelial growth factor (VEGF) and CXCR4, and Western blot (SDF-1α and CXCR4). The bone healing area was scanned by Micro-CT at 12 weeks after operation, and the volume and density of new bone were calculated. RESULTS: X-ray film showed that the Lane-Sandhu scores of bone healing in group B were significantly higher than those in groups A and C at 4, 8, and 12 weeks after operation ( P<0.05). Micro-CT scan showed that the bone defect was repaired in group B and the pulp cavity was re-passed at 12 weeks after operation. The volume and density of new bone were higher in group B than in groups A and C ( P<0.05). HE staining showed that the new bone growth in bone defect area and the degradation of scaffolds were faster in group B than in groups A and C after 4 weeks. The immunohistochemical staining showed that the expressions of VEGF and CXCR4 in 3 groups reached the peak at 4 weeks, and group B was higher than groups A and C ( P<0.05). Western blot analysis showed that the expressions of SDF-1α and CXCR4 in group B were significantly higher than those in groups A and C at 4 and 8 weeks after operation ( P<0.05). CONCLUSION: Axial stress stimulation can promote the expression of SDF-1α in bone defect tissue, activate and regulate the CXCR4 signal collected by marrow mesenchymal stem cells, and accelerate bone regeneration in bone defect area.

[Effect of early use of different doses of enteral nutrition on prognosis of patients with acute respiratory failure].

OBJECTIVE: To observe the impact of initial low-dose (trophic type) enteral nutrition (EN) support on mechanical ventilation (MV) time, the incidence of complications and survival rate in patients with acute respiratory failure (ARF). METHODS: A prospective study was conducted. Forty-four patients with ARF undergoing MV admitted to Department of Critical Care Medicine of Hangzhou Traditional Chinese Medical Hospital from September 2015 to February 2017 were enrolled, and they were divided into a trophic feeding group (n = 23, study group) and a standard-dose feeding group (n = 21, control group). In the two groups, the EN support feeding was given to the patients through a nasogastric tube within 24-hour MV for consecutive 7 days, the protein supply to each one of all of them was 1.2-1.6 g×kg-1×d-1. The study group received EN according to non-protein calories of 41.84-83.68 kJ×kg-1×d-1 to calculate, while the control group accepted EN according to non-protein calories of 104.60-125.50 kJ×kg-1×d-1 to calculate. The serum albumin (Alb) and fasting blood glucose (FBG) levels were measured in two groups 1 day before EN treatment and at 1, 2, 3, 7 days of treatment, and the energy levels in initial 3 days and 7 days of MV and the 24-hour urine creatinine (UCr) level on the 7th day after treatment were recorded. The creatinine-height index (CHI, CHI = actual UCr/standard UCr) was calculated. The incidence of intestinal intolerance (vomiting, gastric retention, diarrhea, gastrointestinal bleeding, etc.) in 7 days of treatment, MV time, the length of stay in ICU, the total length of stay in the hospital and the 28-day incidence of new infections (pulmonary, hematogenous, urinary, abdominal, and other infections) and 60-day survival rate were observed between the two groups. RESULTS: The EN supplies within 3 days and 7 days in the study group were significantly lower than those in the control group [within 3 days (kJ/d): 1 710.58±703.96 vs. 4 152.79±1 334.65, 7 days (kJ/d): 2 471.28±815.50 vs. 5 058.08±875.25, both P < 0.05]; there were no statistically significant differences in CHI after EN therapy for 7 days and serum Alb levels before and after EN between the two groups. FBG level of the study group was significantly lower than that of the control group since the 2nd day of treatment (mmol/L: 8.58±2.37 vs. 10.93±3.75), and continued to the 7th day (mmol/L: 8.96±1.76 vs. 10.97±4.11, both P < 0.05), the incidence of elevated blood glucose was also significantly lower than that of the control group [26.1% (6/23) vs. 66.7% (14/21), P < 0.05]. The incidence of feeding intolerance in the study group was significantly lower than that in the control group from 2 days of treatment till 7 days (26.1% vs. 47.6%, P < 0.05). There were no statistically significant differences in MV time (days: 15.04±6.75 vs. 16.14±8.51), the length of stay in ICU (days: 16.52±6.89 vs. 17.24±7.67), total length of stay in hospital (days: 26.35±9.69 vs. 25.33±7.73), 28-day new infection rate [26.1% (6/23) vs. 42.9% (9/21)] and 60-day survival rate [65.2% (15/23) vs. 66.7% (14/21)] between the study and control groups (all P > 0.05). CONCLUSIONS: Initial trophic EN feeding results in clinical outcomes similar to those of early standard-dose EN feeding in MV patients with ARF, but the former one has less incidence of high blood sugar and more satisfactory gastrointestinal tolerance situation.

A biodegradable antibiotic-eluting PLGA nanofiber-loaded deproteinized bone for treatment of infected rabbit bone defects.

We fabricated a biodegradable antibiotic-eluting poly(d,l)-lactide-co-glycolide nanofiber-loaded deproteinized bone (ANDB) scaffold that provided sustained delivery of vancomycin to repair methicillin-resistant Staphylococcus aureus bone defects. To fabricate the biodegradable ANDB, poly(d,l)-lactide-co-glycolide and vancomycin were first dissolved in 1,1,1,3,3,3-hexafluoro-2-propano. The solution was then electrospun to produce biodegradable antibiotic-eluting membranes that were deposited on the surface of bovine deproteinized cancellous bone. We used scanning electron microscopy to determine the properties of the scaffold. Both elution and high-performance liquid chromatography assays were used to evaluate the in vitro vancomycin release rate from the ANDB scaffold. Three types of scaffolds were co-cultured with bacteria to confirm the in vitro antibacterial activity. The infected bone defect rabbit model was induced by injecting 10(7) colony forming units of a methicillin-resistant Staphylococcus aureus strain into the radial defect of rabbits. Animals were then separated into treatment groups and implanted according to the following scheme: ANDB scaffold in group A, poly(d,l)-lactide-co-glycolide nanofiber-loaded deproteinized bone (NDB) scaffold with intravenous (i.v.) vancomycin in group B, and NDB scaffold alone in group C. Treatment efficacy was evaluated after eight weeks using radiological, microbiological, and histological examinations. In vitro results revealed that biodegradable ANDB scaffolds released concentrations of vancomycin that were greater than the minimum inhibitory concentration for more than four weeks. Bacterial inhibition tests also confirmed antibacterial efficacy lasted for approximately four weeks. Radiological and histological scores obtained in vivo revealed significant differences between groups A, B and C. Importantly, group A had significantly lower bacterial load and better bone regeneration when compared to either group B or C. Collectively, these results show that our fabricated ANDB scaffolds possess: (1) effective bactericidal activity against methicillin-resistant Staphylococcus aureus, (2) the ability to promote site-specific bone regeneration, and (3) the potential for use in the treatment of infected bone defects.

[Treatment of Vancouver type B1 periprosthetic femoral fracture with locked plate and xenogenic bony plate].

OBJECTIVE: To analyze the therapeutic effect of plate cable system and cortical strut bone graft in the prosthesis revision of the total hip arthroplasty for the treatment of Vancouver type B1 periprosthetic femoral fracture. METHODS: A total of 8 patients were selected from January 2006 to January 2013, including 6 males and 2 females, aged from 56 to 74 years old (62.52 years old on average). All the cases were treated by the long plate cable system and appropriate cortical strut bone graft. Harris score was used to evaluate the hip functions before and after the operation. Prosthesis stability and the bony union were assessed by the digital radiography. RESULTS: All the patients were followed up for 45 months on average ranging from 24 to 60 months after operation. All the fractures reached union, and there was no infection, stem loosening, nonunion and malunion. The Harris score was 28.45±5.78 before operation, which was improved to 83.46±10.21 after operation. X ray showed that the prosthesis was stable, and the host bone and bone graft achieved bony union in 7 patients;and the other 1 patient need further operation of revision around the loose stem. CONCLUSIONS: The prosthesis revision of the total hip arthroplasty with the locking plate and cortical strut bone graft used for the Vancouver type B1 periprosthetic proximal femur fractures has the advantages of simple manipulation, less complications, good recovery of the hip function and can improve bone quality to provide favorable conditions for operation of revision.