The road to evolution of ProTx2: how to be a subtype-specific inhibition of human Nav1.7.

The human voltage-gated sodium channel Nav1.7 is a widely proven target for analgesic drug studies. ProTx2, a 30-residue polypeptide from Peruvian green tarantula venom, shows high specificity to activity against human Nav1.7, suggesting its potential to become a non-addictive analgesic. However, its high sensitivity to human Nav1.4 raises concerns about muscle side effects. Here, we engineered three mutants (R13A, R13D, and K27Y) of ProTx2 to evaluate their pharmacological activities toward Nav1.7 and Nav1.4. It is demonstrated that the mutant R13D maintained the analgesic effect in mice while dramatically reducing its muscle toxicity compared with ProTx2. The main reason is the formation of a strong electrostatic interaction between R13D and the negatively charged amino acid residues in DII/S3-S4 of Nav1.7, which is absent in Nav1.4. This study advances our understanding and insights on peptide toxins, paving the way for safer, effective non-addictive analgesic development.

Long-term efficacy, safety and biocompatibility of a novel sirolimus eluting iron bioresorbable scaffold in a porcine model.

Iron is considered as an attractive alternative material for bioresorbable scaffolds (BRS). The sirolimus eluting iron bioresorbable scaffold (IBS), developed by Biotyx Medical (Shenzhen, China), is the only iron-based BRS with an ultrathin-wall design. The study aims to investigate the long-term efficacy, safety, biocompatibility, and lumen changes during the biodegradation process of the IBS in a porcine model. A total of 90 IBSs and 70 cobalt-chromium everolimus eluting stents (EES) were randomly implanted into nonatherosclerotic coronary artery of healthy mini swine. The multimodality assessments including coronary angiography, optical coherence tomography, micro-computed tomography, magnetic resonance imaging, real-time polymerase chain reaction (PCR), and histopathological evaluations, were performed at different time points. There was no statistical difference in area stenosis between IBS group and EES group at 6 months, 1year, 2 years and 5 years. Although the scaffolded vessels narrowed at 9 months, expansive remodeling with increased mean lumen area was found at 3 and 5 years. The IBS struts remained intact at 6 months, and the corrosion was detectable at 9 months. At 5 years, the iron struts were completely degraded and absorbed in situ, without in-scaffold restenosis or thrombosis, lumen collapse, aneurysm formation, and chronic inflammation. No local or systemic toxicity and abnormal histopathologic manifestation were found in all experiments. Results from real-time PCR indicated that no sign of iron overload was reported in scaffolded segments. Therefore, the IBS shows comparable efficacy, safety, and biocompatibility with EES, and late lumen enlargement is considered as a unique feature in the IBS-implanted vessels.

[Mechanism of Fushen Granules treat peritoneal dialysis-related peritonitis by regulating TLR4/NF-κB pathway:based on network pharmacology and animal experiments].

Network pharmacology was employed to probe into the mechanism of Fushen Granules in treating peritoneal dialysis-rela-ted peritonitis(PDRP) in rats. The main active components of Fushen Granules were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and their targets were predicted. PDRP-related targets were retrieved from DisGeNET and other databases. The common targets shared by the drug and the disease were identified by the online tool, and protein-protein interaction(PPI) network of the common targets. The obtained 276 common targets were imported into DAVID for GO function enrichment and KEGG pathway enrichment. The main signaling pathway of Fushen Granules in the treatment of PDRP was predicted as Toll-like receptor 4(TLR4)/nuclear factor(NF)-κB. The rat model of uremia was induced by 5/6 nephrectomy. From two weeks after operation, the rat model of peritoneal dialysis(PD) was established by intraperitoneal injection of 20 mL dialysate with 1.25% glucose every day. The sham operation group and model group received 2 mL normal saline by gavage every day. The rats in Fushen Gra-nules groups were administrated with 2 mL solutions of low-(0.54 g·kg~(-1)), medium-(1.08 g·kg~(-1)) and high-dose(2.16 g·kg~(-1)) Fushen Granules every day. The bifico group received 2 mL(113.4 mg·kg~(-1)) of bifico solution every day. At the end of the 8th week, the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) in each group were measured. The serum levels of hypersensitive C reactive protein(hs-CRP), tumor necrosis factor(TNF)-α, and interleukin(IL)-6 were measured, and the pathological changes in the colon tissue were observed by hematoxylin-eosin(HE) staining. The serum levels of lipopolysaccharide(LPS) and lipopolysaccharide-binding protein(LBP) of rats were measured, and the expression levels of LBP, TLR4, NF-κB p65, inhibitor of κB kinase α(IκBα), TNF-α, and IL-1ß in the colon tissue were determined. Compared with sham operation group, the model group had abnormal structure of all layers of colon tissue, sparse and shorter intestinal villi, visible edema in mucosal layer, wider gap, obvious local inflammatory cell infiltration, significantly decreased body weight(P<0.01), and significantly increased kidney function index(Scr, BUN) content(P<0.01). Serum levels of inflammatory cytokines(hs-CRP, TNF-α, IL-6), LPS and LBP were significantly increased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß were significantly increased(P<0.01), and protein expressions of IκBα were significantly decreased(P<0.01). Compared with model group, intestinal villi damage in colonic tissue of rats in low-, medium-and high-dose Fushen Granules groups and bifico group were alleviated to different degrees, edema in submucosa was alleviated, space was narrowed, and inflammatory cell infiltration in lamina propria was reduced. The contents of renal function index(Scr, BUN) and serum inflammatory factors(hs-CRP, TNF-α, IL-6) were significantly decreased(P<0.05 or P<0.01) in medium-and high-dose Fushen Granules groups and bifico group(P<0.05 or P<0.01). Serum LPS and LBP contents in Fushen Granules group and bifico group were significantly decreased(P<0.01), protein expressions of LBP, TLR4, NF-κB p65, TNF-α and IL-1ß in Fushen Granules group were significantly decreased(P<0.05 or P<0.01), and protein expressions of IκBα were significantly increased(P<0.01). The expression of LBP protein in bifico group was significantly decreased(P<0.01). The results suggest that Fushen Granules can protect the residual renal function of PD rats, reduce the inflammatory response, and protect the colon tissue. Based on network pharmacology, TLR4/NF-κB pathway may be the main signaling pathway of Fushen granule in the treatment of PDRP. The results showed that Fushen Granules could improve intestinal inflammation and protect intestinal barrier to prevent PDRP by regulating the expression of key factors in TLR4/NF-κB pathway in colon of PD rats.

A Dose-Dependent Spatiotemporal Response of Angiogenesis Elicited by Zn Biodegradation during the Initial Stage of Bone Regeneration.

Zinc (Zn) plays a crucial role in bone metabolism and imbues biodegradable Zn-based materials with the ability to promote bone regeneration in bone trauma. However, the impact of Zn biodegradation on bone repair, particularly its influence on angiogenesis, remains unexplored. This study reveals that Zn biodegradation induces a consistent dose-dependent spatiotemporal response in angiogenesis,both in vivo and in vitro. In a critical bone defect model, an increase in Zn release intensity from day 3 to 10 post-surgery is observed. By day 10, the CD31-positive area around the Zn implant significantly surpasses that of the Ti implant, indicating enhanced angiogenesis. Furthermore,angiogenesis exhibits a distance-dependent pattern closely mirroring the distribution of Zn signals from the implant. In vitro experiments demonstrate that Zn extraction fosters the proliferation and migration of human umbilical vein endothelial cells and upregulates the key genes associated with tube formation, such as HIF-1α and VEGF-A, peaking at a concentration of 22.5 µM. Additionally, Zn concentrations within the range of 11.25-45 µM promote the polarization of M0-type macrophages toward the M2-type, while inhibiting polarization toward the M1-type. These findings provide essential insights into the biological effects of Zn on bone repair, shedding light on its potential applications.

Effects of febuxostat on delaying chronic kidney disease progression: a randomized trial in China.

BACKGROUND: A few studies have tested febuxostat for its usefulness in delaying chronic kidney disease (CKD) progression by treating hyperuricemia and results were controversial. Thus, we attempted to conduct a randomized controlled study using the Chinese population with advanced grade of CKD. METHODS: One hundred CKD patients in stages 3 and 4 with asymptomatic hyperuricemia from seven medical centers were included in this prospective randomized controlled study and assigned to the control and febuxostat group, the latter of which received febuxostat to titrate to achieve serum uric acid (SUA) < 6 mg/dL. The observation period was 12 months. The primary outcomes included the event of estimated glomerular filtration rate (eGFR) decline ≥ 30% or 50% from baseline at 12 months, dialysis and death from CKD; secondary outcome was the change in eGFR. Safety analysis was also performed. RESULTS: Forty-seven patients and 45 patients in the febuxostat and control groups, respectively completed the study. Seven of 47 (14.9%) participants reached 30% decline in eGFR in the febuxostat group, while 1 (2.1%) and 2 (4.3%) patients reached 50% decline in eGFR or dialysis. Thirteen (28.9%), 10 (22.2%) and 3 (6.7%) of 45 patients reached primary kidney outcomes separately in the control group. The change in eGFR after 12 months from baseline in the febuxostat group was 0.50 mL/min/1.73 m2, which was significantly higher than that in the control group - 4.46 mL/min/1.73 m2 (p = 0.006). Adverse events did not differ between two groups. CONCLUSIONS: Febuxostat effectively slowed eGFR decline in patients with CKD stages 3 and 4 and asymptomatic hyperuricemia.

Endowing biodegradable Zinc implants with dual-function of antibacterial ability and osteogenic activity by micro-addition of Mg and Ag (≤ 0.1 wt.%).

Infection remains the devastating complications associated with surgical fixation of bones fractured during trauma. In this study, we report a low-alloyed Zn-Mg-Ag that simultaneously has optimized strength degeneration profiles during degradation, outstanding antibacterial efficacy and osteogenic activity. Our results showed that Zn-0.05Mg-0.1Ag alloy had favorable mechanical properties (UTS: 247.8 ± 1.6 MPa, Elong.: 35 ± 2.2 %) and presented a better hold of mechanical integrity than pure Zn during 28 days corrosion, 2.6 % vs. 18.7 % reduction. After one-year of natural aging, the alloy still preserved an elongation of 24.07 ± 3.84 %. As verified by microbial cultures, Zn-0.05Mg-0.1Ag alloy demonstrated high antibacterial performance against Gram-positive and Gram-negative strains, as well as antibiotic-resistant strains (MRSA) in vitro and in vivo due to the synergistic antibacterial actions of Zn2+ and Ag+. Meanwhile, Zn-Mg-Ag alloy also exhibited enhanced viability, osteogenic differentiation, and gene expressions of osteoblasts in vitro, as well as promoted osteogenic activity than pure Zn in the femoral condyle defect repair model. The co-releasing of Zn, Mg and Ag ions did not induce toxic side effects. Collectively, low alloyed Zn-0.05Mg-0.1Ag indicated long-lasting mechanical integrity during degradation, and presented the ability to synergistically inhibit bacteria and promote osteogenesis, possessing tremendous potential in treating implant-associated infections. STATEMENT OF SIGNIFICANCE: Infection after fracture fixation (IAFF) remains the most common and serious side effects of orthopedic surgery. Additionally, widespread antibiotic use contributes to the development of multi-drug resistant bacteria such as methicillin-resistant staphylococcus aureus (MRSA), which exacerbates IAFF treatment and prevention. IAFF treatment and prevention remain clinically challenging, so implants with dual antibacterial and osteogenic functions are in high demand. The antibacterial efficacy and osteogenic activity of low-alloyed Zn-Mg-Ag (≤0.1 wt.% Mg, Ag) alloys were investigated in vitro and in vivo. The results showed that micro addition of Mg and Ag could significantly improve osseointegration function, mechanical properties, and antibacterial performance. These quantification findings shed new light on the development and understanding of dual functional Zn-based orthopedic implants.

A metabolic biomarker panel of restless legs syndrome in peritoneal dialysis patients.

BACKGROUND: Restless legs syndrome (RLS) is a neuromotor disorder, and dialysis patients are more likely to develop RLS. RLS often causes sleep disorders, anxiety and depression in patients. It will increase the risk of death and severely affect the life of patients. At present, RLS has not received enough recognition and attention, and the misdiagnosis rate can reach more than 10%. METHODS: The discovery set selected 30 peritoneal dialysis (PD) patients and 27 peritoneal dialysis patients with RLS (PD-RLS). A metabolomics method based on ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometric method (UPLC-Q-TOF/MS) was used to analyze the differential metabolites of the two groups. 51 PD patients and 51 PD-RLS patients were included in the validation set. The receiver operating characteristic (ROC) analysis was used to evaluate the early diagnostic biomarkers, and the correlation between the differential metabolites and laboratory test indexes was analyzed to explore the biological function of the differential metabolites. RESULTS: Through the integrated analysis, four metabolites can be used as markers for the diagnosis of PD-RLS, including Hippuric acid, Phenylacetylglutamine, N,N,N-Trimethyl-L-alanyl-L-proline betaine and Threonic acid. Through ROC analysis, it is found that they can be used as a metabolic biomarker panel, and the area under the curve of this combination is more than 0.9, indicating that the panel has good diagnostic and predictive ability. CONCLUSION: Metabolomics based on UPLC-Q-TOF/MS technology can effectively identify the potential biomarkers, and provide a theoretical basis for the early diagnosis, prevention and treatment on PD-RLS.

Effect of Microstructure on the Mechanical Properties of Steel Fiber-Reinforced Recycled Concretes.

A steel fiber-reinforced recycled concrete (SFRRC) is a porous material, and its macromechanical properties are affected by its microstructure. To elucidate the change rules and internal mechanisms of the mechanical properties of SFRRCs, the mechanical properties and failure modes of SFRRCs were studied at different water-cement ratio, replacement rate of recycled concrete aggregate (RCA), and steel fiber content. Moreover, the microstructures of the interface transition zones (ITZ) of the SFRRC specimens were tested by scanning electron microscopy and mercury intrusion, and the effect of the microscopic pore structure on the macromechanical properties of SFRRC was analyzed. The research results showed that an appropriate amount of steel fibers could reduce the size and number of cracks in the ITZ and improve the pore structure of an SFRRC. Based on the fractal dimension, porosity and other factors, the quantitative relationship between the macromechanical properties and microscopic pore structure parameters of SFRRCs was established.

Hydroxysafflor yellow A improved retinopathy via Nrf2/HO-1 pathway in rats.

The aim of the study was to investigate the inhibitory effect of hydroxysaff yellow A (HSYA) on diabetic retinopathy (DR). For this, a total of 27 rats were randomly divided into normal control, model, and HSYA groups. The body weight, blood glucose, and blood-retinal barrier damage of the rats were observed and compared. The pathological change of retinal tissue were measured using H&E staining. The apoptosis of retinal tissue ganglion cells was detected by TUNEL. The interleukin (IL)-1ß and tumor necrosis fator (TNF)-α levels were detected using enzyme-linked immunosorbent assay. The level of malondialdehyde (MDA) was detected using thiobarbituric acid method. Superoxide dismutase levels were detected using xanthine oxidase method; Nrf2 and total HO-1 protein expressions were detected using western blot assay; Bcl-2 and P53 protein expression was measured using immunohistochemical staining. The body weight and retinal damage of the HYSA group were significantly improved (p < 0.01, respectively). The apoptosis index of the HYSA group was lower than the model group (p < 0.001). The IL-1ß, TNF-α, and MDA levels of the HYSA group were significantly improved in comparison with those of the model group (p < 0.01, respectively). The Nrf-2, HO-1, Bcl-2, and P53 protein expression of HYSA group was significantly improved (p < 0.001, respectively). In conclusion, HYSA can effectively alleviate the apoptosis of retinal ganglion cells in type 2 diabetic rats and improve the progression of DR.

Efficacy and safety of Zicuiyin decoction on diabetic kidney disease: A multicenter, randomized controlled trial.

BACKGROUD: Zicuiyin (ZCY) decoction created by Xichun Zhang in the Qing dynasty has been used on diabetes mellitus and complications for more than two centuries in China. Huangkui capsule (HKC) is a listed Chinese patent medicine to treat diabetic kidney disease (DKD). To determine whether ZCY is non-inferior to HKC in the treatment of DKD, a multicenter, parallel-control, open-label, randomized clinical trial was conducted. METHODS: In this clinical trial, 88 DKD patients were recruited at three centers in Tianjin from January 2018 to December 2019. They were randomized to receive HKC (2.5 g, TID) or ZCY (crude drug amount 75 g, 150 ml, BID) for eight weeks based on routine treatment. The primary outcome was the change of estimated glomerular filtration rate (eGFR). The secondary outcomes included change of serum creatinine (SCr), urinary albumin excretion rate, 24 h urinary protein, urinary albumin-creatinine ratio, glycosylated hemoglobin A1c, symptom scores, and microbiota compositions profiles. RESULTS: The change of eGFR in HKC and ZCY groups were -7.08 ± 24.65 and 2.57 ± 18.49 ml/min/1.73 m2, respectively (p < 0.05). The 95% lower confidence limit for the difference between the estimated means was 1.93 ml/min/1.73 m2, establishing the superiority of ZCY. Compared to HKC, ZCY could significantly decrease SCr and symptom scores (p < 0.05). There were no significant differences in other outcomes between the two groups (p > 0.05). ZCY ameliorated gut microbiota dysbiosis, including increased Prevotellaceae and Lactobacillaceae and decreased Enterobacteriales, Clostridiaceae and Micrococcaceae. No severe adverse events were reported in any group. CONCLUSIONS: ZCY had better efficacy in improving and protecting kidney function. It would be an alternative option to treat DKD, especially those who decline eGFR and gut microbiota dysbiosis. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR-OON-17012076. Registered July 21, 2017.

Zinc alloy-based bone internal fixation screw with antibacterial and anti-osteolytic properties.

There is no targeted effective treatment for patients undergoing internal fixation surgery/two-stage total joint revision surgery with a high risk of postoperative infection and osteolysis, while the rate of reoperation due to infection and osteolysis remains high. In this study, we report a pioneering application of implants made of biodegradable Zn-Ag alloy with active antibacterial and anti-osteolytic properties in three classical animal models, illustrating antibacterial, anti-osteolysis, and internal fixation for fractures. The antibacterial activity of the Zn-2Ag alloy was verified in a rat femur osteomyelitis prevention model, while the anti-osteolytic properties were evaluated using a mouse cranial osteolysis model. Moreover, the Zn-2Ag based screws showed similar performance in bone fracture fixation compared to the Ti-6Al-4V counterpart. The fracture healed completely after 3 months in the rabbit femoral condyle fracture model. Furthermore, the underlying antibacterial mechanism may include inhibition of biofilm formation, autolysis-related pathways, and antibiotic resistance pathways. Osseointegration mechanisms may include inhibition of osteoclast-associated protein expression, no effect on osteogenic protein expression, and no activation of related inflammatory protein expression. The empirical findings here reveal the great potential of Zn-Ag-based alloys for degradable biomaterials in internal fixation surgery/two-stage total joint revision surgery for patients with a high risk of postoperative infection and osteolysis.

A Network Pharmacology-Based Approach to Investigating the Mechanisms of Fushen Granule Effects on Intestinal Barrier Injury in Chronic Renal Failure.

PURPOSE: Fushen Granule (FSG) is a Chinese medicine prepared by doctors for treating patients with chronic renal failure, which is usually accompanied by gastrointestinal dysfunction. Here, we explore the protective effect of FSG on intestinal barrier injury in chronic renal failure through bioinformatic analysis and experimental verification. METHODS: In this study, information on the components and targets of FSG related to CRF is collected to construct and visualize protein-protein interaction networks and drug-compound-target networks using network pharmacological methods. DAVID is used to conduct gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Then, it is validated by in vitro experiments. In this study, the human intestinal epithelial (T84) cells are used and divided into four groups: control group, model group, FSG low-dose group, and FSG high-dose group. After the experiment, the activity of T84 cells is detected by a MTT assay, and the expressions of tight junction protein ZO-1, claudin-1, nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), malondialdehyde (MDA), and cyclooxygenase-2 (COX-2) are examined by immunofluorescence and/or western blotting. RESULTS: Eighty-six potential chronic renal failure-related targets are identified by FSG; among them, nine core genes are screened. Furthermore, GO enrichment analysis shows that the cancer-related signaling pathway, the PI3K-Akt signaling pathway, the HIF1 signaling pathway, and the TNF signaling pathway may play key roles in the treatment of CRF by FSG. The MTT method showed that FSG is not cytotoxic to uremic toxin-induced injured T84 cells. The results of immunofluorescence and WB indicate that compared with the control group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is decreased and protein expressions level of HO-1, MDA, and COX-2 is increased after urinary toxin treatment. Instead, compared with the model group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is increased and protein expressions level of HO-1, MDA, and COX-2 is decreased after FSG treatment. CONCLUSION: FSG had a protective effect on urinary toxin-induced intestinal epithelial barrier injury in chronic renal failure, and its mechanism may be related to the upregulation of Nrf2/HO-1 signal transduction and the inhibition of tissue oxidative stress and inflammatory responses. Screening CRF targets and identifying the corresponding FSG components by network pharmacological methods is a practical strategy to explain the mechanism of FSG in improving gastrointestinal dysfunction in CRF.

Efficacy and safety of Shenyankangfu Tablet, a Chinese patent medicine, for primary glomerulonephritis: A multicenter randomized controlled trial.

BACKGROUND: Shenyankangfu Tablet (SYKFT) is a Chinese patent medicine that has been used widely to decrease proteinuria and the progression of chronic kidney disease. OBJECTIVE: This trial compared the efficacy and safety of SYKFT, for the control of proteinuria in primary glomerulonephritis patients, against the standard drug, losartan potassium. DESIGN, SETTING, PARTICIPANTS AND INTERVENTION: This was a multicenter, double-blind, randomized, controlled clinical trial. Primary glomerulonephritis patients, aged 18-70 years, with blood pressure ≤ 140/90 mmHg, estimated glomerular filtration rate (eGFR) ≥ 45 mL/min per 1.73 m2, and 24-hour proteinuria level of 0.5-3.0 g, were recruited in 41 hospitals across 19 provinces in China and were randomly divided into five groups: SYKFT, losartan potassium 50 mg or 100 mg, SYKFT plus losartan potassium 50 mg or 100 mg. MAIN OUTCOME MEASURES: The primary outcome was change in the 24-hour proteinuria level, after 48 weeks of treatment. RESULTS: A total of 735 participants were enrolled. The percent decline of urine protein quantification in the SYKFT group after 48 weeks was 8.78% ± 2.56% (P = 0.006) more than that in the losartan 50 mg group, which was 0.51% ± 2.54% (P = 1.000) less than that in the losartan 100 mg group. Compared with the losartan potassium 50 mg group, the SYKFT plus losartan potassium 50 mg group had a 13.39% ± 2.49% (P < 0.001) greater reduction in urine protein level. Compared with the losartan potassium 100 mg group, the SYKFT plus losartan potassium 100 mg group had a 9.77% ± 2.52% (P = 0.001) greater reduction in urine protein. With a superiority threshold of 15%, neither was statistically significant. eGFR, serum creatinine and serum albumin from the baseline did not change statistically significant. The average change in TCM syndrome score between the patients who took SYKFT (-3.00 [-6.00, -2.00]) and who did not take SYKFT (-2.00 [-5.00, 0]) was statistically significant (P = 0.003). No obvious adverse reactions were observed in any group. CONCLUSION: SYKFT decreased the proteinuria and improved the TCM syndrome scores of primary glomerulonephritis patients, with no change in the rate of decrease in the eGFR. SYKFT plus losartan potassium therapy decreased proteinuria more than losartan potassium therapy alone. TRIAL REGISTRATION NUMBER: NCT02063100 on ClinicalTrials.gov.

Involvement of CB2 signalling pathway in the development of osteoporosis by regulating the proliferation and differentiation of hBMSCs.

The aim of the present study was to explore the potential mechanism underlying the involvement of CB2 in osteoporosis. Micro-CT was utilized to examine femur bone architecture. Also, real-time PCR and Western blot analysis were utilized to detect the effect of 2-AG on the expression of CB2 and Notch, or the interaction between CB2 and Notch 2. 2-AG treatment up-regulated BMD, Tb.Sp and SMI in OVX mice, whereas proportion of bone volume in total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and bone mineral density (BMD) were decreased in 2-AG-treated OVX mice. Accordingly, 2-AG administration up-regulated Notch 1 expression in OVX mice but had no effect on CB2 and Notch 2 expression. Meanwhile, 2-AG administration promoted the differentiation of hBMSCs in OVX mice, while exhibiting no effect on the proliferation of hBMSCs. Furthermore, in the cellular models, 2-AG treatment also up-regulated Notch 1 expression but had no effect on CB2 and Notch 2 expression, while Notch 1 shRNA had no effect on CB2 and Notch 2 expression. 2-AG promoted cell proliferation and differentiation, which were inhibited by Notch 1 shRNA. NICD had no effect on CB2 level but increased Notch 1 expression, and CB2 shRNA decreased CB2 and Notch 1 expression. Finally, CB2 shRNA inhibited cell proliferation and differentiation, whereas NICD promoted proliferation and differentiation of hBMSCs. Our results provided further evidence for the association of CB2 gene with BMD and osteoporosis, and identified CB2 as a promising target for the treatment of osteoporosis.

Identification of biomarkers and construction of a microRNA­mRNA regulatory network for clear cell renal cell carcinoma using integrated bioinformatics analysis.

With the recent research development, the importance of microRNAs (miRNAs) in renal clear cell carcinoma (CCRCC) has become widely known. The purpose of this study is to screen out the potential biomarkers of renal clear cell carcinoma (CCRCC) by microarray analysis. The miRNA chip (GSE16441) and mRNA chip (GSE66270) related to CCRCC were downloaded from the Gene Expression Omnibus (GEO) database. After data filtering and pretreating, R platform and a series of analysis tools (funrich3.1.3, string, Cytoscape_ 3.2.1, David, etc.) were used to analyze chip data and identify the specific and highly sensitive biomarkers. Finally, by constructing the miRNA -mRNA interaction network, it was determined that five miRNAs (hsa-mir-199a-5p, hsa-mir-199b-5p, hsa-mir-532-3p and hsa-mir-429) and two key genes (ETS1 and hapln1) are significantly related to the overall survival rate of patients.

Biodegradable Zn-Sr alloy for bone regeneration in rat femoral condyle defect model: In vitro and in vivo studies.

Bone defects are commonly caused by severe trauma, malignant tumors, or congenital diseases and remain among the toughest clinical problems faced by orthopedic surgeons, especially when of critical size. Biodegradable zinc-based metals have recently gained popularity for their desirable biocompatibility, suitable degradation rate, and favorable osteogenesis-promoting properties. The biphasic activity of Sr promotes osteogenesis and inhibits osteoclastogenesis, which imparts Zn-Sr alloys with the ideal theoretical osteogenic properties. Herein, a biodegradable Zn-Sr binary alloy system was fabricated. The cytocompatibility and osteogenesis of the Zn-Sr alloys were significantly better than those of pure Zn in MC3T3-E1 cells. RNA-sequencing illustrated that the Zn-0.8Sr alloy promoted osteogenesis by activating the wnt/ß-catenin, PI3K/Akt, and MAPK/Erk signaling pathways. Furthermore, rat femoral condyle defects were repaired using Zn-0.8Sr alloy scaffolds, with pure Ti as a control. The scaffold-bone integration and bone ingrowth confirmed the favorable in vivo repair properties of the Zn-Sr alloy, which was verified to offer satisfactory biosafety based on the hematoxylin-eosin (H&E) staining and ion concentration testing of important organs. The Zn-0.8Sr alloy was identified as an ideal bone repair material candidate, especially for application in critical-sized defects on load-bearing sites due to its favorable biocompatibility and osteogenic properties in vitro and in vivo.

Biodegradable Zn-Cu alloys show antibacterial activity against MRSA bone infection by inhibiting pathogen adhesion and biofilm formation.

Bone and joint-related infections remain the primary and most critical complications of orthopedic surgery. We have innovatively prepared Zn-Cu alloys to achieve outstanding material and antibacterial properties. In this study, we systematically assessed the material properties and antibacterial activity of these Zn-Cu alloys. Our results showed that the Zn-2Cu alloy had the best mechanical properties, biocompatibility, and osteogenic properties. Findings of microbial cultures, CLSM, SEM, and TEM indicated that Zn-2Cu alloy can inhibit both coagulase-positive and coagulase-negative staphylococci, as well as antibiotic-resistant strains (MRSA and MRSE), by preventing the bacteria adhesion and the biofilm formation. Zn-2Cu alloy could broadly affect the expression of MRSA genes associated with adhesion, autolysis, biofilm formation, virulence, and drug resistance. A rat femur intramedullary nail infection-prevention model was established and the Zn-2Cu alloy-treated group showed significant antibacterial activity against MRSA and reduced the inflammatory toxic side-effects and infection-related bone loss. Collectively, our results indicate the potential utility of Zn-Cu alloy implants with 2 wt% Cu in treating orthopedic infections. Statement of significance: Osteomyelitis is a serious complication of orthopedic surgeries. Wide use of antibiotics contributes to the appearance of multi-drug resistant strains like methicillin-resistant staphylococcus aureus (MRSA). Alternatively, anti-osteomyelitis implants with broad-spectrum antibacterial properties can be favorable. Here, the antibacterial performance of biodegradable Zn-Cu alloys was evaluated with four different bacteria strains including antibiotic-resistant strains (MRSA and MRSE). Zn-Cu alloys exert excellent bacterial killing capability in all strains. In a rat femur infection model, the alloy showed significant antibacterial activity against MRSA and reduced inflammatory toxic side-effects as well as infection-related bone loss. The antibacterial property of Zn-2Cu alloy was associated with inhibition of gene expression related to wall synthesis, adhesion, colonization, biofilm formation, autolysis, and secretion of virulence factors in MRSA.

Insights into the Role of Renal Biopsy in Patients with T2DM: A Literature Review of Global Renal Biopsy Results.

INTRODUCTION: Renal biopsy performed in patients with type 2 diabetes mellitus (T2DM) for atypical or suspected diabetic kidney disease (DKD) reveals one of three possibilities: diabetic nephropathy (DN, pathological diagnosis of DKD), nondiabetic kidney disease (NDKD) and DN plus NDKD (mixed form). NDKD (including the mixed form) is increasingly being recognized worldwide. With the emerging concept of DKD and the complexity of routine application of renal biopsy, the identification of "clinical indicators" to differentiate DKD from NDKD has been an area of active research. METHODS: The PubMed database was searched for relevant articles mainly according to the keyword search method. We reviewed prevalence of the three types of DKD and different pathological lesions of NDKD. We also reviewed the clinical indicators used to identify DKD and NDKD. RESULTS: The literature search identified 40 studies (5304 data) worldwide between 1977 and 2019 that looked at global renal biopsy and pathological NDKD lesions. The overall prevalence rate of DN, NDKD and DN plus NDKD is reported to be 41.3, 40.6 and 18.1%, respectively. In Asia, Africa (specifically Morocco and Tunisia) and Europe, the most common isolated NDKD pathological type is membranous nephropathy, representing 24.1, 15.1 and 22.6% of cases, respectively. In contrast, focal segmental glomerulosclerosis is reported to be the primary pathological type in North America (specifically the USA) and Oceania (specifically New Zealand), representing 22% and 63.9% of cases, respectively. Tubulointerstitial disease accounts for a high rate in the mixed group (21.7%), with acute interstitial nephritis being the most prevalent (9.3%), followed by acute tubular necrosis (9.0%). Regarding clinical indicators to differentiate DKD from NDKD, a total of 14 indicators were identified included in 42 studies. Among these, the most commonly studied indicators included diabetic retinopathy, duration of diabetes, proteinuria and hematuria. Regrettably, indicators with high sensitivity and specificity have not yet been identified. CONCLUSION: To date, renal biopsy is still the gold standard to diagnose diabetes complicated with renal disease, especially when T2DM patients present atypical DKD symptoms (e.g. absence of diabetic retinopathy, shorter duration of diabetes, microscopic hematuria, sub-nephrotic range proteinuria, lower glycated hemoglobin, lower fasting blood glucose). We conclude that renal biopsy as early as possible is of great significance to enable personalized treatment to T2DM patients.

Antibacterial and antibiofilm effects of flufenamic acid against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are one of the most serious surgery complications, and their prevention is of utmost importance. Flufenamic acid is a non-steroid anti-inflammatory drug approved for clinical use to relieve inflammation and pain in rheumatoid arthritis patients. In this study, we explored the antibacterial efficacy of flufenamic acid and the mechanisms underlying this effect. By using minimal inhibitory concentration (MIC), time-kill, resistance induction assays, and the antibiotic synergy test, we demonstrated that flufenamic acid inhibited the growth of methicillin-resistant staphylococci and did not induce resistance when it was used at the MIC. Furthermore, flufenamic acid acted synergistically with the beta-lactam antibiotic oxacillin and did not show significant toxicity toward mammalian cells. The biofilm inhibition assay revealed that flufenamic acid could prevent biofilm formation on medical implants and destroy the ultrastructure of the bacterial cell wall. RNA sequencing and quantitative RT-PCR indicated that flufenamic acid inhibited the expression of genes associated with peptidoglycan biosynthesis, beta-lactam resistance, quorum sensing, and biofilm formation. Furthermore, flufenamic acid efficiently ameliorated a local infection caused by MRSA in mice. In conclusion, flufenamic acid may be a potent therapeutic compound against MRSA infections and a promising candidate for antimicrobial coating of implants and surgical devices.

Hirudin Ameliorates Renal Interstitial Fibrosis via Regulating TGF-ß1/Smad and NF-κB Signaling in UUO Rat Model.

PURPOSE: Hirudin, a polypeptide structure containing 65 amino acids, is a potent natural thrombin inhibitor with anticoagulant property extracted from Hirudo medicinalis. It has been reported to have anti-inflammatory and antifibrotic property. Here we explored the renoprotective effect of hirudin on unilateral ureteral obstruction (UUO) induced renal interstitial fibrosis (RIF). METHODS: Rats were randomly divided into five groups: sham group, UUO alone group, and three UUO + hirudin-treatment groups (10, 20, or 40 IU/kg/d, for 14 continuous days). At the end of the experiment period, animals were sacrificed. Pathologic changes in renal specimens were observed using hematoxylin and eosin (HE) staining and Masson staining. The expressions of collagen III (Col III), fibronectin (FN), α-smooth muscle actin (α-SMA), protease-activated receptor 1 (PAR-1), and proteins in the TGF-ß1/Smad and NF-κB pathways in renal tissues were examined by immunohistochemistry and/or Western blotting. RESULTS: HE and Masson staining showed that hirudin-treated UUO rats had lower extent of renal injury and deposition of extracellular matrix (ECM) in renal interstitium than those in the UUO group. The results of immunohistochemistry and WB indicated decreased protein expressions of Col III, FN, α-SMA, PAR-1, and inflammatory markers such as tumor necrosis factor-α and interleukin-6 after hirudin treatment. Furthermore, hirudin reduced the expressions of transforming growth factor ß1 (TGF-ß1), phosphorylated-Smad2, and phosphorylated-Smad3 in the UUO model. In parallel, we found inhibited nuclear factor-κB (NF-κB) signaling after hirudin treatment, with downregulated protein expressions of P65, phosphorylated-P65, and phosphorylated-iκBα and increased iκBα. CONCLUSION: Hirudin improves kidney injury and suppresses inflammatory response and ECM accumulation in UUO rats; its underlying mechanism may be associated with the inhibition of TGF-ß1/Smad and NF-κB signaling.