A novel 3D printed bioactive scaffolds with enhanced osteogenic inspired by ancient Chinese medicine HYSA for bone repair.

Some traditional Chinese medicine (TCM) has been applied in bone repair, however, hydroxy-safflower yellow A (HYSA), one composition of safflower of the typical invigorating the circulation of TCM, has little been studied in orthopedics field for osteogenesis and angiogenesis clinically. Herein, we hypothetically speculated that the synthetic bioactive glasses (BG, 1393) scaffolds carried HYSA by a 3D print technique could enhance osteogenic repair properties. Notably, scaffolds coating chitosan/sodium alginate endowed with excellent drug control release ability, and significantly improved the BG mechanical strength. HYSA was loaded into BG scaffolds by coating chitosan/sodium alginate film, and the osteogenesis and angiogenesis of the HYSA/scaffolds were evaluated in vitro and in vivo. In vitro the cell culture results exhibited that the high dose of HYSA (0.5 mg/ml) loaded scaffolds can promote the proliferation of bone marrow stromal cells (rBMSCs) and migration, tubule formation of human umbilical vein endothelial cells (HUVECs). The active alkaline phosphatase (ALP) of rBMSCs can also be improved by the high dose of HYSA/scaffolds. Results of qRT-PCR and Western blot indicated that the high dose of HYSA/scaffolds can up-regulate ALP, OCN, OPN and RUNX-2 expression and relative protein secretion of the HIF-1α and BMP-2. In the animal experiment, the high dose of HYSA/scaffolds has a significantly better capacity to promote new bone formation than the undoped scaffolds at 8 weeks post-surgery. Thus, our results claimed that the novel HYSA/scaffolds hold the substantial potential to be further developed as effective and safe bone tissue engineering biomaterials for bone regeneration by combining enhanced osteogenesis and angiogenesis.

A naturally occurring point mutation in the hyaluronidase gene (hysA1) of Staphylococcus aureus UAMS-1 results in reduced enzymatic activity.

Hyaluronic acid is a high-molecular-weight polysaccharide that is widely distributed in animal tissues. Bacterial hyaluronidases degrade hyaluronic acid as secreted enzymes and have been shown to contribute to infection. Staphylococcus aureus UAMS-1 is a clinical isolate that codes for two hyaluronidases (hysA1 and hysA2). Previous research has shown the presence of a full-length HysA1 protein from the S. aureus UAMS-1 strain with no evidence of enzymatic activity. In this study, the coding and upstream promoter regions of hysA1 from the S. aureus UAMS-1 strain were cloned, sequenced, and compared to the hysA1 gene from the S. aureus Sanger 252 strain. A single base change resulting in an E480G amino acid change was identified in the hysA1 gene from the S. aureus UAMS-1 strain when compared to the hysA1 gene from S. aureus Sanger 252. A plasmid copy of hysA1 from S. aureus Sanger 252 transduced into an S. aureus UAMS-1 hysA2 deletion mutant strain restored near wild-type levels of enzymatic activity. Homology modeling of the HysA1 hyaluronidase was performed with SWISS-MODEL using hyaluronidase from Streptococcus pneumoniae as the template, followed by a series of structural analyses using PyMOL, PLIP, PDBsum, and HOPE servers. This glutamic acid is highly conserved among hyaluronidases from Staphylococcus and other gram-positive bacteria. A series of structural analyses suggested that Glu-480 in HysA1 is critically responsible for maintaining the structural and functional ensemble of the catalytic and tunnel-forming residues, which are essential for enzyme activity. The missense mutation of Glu-480 to Gly introduces a loss of side chain hydrogen bond interactions with key residues Arg-360 and Arg-364, which are responsible for the tunnel topology, resulting in displacement of the substrate from an ideal position for catalysis through a localized conformational change of the active site. There is a high degree of relatedness among several gram-positive bacterial hyaluronidases; the loss of enzymatic activity of HysA1 in the S. aureus UAMS-1 strain is most likely caused by the mutation identified in our study. The role of hyaluronidase in staphylococcal infection and the redundancy of this gene are yet to be determined.

Diversity and Standard Nomenclature of Staphylococcus aureus Hyaluronate Lyases HysA and HysB.

Bacterial hyaluronate lyases (Hys) are enzymes that degrade hyaluronic acid in their host and are known to contribute to the pathogenesis of several illnesses. The first two identified Hys genes in Staphylococcus aureus were registered as hysA1 and hysA2. However, their annotations have been mistakenly reversed in some registered assembly data, and different abbreviations (hysA and hysB) in some reports complicates comparative analysis of Hys proteins. We investigated the hys loci of S. aureus genome sequences registered in public databases, analyzed the homology, and defined hysA as hys located in the core genome surrounded by a lactose metabolic operon and a ribosomal protein cluster present in almost all strains and hysB as that located on the genomic island νSaß of the accessory genome. Homology analysis of the amino acid sequences of HysA and HysB revealed that they are conserved among clonal complex (CC) groups with a few exceptions. Thus, we propose a new nomenclature for S. aureus Hys subtypes: HysACC*** for HysA and HysBCC*** for HysB, with the asterisks representing the clonal complex number of the S. aureus strain producing the Hys subtype. The application of this proposed nomenclature will facilitate the intuitive, straightforward, and unambiguous designation of Hys subtypes and contribute to enhancing comparative studies in this regard. IMPORTANCE Numerous whole-genome sequence data for Staphylococcus aureus harboring two hyaluronate lyase (Hys) genes have been registered. However, the assigned gene names for hysA1 and hysA2 are incorrect in some assembled data, and in some cases, the genes are annotated differently as hysA and hysB. This creates confusion with respect to the nomenclature of Hys subtypes and complicates analysis involving Hys. In this study, we compared the homology of Hys subtypes and observed that to some extent, their amino acid sequences are conserved in each clonal complex group. Hys has been implicated as an important virulence factor, but relative sequence heterogeneity among S. aureus clones raises the question of whether Hys activities are different among these clones. Our proposed Hys nomenclature will facilitate comparison of the virulence of Hys, as well as discussions of the subject.

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.

Pharmacokinetics study of main effective constituents in Danhong Injection after compatibilities in rats with cerebral ischemia reperfusion injury / 中草药

Objective: Using total quantum statistical moment analysis to evaluate the influence of the main effective constituents in Danhong Injection on the total pharmacokinetic parameters after orthogonal design to research internal compatibility rule of traditional Chinese medicine (TCM) compound. Methods: Using modified middle cerebral artery occlusion (MCAO) method to establish rat disease model of focal cerebral ischemia-reperfusion (I/R) injury. According to the orthogonal design L9 (34) composed of the main effective constituents (danshensu, protocatechuic aldehyde, salvianolic acid B, and HYSA) of Danhong Injection, nine combinations with different dosage ratios were formed, which were used for iv administration in MCAO rats to study the pharmacokinetics of effective constituents after compatibility. Using non-compartmental model of DAS 3.2.6 software to fit the pharmacokinetic parameters and using total quantum statistical moment analysis method to calculate the main total quantum statistical moment parameters. The influence of different compatibilities on the total pharmacokinetics parameters was analyzed by orthogonal test. Results: There were different effects of the main effective constituents with orthogonal compatibilities on the total quantum statistical moment parameters. Danshensu had the strongest effect on the total AUC and had a significant effect (P < 0.05). A3B3C2D1 was the preferred dose combination for AUCt. HYSA had the strongest effect on the total MRT and its optimal ingredient composition was A1B3C1D1. Conclusion: The total quantum statistical moment analysis could combine the pharmacokinetics of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B, and HYSA, and express the total pharmacokinetic behaviors. The total quantum statistical moment analysis method can be used to study the pharmacokinetics of multi-component in TCM compound prescription, which can provide the method for research on internal compatibility rule for TCM compound.

In vivo pharmacokinetic study on active constituents in Danhong Injection against cerebral ischemia of rats / 中草药

Objective: To develop an HPLC-DAD method for the simultaneous determination of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B, and hydroxysafflor yellow A (HYSA) in cerebral ischemic injury of rats, in order to provide an accurate and reliable analytical method to study the pharmacokinetics. Methods: The middle cerebral artery occlusion (MCAO) model was established, in which all the rats were iv injected with the active constituents in Danhong Injection simultaneously (danshensu, protocatechuic aldehyde, salvianolic acid B, and HYSA). The analysis of the measured components' plasma concentration was carried at 30℃ on the reversed-phase column of Agilent Eclipse XDB-C18 (150 mm×4.6 mm, 5 μm) and eluted with acetonitrile and water containing 0.4% phosphate acid as mobile phase in gradient mode. Detection wavelengths were 280 nm for danshensu, protocatechuic acid, vanillic acid, salvianolic acid B and 403 nm for HYSA. The propyl p-hydroxybenzoate was used as the internal standard (IS). Results: Protocatechuic aldehyde rapidly metabolized in MCAO rats, but its metabolites, protocatechuic acid and vanillic acid, were easily detected. The linear ranges of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B, and HYSA were 0.35-140 (R2=0.9998), 0.15-60 (R2=0.9990), 0.05-20 (R2=0.9988), 0.25-100 (R2=0.9996), and 0.075-30 mg/L (R2=0.9985), respectively. The mean recoveries were between 80% and 120% and the RSD value of intra-day and inter-day was less than 10%. The results of stability meet the requirements for biopharmaceutical analysis. The main pharmacokinetic parameters of danshensu, protocatechuic acid, vanillic acid, salvianolic acid B and HYSA were as follows: AUC0~∞ were (1143.862±230.840), (427.024±59.293), (135.785±47.631), (418.631±66.242), (288.788±87.809) mg·min/L and t1/2z were (71.496±29.067), (82.379±26.279), (40.331±6.006), (125.164±59.709), (177.577±112.836) min. Conclusion: The method is simple, rapid, precise and stable, which can be used to the pharmacokinetic studies on the compatibilities of danshensu, protocatechuic aldehyde, salvianolic acid B, and HYSA in MCAO rats.

Effects of hydroxysafflor yellow A on iNOS expression in rats of focal cerebral ischemic injury / 中国药理学通报

Aim This was to study the effects of hydroxysafflor yellow A(HYSA)on iNOS expression in rats following focal cerebral ischemia. Methods Rat cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion. The expression levels of iNOS were examined using immunohistochemical method. The neurological outcome and the cerebral infarct area were evaluated. Results The expression levels of iNOS in HYSA groups were significantly lower than those in ischemic model group. Treatment with HYSA also decreased the cerebral infarct area and the neurological deficit score. Conclusion HYSA induced down regulation of iNOS expression, which may mediate the protective effect of HYSA on cerebral ischemia.