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Objective. This study is designed to find out the molecular targets of effective Chinese medicine Ziyin Mingmu pills (ZMPs) in treating age-related macular degeneration (AMD) based on network pharmacology and experimental data. Methods. A comprehensive network pharmacology strategy that consists of three sequential modules (drug-disease target molecular docking, enrichment analysis, and external verification) was carried out to identify potential targets of ZMPs acting on AMD. Results. The active ingredients of ZMPs targeting 66 genes have effects on the process of AMD. GO and KEGG pathway enrichment analyses suggested that response to oxidative stress, regulation of angiogenesis, and lipid and atherosclerosis might serve as the most important signaling pathways in ZMPs for AMD treatment. Combined with the GSE29801 dataset for further analysis, two key genes, EGFR and VEGFA, were identified. Immune infiltration analysis showed that there was a strong association between EGFR and immune cell content. In addition, images were acquired following 24 h in the scratch experiment showed that ZMPs can reduce the percentage of wound healing distance. The Western blot assay found that ZMPs increased the expression of EGFR and decreased the expression of VEGFA. Conclusion. This study sheds light on some mechanisms of ZMP therapy for AMD, particularly the effect of ZMP on the oxidative stress in RPE and cell survival and angiogenesis in AMD. We propound ZMPs as a promising strategy to intervene in the process of AMD.

Peripheral arterial disease (PAD) is a progressive atherosclerotic disorder characterized by blockages of the arteries supplying the lower extremities. Ischemia initiates oxidative damage and mitochondrial dysfunction in the legs of PAD patients, causing injury to the tissues of the leg, significant decline in walking performance, leg pain while walking, and in the most severe cases, nonhealing ulcers and gangrene. Current clinical trials based on cells/stem cells, the trophic factor, or gene therapy systems have shown some promising results for the treatment of PAD. Biomaterial matrices have been explored in animal models of PAD to enhance these therapies. However, current biomaterial approaches have not fully met the essential requirements for minimally invasive intramuscular delivery to the leg. Ideally, a biomaterial should present properties to ameliorate oxidative stress/damage and failure of angiogenesis. Recently, we have created a thermosensitive hyaluronic acid (HA) hydrogel with antioxidant capacity and skeletal muscle-matching stiffness. Here, we further optimized HA hydrogels with the cell adhesion peptide RGD to facilitate the development of vascular-like structures in vitro. The optimized HA hydrogel reduced intracellular reactive oxygen species levels and preserved vascular-like structures against H2O2-induced damage in vitro. HA hydrogels also provided prolonged release of the vascular endothelial growth factor (VEGF). After injection into rat ischemic hindlimb muscles, this VEGF-releasing hydrogel reduced lipid oxidation, regulated oxidative-related genes, enhanced local blood flow in the muscle, and improved running capacity of the treated rats. Our HA hydrogel system holds great potential for the treatment of the ischemic legs of patients with PAD.

Background Diabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway. Methods Network pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC). Results Overproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor. Conclusions SDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

Xiang Li,1– 3 Yijing Yang,2,3 Yan Song,1– 3 Fujiao Nie,1– 3 Chaojun Fu,2,3 Yuhui Qin2– 4 1Ophthalmology Department, the First Affiliated Hospital, Hunan University of Chinese Medicine, Changsha, 410007, People’s Republic of China; 2Hunan Provincial Key Laboratory for the Prevention and Treatment of Ophthalmology and Otolaryngology Diseases with Chinese Medicine, Hunan University of Chinese Medicine, Changsha, 410208, People’s Republic of China; 3Hunan Engineering Technological Research Center for the Prevention and Treatment of Otolaryngologic Disease and Protection of Visual Function with Chinese Medicine, Changsha, 410208, People’s Republic of China; 4Institute of Chinese Medicine of Hunan Province, Changsha, 410006, People’s Republic of ChinaCorrespondence: Yuhui Qin, Institute of Chinese Medicine of Hunan Province, Lushan Road, Yuelu District, Changsha, 410006, People’s Republic of China, Tel +86 13873120865, Email 1243695133@qq.comPurpose: To evaluate the effects of Shuangdan Mingmu (SDMM) capsule on diabetic retinopathy in rats by regulating miRNAs.Materials and Methods: Streptozotocin (STZ) (50 mg/kg) was successfully used to induce diabetes in male Sprague-Dawley rats, which were randomly assigned to a group taking SDMM capsules (“diabetic+SDMM”) or a control group (“diabetic”), and the normal group (n=10/group). The diabetic+SDMM capsule group received 1.89g/kg/d of SDMM capsule by gavage, whereas the other groups received the same amount of distilled water. After 12-weeks of gavage, the retina was removed from all rats for histopathological analysis, and miRNA sequencing experiments were carried out to identify the differential expression of miRNAs. These results were then confirmed by quantitative real-time polymerase chain reaction (qRT-PCR).Results: SDMM capsules improved retinal morphology, restored the number of cells in the ganglion cell layer (p< 0.0001) and reduced apoptosis in all retinal layers (p values in the outer nuclear layers, inner nuclear layers and ganglion cell layers 0.0001, 0.0147, 0.0034, respectively). In addition, miRNA expression was changed in rats taking SDMM capsules. Compared with the diabetic group, six miRNAs were up-regulated and four miRNAs were down-regulated in the diabetic+SDMM capsule group. The qRT-PCR validation results showed that the expression levels of miR-450b-5p, miR-1249 and miR-155-5p were consistent with the trend of miRNA sequencing results, and were all up-regulated after SDMM capsule treatment. Target gene prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed miRNAs showed that these pathways were mainly concentrated in the focal adhesions and PI3K/Akt, MAPK, and neural factor signaling pathways.Conclusion: SDMM capsules may prevent and treat diabetic retinopathy by regulating the expression of miR-450b-5p, miR-1249 and miR-155-5p.Keywords: SDMM capsule, diabetic retinopathy, traditional Chinese medicine, miRNAs, bioinformatics analysis

Peripheral arterial disease (PAD) is initiated by progressive atherosclerotic blockages of the arteries supplying the lower extremities. The most common presentation of PAD is claudication (leg pain and severe walking limitation), with many patients progressing to limb threatening ischemia and amputation. Biomaterial approaches are just beginning to be explored in the therapy of PAD with different materials now being evaluated for the delivery of cells or growth factors in animal models of PAD. A biomaterial matrix optimized for minimally invasive injection in the ischemic leg muscles of patients with PAD is urgently needed. There are several important requirements for optimal delivery, retention, and performance of a biomaterial matrix in the mechanically, histologically, and biochemically dynamic intramuscular environment of the PAD leg. Ideally, the material should have mechanical properties matching those of the recipient muscle, undergo minimal swelling, and should introduce properties that can ameliorate the mechanisms operating in PAD like oxidative stress and damage. Here we have developed an injectable, antioxidative, and thermosensitive hydrogel system based on hyaluronic acid (HA). We first synthesized a unique crosslinker of disulfide-modified poloxamer F127 diacrylate. This crosslinker led to the creation of a thermosensitive HA hydrogel with minimal swelling and muscle-matching mechanical properties. We introduced unique disulfide groups into hydrogels which functioned as an effective reactive oxygen species scavenger, exhibited hydrogen peroxide (H2O2)-responsive degradation, and protected cells against H2O2-induced damage. Our antioxidative thermosensitive HA hydrogel system holds great potential for the treatment of the ischemic legs of patients with PAD.

Following traumatic or ischemic brain injury, rapid cell death and extracellular matrix degradation lead to the formation of a cavity at the brain lesion site, which is responsible for prolonged neurological deficits and permanent disability. Transplantation of neural stem/progenitor cells (NSCs) represents a promising strategy for reconstructing the lesion cavity and promoting tissue regeneration. In particular, the promotion of neuronal migration, organization, and integration of transplanted NSCs is critical to the success of stem cell-based therapy. This is particularly important for the cerebral cortex, the most common area involved in brain injuries, because the highly organized structure of the cerebral cortex is essential to its function. Biomaterials-based strategies show some promise for conditioning the lesion site microenvironment to support transplanted stem cells, but the progress in demonstrating organized cell engraftment and integration into the brain is very limited. An effective approach to sufficiently address these challenges has not yet been developed. Here, we have implemented a digital light-processing-based 3D printer and printed hydrogel scaffolds with a designed shape, uniaxially aligned microchannels, and tunable mechanical properties. We demonstrated the capacity to achieve high shape precision to the lesion site with brain tissue-matching mechanical properties. We also established spatial control of bioactive molecule distribution within 3D printed hydrogel scaffolds. These printed hydrogel scaffolds have shown high neuro-compatibility with aligned neuronal outgrowth along with the microchannels. This study will provide a biomaterial-based approach that can serve as a protective and guidance vehicle for transplanted NSC organization and integration for brain tissue regeneration after injuries.

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Objective Vitamin K2 (MK-4, menaquinone 4) plays an important role in osteoprotection. The present study aimed to examine the effect of MK-4 on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in vitro and probed the potential signaling pathway. Design PDLSCs were isolated from extracted premolars by tissue block culture method and were identified by flow cytometry. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to determine the effect of MK-4 on the proliferation of PDLSCs. Alkaline phosphatase (ALP) activity was analyzed quantitatively, and extracellular matrix mineralization was examined by Alizarin Red S staining. The mRNA and protein expression levels of ALP, Runx Family Transcription Factor 2 (Runx2), osteocalcin (OCN), and Sp7 Transcription Factor (SP7; Osterix) were measured by qRT-PCR and Western blot. In addition, after adding the inhibitor XAV-939, Western blot was used to assess the correlation with the Wnt/β-catenin signaling pathway. The above results were obtained by observing at least three fields randomly, and each experiment was repeated at least three times. Results This study found that 10−5 M MK-4 significantly promoted the osteogenic differentiation of PDLSCs. Gene and protein expression levels of ALP, Runx2, OCN, and Osterix were all upregulated compared with control. Remarkably, after blocking the Wnt/β-catenin signaling pathway with XAV-939, the effect of MK-4 was apparently reversed. Conclusion These results demonstrate that MK-4 can promote the osteogenic differentiation of PDLSCs, which is likely related to the activation of the Wnt/β-catenin signaling pathway.

Objective To investigate the effect of resveratrol (RSV) on orthodontic tooth movement (OTM) and orthodontic induced root resorption (OIRR) in rats. Methods Thirty-six male Wistar rats used in this study were randomly divided into three groups of 12 animals each. All test subjects underwent a 50 g orthodontic force each, generated from a nickel-titanium closed-coil spring. The control group were fed carboxymethylcellulose (CMC) while rats in other two groups were fed 5 mg/kg/d RSV or 10 mg/kg/d RSV (dissolved in CMC). After 14 days of OTM, all rats were sacrificed, after which each group was randomly divided into two subgroups (6 test subjects in each subgroup). One subgroup was used to measure the amount of OTM and assessed by hematoxylin and eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry staining of Receptor Activator of Nuclear Factor-κ B Ligand (RANKL), Osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX2), as well as Osteocalcin (OCN). The second subgroup was used to analyze OIRR via scanning electron microscopy. Results Compared with the control group, the RSV groups showed a significant decrease in the distance of OTM and the OIRR ratio (p＜0.05). The number of TRAP positive osteoclasts and the expression of RANKL in periodontal tissue of the RSV groups were significantly inhibited (p＜0.01) while the expression of OPG, RUNX2, and OCN were remarkably promoted (p＜0.05). The effect of 10 mg/kg/d RSV group was more obvious than that of 5 mg/kg/d RSV group (p＜0.05). Conclusions RSV could reduce the extent of OTM and root resorption areas.

Objective The aim of this study was to evaluate the influence of matrine on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) as well as on bone metabolism in a rat rapid maxillary expansion (RME) model. Methods In in vitro experiments, rat BMSCs were adopted and cell proliferation of BMSCs was measured. Meanwhile, the osteogenic differentiation of BMSCs was detected by alkaline phosphatase (ALP) activity assay, Alizarin red S staining and gene expression. In vivo bone regeneration was analyzed in a rat RME model. Eighteen rats were divided into 3 groups: one group without any treatment, one group receiving only RME, and a group with RME and matrine treatment. After 2 weeks, new bone formation was detected by Micro-CT and histology. Immunohistochemical staining was used to evaluate ALP and BMP2 expression. Results Overall, we found that matrine upregulated cell proliferation dose-dependently. Also, ALP activity and mineralized matrix generation were enhanced. Moreover, the osteoblast-related gene expression (ALP, bone sialoprotein and osteocalcin) by BMSCs was also promoted. Micro-CT revealed that matrine significantly promoted in vivo bone formation after 2 weeks. Concomitantly, histological examination of haematoxylin-eosin, safranin-O and toluidine blue staining confirmed these findings. In addition, the levels of ALP and BMP2 in the palatal suture tissues of rats with matrine treatment were the highest among three groups. Conclusion This work suggests that matrine regulates osteogenesis and enhances bone regeneration. Matrine treatment may be beneficial in improving the stability of maxillary expansion.