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Huihao Zhang,1,* Sai Yao,1,* Zhiguo Zhang,1,* Chengcong Zhou,1 Fangda Fu,1 Yishan Bian,1 Huan Luo,2 Yan Li,1 Shuxin Yan,1 Yuying Ge,1 Yuying Chen,3 Kunyu Zhan,4 Yanzhi Ge,1 Zuxiang Chen,1 Ming Yue,5 Xiaofeng Li,6 Weibin Du,1,7 Hongting Jin,1 Peijian Tong,1 Hongfeng Ruan,1,8 Chengliang Wu1 1Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China; 2Department of Pharmacy, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3The Fourth Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China; 4The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China; 5Department of Physiology, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China; 6Department of Orthopedics and Traumatology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China; 7Research Institute of Orthopedics, The Affiliated JiangNan Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China; 8Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hongfeng Ruan; Peijian TongInstitute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, People’s Republic of ChinaEmail rhf@zcmu.edu.cn; peijiantongzjtcm@163.comPurpose: To explore the pharmacological mechanisms of Liuwei Dihuang Decoction (LWDHD) against intervertebral disc (IVD) degeneration (IVDD) via network pharmacology analysis combined with experimental validation.Methods: First, active ingredients and related targets of LWDHD, as well as related genes of IVDD, were collected from public databases. The protein–protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed to predict the core targets and pathways of LWDHD against IVDD. Secondly, the IVDD model of mice treated with LWDHD was selected to validate the major targets predicted by network pharmacology.Results: By searching the intersection of the active ingredient targets and IVDD targets, a total of 110 targets matched the related targets of 30 active ingredients in LWDHD and IVDD were retrieved. PPI network analysis indicated that 17 targets, including Caspase-3, IL-1β, P53, etc., were hub targets. GO and KEGG enrichment analyses showed that the apoptosis pathway was enriched by multiple targets and served as the target for in vivo experimental study validation. The results of animal experiments revealed that LWDHD administration not only restored the decrease in disc height and abnormal degradation of matrix metabolism in IVDD mice but also reversed the high expression of Bax, Caspase-3, IL-1β, P53, and low expression of Bcl-2, thereby inhibiting the apoptosis of IVD tissue and ameliorating the progression of IVDD.Conclusion: Using a comprehensive network pharmacology approach, our findings predicted the active ingredients and potential targets of LWDHD intervention for IVDD, and some major target proteins involved in the predictive signaling pathway were validated experimentally, which gave us a new understanding of the pharmacological mechanism of LWDHD in treating IVDD at the comprehensive level.Keywords: intervertebral disc degeneration, Liuwei Dihuang decoction, network pharmacology, pharmacological mechanisms, apoptosis

Zhiguo Zhang,1,2,* Fangda Fu,1,2,* Yishan Bian,1,2,* Huihao Zhang,1,2 Sai Yao,1,2 Chengcong Zhou,1,2 Yuying Ge,2 Huan Luo,3 Yuying Chen,4 Weifeng Ji,1 Kun Tian,1 Ming Yue,5 Weibin Du,6 Hongting Jin,1 Peijian Tong,1 Chengliang Wu,1,2 Hongfeng Ruan1,2 1Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou, People’s Republic of China; 2The First Clinical Medical College of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 3Department of Pharmacy, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 4The Fourth Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 5Department of Physiology, College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; 6Research Institute of Orthopedics, The Affiliated Jiangnan Hospital of Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hongfeng Ruan; Chengliang Wu, Institute of Orthopaedics and Traumatology, The First Affiliated Hospital of Zhejiang Chinese Medical University, 548 Binwen Road, Hangzhou, 310053, People’s Republic of China, Fax +86 571 86613684, Email rhf@zcmu.edu.cn; wcl@zcmu.edu.cnBackground: With the rapid growth of the elderly population, the incidence of osteoarthritis (OA) increases annually, which has attracted extensive attention in public health. The roles of dietary intake in controlling joint disorders are perhaps one of the most frequently posed questions by OA patients, while the information about the interaction between dietary intake and OA based on scientific research is limited. α-Chaconine is the richest glycoalkaloid in eggplants such as potatoes. Previous evidence suggests that α-Chaconine is a toxic compound to nervous and digestive systems with potentially severe and fatal consequences for humans and farm animals, but its effect on OA development remains obscure.Objective: To determine whether α-Chaconine deteriorates OA progression through sensory innervation and chondrocyte pyroptosis via regulating nuclear factor-κB (NF-κB) signaling, providing evidence for a possible linkage between α-Chaconine and OA progression.Methods: We established a mouse OA model by destabilization of medial meniscus (DMM) surgery and then intra-articular injection of 20 or 100 μM α-Chaconine into the OA mice for 8 and 12 weeks. The severity of OA progression was evaluated by histological staining and radiographic analyses. The expressions of matrix metabolic indicators, Col2, Mmp3, and Mmp13, as well as pyroptosis-related proteins, Nlrp3, Caspase-1, Gsdmd, IL-1β, IL-18, were determined by immunohistochemistry. And the changes in sensory nerve ingrowth and activity of NF-κB signaling were determined by immunofluorescence.Results: We found that α-Chaconine could exacerbate mouse OA progression, resulting in subchondral sclerosis, osteophyte formation, and higher OARSI scores. Specifically, α-Chaconine could augment cartilage matrix degradation and induce chondrocyte pyroptosis and nerve ingrowth. Mechanistical analysis revealed that α-Chaconine stimulated NF-κB signaling by promoting I-κB α phosphorylation and p65 nuclear translocation.Conclusion: Collectively, our findings raise the possibility that α-Chaconine intake can boost chondrocyte pyroptosis and nerve ingrowth to potentiate OA progression by activating NF-κB signaling.Graphical Abstract: Keywords: α-Chaconine, osteoarthritis, pyroptosis, nerve ingrowth, NF-κB signaling

Cadmium (Cd) is a ubiquitous toxic metal and environmental pollutant. Increasing studies have shown that Cd exposure increases the incidence of various endocrine system diseases, including thyrotoxicity reflected by thyroid structural damage and endocrine toxicity. However, the observed outcomes are complex and conflicting, leading to the mechanism of Cd-induced thyrotoxicity remaining obscure. In this study, 4-week-old male C57BL/6 mice were given 2 or 7 mg/kg Cadmium Chloride (CdCl

Aberrant mechanical factor is one of the etiologies of the intervertebral disc (IVD) degeneration (IVDD). However, the exact molecular mechanism of spinal mechanical loading stress-induced IVDD has yet to be elucidated due to a lack of an ideal and stable IVDD animal model. The present study aimed to establish a stable IVDD mouse model and evaluated the effect of aberrant spinal mechanical loading on the pathogenesis of IVDD. Eight-week-old male mice were treated with lumbar spine instability (LSI) surgery to induce IVDD. The progression of IVDD was evaluated by μCT and Safranin O/Fast green staining analysis. The metabolism of extracellular matrix, ingrowth of sensory nerves, pyroptosis in IVDs tissues were determined by immunohistological or real-time PCR analysis. The apoptosis of IVD cells was tested by TUNEL assay. IVDD modeling was successfully produced by LSI surgery, with substantial reductions in IVD height, BS/TV, Tb.N. and lower IVD score. LSI administration led to the histologic change of disc degeneration, disruption of the matrix metabolism, promotion of apoptosis of IVD cells and invasion of sensory nerves into annulus fibrosus, as well as induction of pyroptosis. Moreover, LSI surgery activated Wnt signaling in IVD tissues. Mechanical instability caused by LSI surgery accelerates the disc matrix degradation, nerve invasion, pyroptosis, and eventually lead to IVDD, which provided an alternative mouse IVDD model.

Background Whole body vibration (WBV) is a non-pharmaceutical therapy that has been widely incorporated into clinical practice for musculoskeletal disorders, including low back pain (LBP). Intervertebral disc (IVD) degeneration (IVDD) is clinically associated with LBP and is known as the main cause for LBP. However, cumulative evidence also suggested WBV might have an adverse impact on IVDs. Moreover, previous studies have been focusing on the effects of WBV on healthy mice, rather than those suffering from IVDD. Thus, uncertainties still exist concerning the effects of WBV on IVDs undergoing IVDD. This study was aiming to evaluate the effects of WBV intervention on the development and progression of IVDD mouse model induced by lumbar spine instability (LSI) surgery. Methods LSI surgery, by resecting the lumbar 3 rd -5 th spinous processes along with the supraspinous and interspinous ligaments, was conducted in 10-week-old male mice which then received WBV treatment (1 h per day, 5 days per week, at 3 Hz with peak acceleration at 0.4 g) or sham treatment. The progression of IVDD was evaluated by MRI, μCT and histological analyses after WBV treatment. The matrix metabolism, distribution of sensory nerves, pyroptosis in IVDs tissues were determined by immunohistological analysis or real-time PCR. The apoptosis of IVD cells was detected by TUNEL assay. Results LSI surgery was successful in producing IVDD modeling. WBV caused decreases in IVD height and annulus fibrosus (AF) score, as well as increased numbers of apoptotic cells in IVD tissues. WBV contributed to sensory innervation into AF and upregulation of Adamts5 and MMP3 expression in IVDD mice received LSI surgery. In addition, WBV treatment triggered earlier activation of Wnt/β-catenin signaling in IVDD mice with WBV treatment compared with those without WBV treatment. Unexpectedly, WBV significantly attenuated Caspase-1 and IL-1β expression in AF. Conclusions Collectively, our findings demonstrate that WBV treatment may worsen the development of ongoing IVDD. Decrease of IL-1β expression after WBV intervention may partially account for patient self-reported pain relief after WBV treatment in some previous trials. This study may help us better understand the effects of WBV intervention on patients experiencing LBP resulting from the degeneration of lumbar IVDs.

Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes.

In order to obtain and maintain competitive advantages to adapt to the changes of e-business environment, e-commerce enterprise must choose and adjust their niche reasonably. Some optimization principles and strategies of e-commerce enterprise niche are put forward. By introducing niche and business niche, the concept of e-commerce enterprise niche is defined. It means the specific position that e-commerce enterprise occupies when it is in the interaction with e-commerce ecology environment and other organisms of the e-business world. It should follow some principles to optimize e-commerce enterprise niche, such as positioning clearly, proper width, multi-dimensional anastomosis, and overall coordination. E-commerce enterprise should get a clear and reasonable position to form operating advantages, and be dislocated appropriately to avoid excessive competition, and coordinate to possess the same niche to seek symbiosis and mutual benefit, and fill the vacancy to create new growth point, and change their niche timely to pace with times.