Background: In this study, we have investigated the potential regulatory mechanisms of IL‐35 to relieve lupus nephritis (LN) through regulating Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway in mesangial cells.Results: Among 105 significant differentially expressed proteins (DEPs) between juvenile systemic lupus erythematosus (JSLE) patients with LN and healthy controls, LAIR1, PDGFRβ, VTN, EPHB4, and EPHA4 were downregulated in JSLE‐LN. They consist of an interactive network with PTPN11 and FN1, which involved in IL‐35‐related JAK/STAT signaling pathway. Besides, urinary LAIR1 was significantly correlated with JSLE‐LN clinical parameters such as SLEDAI‐2K, %CD19+ B, and %CD3+ T cells. Through bioinformatics analysis of co‐immunoprecipitation with mass spectrometry results, including GO, KEGG, and STRING, five genes interacted with Lair1 were upregulated by IL‐35, but only Myh10 was downregulated. Therefore, we presumed an interactive network among these DEPs, JAK/STAT, and IL‐35. Moreover, the downregulated phosphorylated (p)‐STAT3, p‐p38 MAPK, and p‐ERK, and the upregulated p‐JAK2/p‐STAT1/4 in IL‐35 overexpressed mesangial cells, and RNA‐sequencing results validated the potential regulatory mechanisms of IL‐35 in alleviating JSLE‐LN disease. Moreover, the relieved histopathological features of nephritis including urine protein and leukocyte scores, a decreased %CD90⁺αSMA⁺ mesangial cells and pro‐inflammatory cytokines, the inactivated JAK/STAT signals and the significant upregulated Tregs in spleen, thymus and peripheral blood were validated in Tregs and IL‐35 overexpression plasmid‐treated lupus mice.Conclusions: Our study provided a reference proteomic map of urinary biomarkers for JSLE‐LN and elucidated evidence that IL‐35 may regulate the interactive network of LAIR1‐PTPN11‐JAK‐STAT‐FN1 to affect JAK/STAT and MAPK signaling pathways to alleviate inflammation in JSLE‐LN. This finding may provide a further prospective mechanism for JSLE‐LN clinical treatment.

According to the Chinese medicine, magnoflorine exerted significant anti-inflammatory effects and potentially promoted synthesis of proteoglycans in chondrocytes to reverse the progression of rheumatoid arthritis. However, the latent beneficial effect of magnoflorine for the treatment of traumatic osteoarthritis (OA) is still unknown. Therefore, we aim to demonstrate the efficacy of magnoflorine combined with HA-gel in attenuating cartilage degeneration in anterior cruciate ligament transection (ACLT) induced OA rat model. We found that the histological results showed the elevated cartilage matrix, chondrogenic signals and chondroprogenitor cells in HA-gel + magnoflorine treatment. HA-gel + magnoflorine treatment resulted in a decreased modified Mankin's score, and a higher volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and HC/Sum (whole cartilage), compared to ACLT and HA-gel groups. Furthermore, both the volume ratios of HC/Sum and HC/CC were negatively correlated with modified Mankin's scores. Finally, HA-gel + magnoflorine could significantly increase the BV/TV, Tb.Th, and decrease the Tb.Pf, Po(tot), Conn.Dn and Tb.Sp. In vitro, 50 μg/ml magnoflorine treatment could significantly increase the viability, S-phase, migration rate and chondrogenesis of chondroprogenitor cells. There were significant downregulations of MAPK/NF-κB signaling, and upregulations of chondrogenic signals in 50 μg/ml magnoflorine treatment. There were significant downregulations of proinflammatory cytokines and upregulation of IL-10 in HA-gel + magnoflorine treated group. Therefore, our study elucidated a protective effect of HA-gel + magnoflorine on attenuating cartilage degradation and maintaining SCB stabilization in ACLT induced OA.

Radix Salviae Miltiorrhizae (RSM), a well-known traditional Chinese medicine, has been shown to inhibit tumorigenesis in various human cancers. However, the anticancer effects of RSM on human hepatocellular carcinoma (HCC) and the underlying mechanisms of action remain to be fully elucidated. In this study, we aimed to elucidate the underlying molecular mechanisms of RSM in the treatment of HCC using a network pharmacology approach. In vivo and in vitro experiments were also performed to validate the therapeutic effects of RSM on HCC. In total, 62 active compounds from RSM and 72 HCC-related targets were identified through network pharmacological analysis. RSM was found to play a critical role in HCC via multiple targets and pathways, especially the EGFR and PI3K/AKT signaling pathways. In addition, RSM was found to suppress HCC cell proliferation, and impair cancer cell migration and invasion in vitro. Flow cytometry analysis revealed that RSM induced cell cycle G2/M arrest and apoptosis, and western blot analysis showed that RSM up-regulated the expression of BAX and down-regulated the expression of Bcl-2 in MHCC97-H and HepG2 cells. Furthermore, RSM administration down-regulated the expression of EGFR, PI3K, and p-AKT proteins, whereas the total AKT level was not altered. Finally, the results of our in vivo experiments confirmed the therapeutic effects of RSM on HCC in nude mice. We provide an integrative network pharmacology approach, in combination with in vitro and in vivo experiments, to illustrate the underlying therapeutic mechanisms of RSM action on HCC.

Background. To examine the potential therapeutic targets of Chinese medicine formula San-Miao-San (SMS) in the treatment of osteoarthritis (OA), we analyzed the active compounds of SMS and key targets of OA and investigated the interacting pathways using network pharmacological approaches and molecular docking analysis. Methods. The active compounds of SMS and OA-related targets were searched and screened by TCMSP, DrugBank, Genecards, OMIM, DisGeNet, TTD, and PharmGKB databases. Venn analysis and PPI were performed for evaluating the interaction of the targets. The topological analysis and molecular docking were used to confirm the subnetworks and binding affinity between active compounds and key targets, respectively. The GO and KEGG functional enrichment analysis for all targets of each subnetwork were conducted. Results. A total of 57 active compounds and 203 targets of SMS were identified by the TCMSP and DrugBank database, while 1791 OA-related targets were collected from the Genecards, OMIM, DisGeNet, TTD, and PharmGKB databases. By Venn analysis, 108 intersection targets between SMS targets and OA targets were obtained. Most of these intersecting targets involve quercetin, kaempferol, and wogonin. Moreover, intersecting targets identified by PPI analysis were introduced into Cytoscape plug-in CytoNCA for topological analysis. Hence, nine key targets of SMS for OA treatment were obtained. Furthermore, the potential binding conformations between active compounds and key targets were found through molecular docking analysis. According to the DAVID enrichment analysis, the main biological processes of SMS in the treatment of OA include oxidative stress, response to reactive oxygen species, and apoptotic signaling pathways. Finally, we found wogonin, the key compound in SMS, might play a pivotal role on Toll-like receptor, IL-17, TNF, osteoclast differentiation, and apoptosis signaling pathways through interacting with four key targets. Conclusions. Therefore, this study elucidated the potential active compounds and key targets of SMS in the treatment of OA based on network pharmacology.

To investigate the mitigate efficacy of Chinese medicine Lingzhi (LZ) and San-Miao-San (SMS) combined with hyaluronic acid (HA)-gel in attenuating cartilage degeneration in traumatic osteoarthritis (OA). The standardized surgery of anterior cruciate ligament transection (ACLT) was made from the medial compartment of right hind limbs of 8-week-old female SD rats and resulted in a traumatic OA. Rats (n ​= ​5/group) were treated once intra-articular injection of 50 ​μl HA-gel, 50 ​μl HA-gel+50 ​μg LZ-SMS, 50 ​μl of saline+50 ​μg LZ-SMS and null (ACLT group) respectively, except sham group. Limbs were harvested for μCT scan and histopathological staining 3-month post-treatment. Inflammatory cytokines from plasma and synovial fluid were detected using Immunology Multiplex Assay kit. The putative targets of active compounds in LZ-SMS and known therapeutic targets for OA were combined to construct protein–protein interaction network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was adopted to predict the potential targets and signaling pathway of LZ-SMS in OA through the tool of DAVID Bioinformatics. In vivo, HA-gel ​+ ​LZ-SMS treatment resulted in a higher volume ratio of hyaline cartilage (HC)/calcified cartilage (CC) and HC/Sum (total volume of cartilage), compared to ACLT and HA-gel groups. In addition, histological results showed the elevated cartilage matrix, chondrogenic and osteoblastic signals in HA-gel ​+ ​LZ-SMS treatment. Treatment also significantly altered subchondral bone (SCB) structure including an increase in BV/TV, Tb.Th, BMD, Conn.Dn, Tb.N, and DA, as well as a significant decrease in Tb.Sp and Po(tot), which implied a protective effect on maintaining the stabilization of tibial SCB microstructure. Furthermore, there was also a down-regulated inflammatory cytokines and upregulated anti-inflammatory cytokine IL-10 in HA+LZ-SMS group. Finally, 64 shared targets from 37 active compounds in LZ-SMS related to the core genes for the development of OA. LZ-SMS has a putative role in regulating inflammatory circumstance through influencing the MAPK signaling pathway. Our study elucidated a protective effect of HA-gel ​+ ​LZ-SMS in mitigating cartilage degradation and putative interaction with targets and signaling pathway for the development of traumatic OA. Our results provide a biological rationale for the use of LZ-SMS as a potential candidate for OA treatment.