Protosappanin A Protects DOX-Induced Myocardial Injury and Cardiac Dysfunction by Targeting ACSL4/FTH1 Axis-Dependent Ferroptosis.

Doxorubicin (DOX) is an effective anticancer agent, but its clinical utility is constrained by dose-dependent cardiotoxicity, partly due to cardiomyocyte ferroptosis. However, the progress of developing cardioprotective medications to counteract ferroptosis has encountered obstacles. Protosappanin A (PrA), an anti-inflammatory compound derived from hematoxylin, shows potential against DOX-induced cardiomyopathy (DIC). Here, it is reported that PrA alleviates myocardial damage and dysfunction by reducing DOX-induced ferroptosis and maintaining mitochondrial homeostasis. Subsequently, the molecular target of PrA through proteome microarray, molecular docking, and dynamics simulation is identified. Mechanistically, PrA physically binds with ferroptosis-related proteins acyl-CoA synthetase long-chain family member 4 (ACSL4) and ferritin heavy chain 1 (FTH1), ultimately inhibiting ACSL4 phosphorylation and subsequent phospholipid peroxidation, while also preventing FTH1 autophagic degradation and subsequent release of ferrous ions (Fe2+) release. Given the critical role of ferroptosis in the pathogenesis of ischemia-reperfusion (IR) injury, this further investigation posits that PrA can confer a protective effect against IR-induced cardiac damage by inhibiting ferroptosis. Overall, a novel pharmacological inhibitor is unveiled that targets ferroptosis and uncover a dual-regulated mechanism for cardiomyocyte ferroptosis in DIC, highlighting additional therapeutic options for chemodrug-induced cardiotoxicity and ferroptosis-triggered disorders.

The Effect of an Extract of Sappanwood, Protosappanin A and Protosappanin B on Osteogenesis in Periodontitis.

BACKGROUND: Sappanwood is widely used in the prevention and treatment in diseases due to its ability to seal blood vessels, dissipate stasis, and relieve pain. Important monomer components of sappanwood, Protosappanin A (PA) and Protosappanin B (PB) have anti-tumour and antimicrobial medicinal properties. This study investigated the anti-inflammatory and osteogenic differentiation effects of a crude extract of Sappanwood (ESP), PA and PB against periodontitis in periodontal ligament stem cells (PDLSCs). METHODS: Oil Red O staining was used to assess the ability of adipocytes to differentiate. Alizarin Red staining was used to assess the capacity to differentiate into osteoblasts. Third-passage PDLSCs were grown in either basic medium alone or basic media with varying doses of ESP (0.0625 mg/mL, 0.03125 mg/mL and 0.125 mg/mL), PA and PB (2.5 µM, 5 µM, 10 µM). The CCK-8 assay was used to measure cell proliferation. Real Time PCR (RT-qPCR) and Enzyme-Linked Immunosorbnent Assay (ELISA) assay were used to measure gene expression. The capacity to differentiate into osteoblasts was evaluated using Alizarin Red staining, and Alkaline Phosphatase (ALP) staining and activity. RESULTS: The development of lipid droplets and mineralized nodules was examined using Oil Red O staining and Alizarin Red staining. Flow cytometry revealed that PDLSCs were CD29 (98.23%) and CD44 (98.81%) positive, but CD34 (0.16%) and CD45 (0.09%) negative. CCK-8 assay showed that ESP at three concentrations (0.03125 mg/mL, 0.0625 mg/mL and 0.125 mg/mL) and 2.5 µM, 5 µM and 10 µM PA and PB had no cytotoxicity at 5 and 7 days (p < 0.05). qRT-PCR and ELISA assay indicated that ESP, PA and PB downregulated the inflammatory cytokines IL-8, IL-6, IL-1ß, IL-10 and IL-4 and elevated the mRNA expression of osteogenesis cytokines RUNX2 , OSX and OCN in PDLSCs (p < 0.05). Alizarin red staining, and ALP staining and activity showed that ESP, PA and PB increased mineralized nodules and the ALP content of in PDLSCs (p < 0.05). CONCLUSIONS: ESP, PA and PB can reduce the inflammatory response and amplify the osteogenic differentiation of PDLSCs. Therefore, ESP, PA and PB may have potential pharmacological effects in controlling the progression of periodontitis and promoting periodontal tissue regeneration.

Protosappanin A Maintains Neuronal Mitochondrial Homeostasis through Promoting Autophagic Degradation of Bax.

Cerebral ischemia is accompanied by mitochondrial integrity destruction. Thus, reversion of mitochondrial damage holds great potential for cerebral ischemia therapy. As a crucial Bcl-2 family member, pro-apoptotic Bax protein is a main effector of mitochondrial permeabilization and plays an important role in mitochondrial homeostasis. However, there is still a lack of an effective cerebral protective strategy through selectively targeting Bax. In this study, we reported that natural small-molecule protosappanin A (PTA) showed a significant mitochondrial protective effect on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cells injury through increasing ATP production and maintaining mitochondrial DNA (mtDNA) content. The mechanism study revealed that PTA selectively induced pro-apoptotic protein Bax degradation, without affecting other Bcl-2 family members such as Bcl-2, Bcl-xl, Bad, Puma, Bid, Bim, and Bik. In addition, we found that PTA promoted the association of autophagosomal marker LC3B to Bax for its degradation via an autophagy-dependent manner but not the ubiquitin-proteasome pathway. Collectively, our findings offered a new pharmacological strategy for maintaining mitochondrial function by inducing autophagic degradation of Bax and also provided a novel drug candidate against ischemic neuronal injury.

Protosappanin-A and oleanolic acid protect injured podocytes from apoptosis through inhibition of AKT-mTOR signaling.

Protosappanin-A (PrA) and oleanolic acid (OA), which are important effective ingredients isolated from Caesalpinia sappan L., exhibit therapeutic potential in multiple diseases. This study focused on exploring the mechanisms of PrA and OA function in podocyte injury. An in vitro model of podocyte injury was induced by the sC5b-9 complex and assays such as cell viability, apoptosis, immunofluorescence, quantitative real-time polymerase chain reaction, and western blot were performed to further investigate the effects and mechanisms of PrA and OA in podocyte injury. The models of podocyte injury were verified to be successful as seen through significantly decreased levels of nephrin, podocin, and CD2AP and increased level of desmin. The sC5b-9-induced podocyte apoptosis was inhibited in injured podocytes treated with PrA and OA, accompanied by increased protein levels of nephrin, podocin, CD2AP, and Bcl2 and decreased levels of desmin and Bax. The p-AKT/p-mTOR levels were also reduced by treatment of PrA and OA while AKT/mTOR was unaltered. Further, the effects of PrA and OA on injured podocytes were similar to that of LY294002 (a PI3K-AKT inhibitor). PrA and OA were also seen to inhibit podocyte apoptosis and p-AKT/p-mTOR levels induced by IGF-1 (a PI3K-AKT activator). Our data demonstrate that PrA and OA can protect podocytes from injury or apoptosis, which may occur through inhibition of the abnormal activation of AKT-mTOR signaling.

Protosappanin A protects against experimental autoimmune myocarditis, and induces metabolically reprogrammed tolerogenic DCs.

Autoimmune myocarditis is an immune-mediated myocardial injury that evolves into dilated cardiomyopathy (DCM). Protosappanin A (PrA), an immunosuppressive compound, induces immune tolerance in cardiac transplantation. However, whether PrA confers protective immunosuppression on experimental autoimmune myocarditis (EAM) is unknown. In this study, PrA treatment remarkably suppressed cardiac inflammatory cell infiltration and ameliorated cardiac remodeling in EAM mice. Additionally, PrA treatment reduced splenic T cells response, and induced expansion of immunosuppressive regulatory T cells (Tregs). Meanwhile, PrA induced the splenic dendritic cells (DCs) into a tolerogenic state with reduced co-stimulatory molecules, increased the production of tolerogenic cytokines in vivo. PrA also reprogrammed the metabolism of splenic DCs to a more glycolytic phenotype. To further investigate the effect of PrA on the functional and metabolic phenotype of DCs, the compound was added into the in vitro culture of MyHC-α-loaded DCs. These cells switched to a tolerogenic state and a metabolic profile similar to that found in cells during in ex vivo experiments. Treatment with glycolytic inhibitor 2-DG significantly reversed PrA-mediated DC tolerogenic properties, suggesting that glycolysis is indispensable for PrA-conditioned DCs to maintain their tolerogenic properties. Notably, PrA-conditioned DC vaccinations dampened EAM progress, and promoted Tregs expansion. Similarly, tolerogenic and metabolic patterns were also observed in PrA-modified human DC. In conclusion, PrA endows DC with a tolerogenic profile via glycolytic reprogramming, thereby inducing expansion of immunosuppressive Tregs, and preventing EAM progress. Our results suggested that PrA may confer immunosuppressive and protective effects on EAM by metabolically reprogramming DCs, which could contribute to the development of a new potential immunotherapy for the treatment of EAM and immune-related disorders.

Protosappanin A protects against atherosclerosis via anti- hyperlipidemia, anti-inflammation and NF-κB signaling pathway in hyperlipidemic rabbits.

OBJECTIVES: Protosappanin A (PrA) is an effective and major ingredient of Caesalpinia sappan L. The current study was aimed to explore the effect of PrA on atherosclerosis (AS). MATERIALS AND METHODS: Firstly, the experimental model of AS was established in rabbits by two-month feeding of high fat diet. Then, the rabbits were randomly divided into five groups and treated with continuous high lipid diet (model control), high lipid diet containing rosuvastatin (positive control), 5 mg/kg PrA (low dose) or 25 mg/kg PrA (high dose). RESULTS: Our results showed that PrA markedly alleviated AS as indicated by hematoxylin/eosin (HE) staining. PrA also reduced hyperlipidemia (as demonstrated by the serum levels of total blood cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL)) in a time and dose-dependent manner, and decreased inflammation (as indicated by the serum levels of matrix metalloproteinase-9 [MMP-9], interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α]). Moreover, PrA significantly inactivated nuclear factor kappa B (NF-κB) signaling as indicated by nuclear NF-κB p65 protein expression, as well as the mRNA expression and serum levels of downstream genes, interferon-γ (IFN-γ) and interferon-gamma-inducible protein 10 (IP10). CONCLUSION: This study proved that PrA might protect against atherosclerosis via anti-hyperlipidemia, anti-inflammation and NF-κB signaling pathways in hyperlipidemic rabbits.

Protosappanin A exerts anti-neuroinflammatory effect by inhibiting JAK2-STAT3 pathway in lipopolysaccharide-induced BV2 microglia.

Microglial activation and resultant neuroinflammatory response are implicated in various brain diseases including Alzheimer's disease and Parkinson's disease. Treatment with anti-neuroinflammatory agents could provide therapeutic benefits for such disorders. Protosappanin A (PTA) is a major bioactive ingredient isolated from Caesalpinia sappan L.. In this work, the anti-neuroinflammatory effects of PTA on LPS-stimulated BV2 cells were investigated and the underlying mechanisms were explored. Results showed that PTA significantly inhibited the production of TNF-α and IL-1ß in LPS-activated BV2 microglia. Moreover, the mRNA expressions of IL-6, IL-1ß, and MCP-1 were reduced by PTA in a dose-dependent manner. Furthermore, PTA suppressed JAK2/STAT3-dependent inflammation pathway through down-regulating the phosphorylation of JAK2 and STAT3, as well as STAT3 nuclear translocation against LPS treatment. These observations suggested a novel role for PTA in regulating LPS-induced neuroinflammatory injuries.

Protosappanin a inhibits osteoclastogenesis via reducing oxidative stress in RAW264.7 cells.

Protosappanin A (PrA), obtained from the traditional Chinese herbal medicine, Caesalpinia sappan L. (Lignum Sappan), possesses a lot of pharmaceutical activities. Typically, it is a potent antioxidant. This study makes an effort to test its protective effects against osteoporosis by partially reducing oxidative stress in RAW264.7 cells and a mouse ovariectomized (OVX) osteoporosis model. The influence that PrA affected on osteoclastic proliferation and differentiation under oxidative status was investigated. Our results revealed that PrA significantly inhibited the proliferation of RAW264.7 cells in oxidative stress conditions. Moreover, it suppressed some osteoclastic markers by TRAP staining, bone section assay and quantitative real-time PCR. PrA decreased reactive oxygen species (ROS) generation in RAW264.7 cells. In vivo, our results demonstrated that PrA supplementation improved some serum oxidative markers, including malondialdehyde (MDA) and reduced glutathione (GSH), and inhibited some osteoclastic markers, such as CTX-1 and TRAP. Importantly, it ameliorated the micro-architecture of trabecular bones by micro-CT assay. In summary, these findings showed that protection by PrA against osteoporosis is associated with a reduction in oxidative stress, suggesting that PrA may be useful in bone resorption related diseases, especially osteoporosis.

Protosappanin A exerts anti-neuroinflammatory effect by inhibiting JAK2-STAT3 pathway in lipopolysaccharide-induced BV2 microglia / 中国天然药物

Microglial activation and resultant neuroinflammatory response are implicated in various brain diseases including Alzheimer's disease and Parkinson's disease. Treatment with anti-neuroinflammatory agents could provide therapeutic benefits for such disorders. Protosappanin A (PTA) is a major bioactive ingredient isolated from Caesalpinia sappan L.. In this work, the anti-neuroinflammatory effects of PTA on LPS-stimulated BV2 cells were investigated and the underlying mechanisms were explored. Results showed that PTA significantly inhibited the production of TNF-α and IL-1β in LPS-activated BV2 microglia. Moreover, the mRNA expressions of IL-6, IL-1β, and MCP-1 were reduced by PTA in a dose-dependent manner. Furthermore, PTA suppressed JAK2/STAT3-dependent inflammation pathway through down-regulating the phosphorylation of JAK2 and STAT3, as well as STAT3 nuclear translocation against LPS treatment. These observations suggested a novel role for PTA in regulating LPS-induced neuroinflammatory injuries.

Protosappanin A increases the sensitivity of gastric cancer SGC-7901 cells to radiotherapy / 实用肿瘤学杂志

Objective In this study,Protosappanin A,Caesalpinia Sappan L extract and Cisplatin were combined with radiotherapy in gastric cancer cell SGC-7901 to investigate whether the Protosappanin A could in-crease radiosensitivity( SER) in gastric cancer SGC-7901 cells. This will be medication to create new areas of in-novation in the future. Methods The cell proliferation of SGC-7901 cells was detected by MTT assay. The rela-tionship between the effect of the Protosappanin A on cell proliferation and the time of action was determined. Caesalpinia Sappan L extract and Cisplatin were as controls. The fitted cell survival curve and clonal formation as-says were used to determine the SER to analyze the sensitizative effect of Protosappanin A. Results Protosappa-nin A could inhibit the growth of SGC-7901 cells,and its inhibitory effect is relatively weak. Its cytotoxicity has a time-and concentration-dependent manner. Cellular morphological changes were observed accompanying with increased concentrations and time treatments of Protosappanin A. Clonal formation experiment showed that the Protosappanin A significantly increased the radiosensitivity of SGC-7901 cells when compared to the radioactive group. They showed a statistically difference. Conclusion The inhibitory effect of the Protosappanin A on SGC-7901 cells in a concentration and time-dependent manner. Protosappanin A combined radiotherapy can improve the radiosensitization of cells,both of which may have synergistic anti-tumor effect.

Chemical constituents from ethyl acetate extract of Sappan Lignum / 中草药

Objective: To study the chemical constituents in Sappan Lignum (the core material of Caesalpinia sappan). Methods: The chemical constituents of Sappan Lignum were isolated by different column chromatographic techniques, including silica gel and Sephadex LH-20 columns. The structures of these compounds were identified by a comprehensive analysis on the spectroscopic data. Results: Fifteen compounds were isolated from the EtOAc extract of Sappan Lignum. The compounds were identified as protosappanin A (1), 3,7-dihydroxychroman-4-one (2), 7,3',4'-trihydroxy-3-benzyl-2H-chromene (3), bonducellin (4), 3'-deoxysappanol (5), 3'-deoxy-4-O-methylepisappanol (6), 3-deoxysappanchalcone (7), 3,8,9-thihydroxy-6H-benzo[c] chromen- 6-one (8), 3,9-dihydroxy-8-methoxydibenzo [b, d] pyran-6-one (9), (-)-syringaresinol (10), dibutyl phthalate (11), β-sitosterol (12), β-daucosterol (13), stigmasterol (14), and 1-heneicosanol (15). Conclusion: Compounds 9, 11, and 12 are first isolated from the plants of Caesalpinia Linn. and compound 4 is first isolated from this plant.