Ziyuglycoside II, a triterpene glycoside compound in Sanguisorbae officinalis l. extract, suppresses metastasis in osteosarcoma via CBX4-mediated Wnt/ß-catenin signal pathway.

BACKGROUND: Osteosarcoma (OS), the most prevalent primary bone malignancy, exhibits rapid growth and a high tendency for lung metastasis, posing significant treatment challenges. Ziyuglycoside II (ZGS II), a main active compound derived from Sanguisorba officinalis l., has shown potential in cancer treatment. However, the effects of ZGS II and its potential mechanism in OS remain elusive. PURPOSE: This study aims to explore the anti-metastatic potential of ZGS II in OS, offering a novel therapeutic strategy for improved patient outcomes. METHODS: Cell viability and proliferation was detected by cell counting kit-8 (CCK-8) and clone formation assay, respectively. Transwell and wound-healing assay were applied to evaluate the potential metastatic abilities of OS cells in vitro. More critically, the chromobox protein homolog 4 (CBX4) and Wnt/ß-catenin signaling pathway was investigated utilizing Western blotting, immunohistochemistry, shRNA knockdown and immunofluorescence. An orthotopic metastasis mouse model was utilized to evaluate the efficacy of ZGS II in suppressing OS metastasis in vivo, with molecular docking studies conducted to elucidate the interaction between ZGS II and the CBX4 protein. RESULTS: Our study demonstrated the potent inhibitory effects of ZGS II on OS cell proliferation and induced apoptosis in vitro, as evidenced by decreased cell viability, enhanced caspase-3 activation, and mitochondrial dysfunction. Furthermore, using an orthotopic metastasis mouse model, we illustrated that ZGS II effectively suppressed tumor growth and lung metastasis in vivo. Notably, our investigation revealed that the antitumor action of ZGS II is dependent on the reduction of CBX4 levels, leading to the attenuation of the Wnt/ß-catenin signaling pathway activation. Molecular docking analyses supported this pathway's suppression, showing that ZGS II has the capability to directly bind and disrupt CBX4 function. To further confirm this mechanism, we utilized shRNA to silence CBX4 in OS cells, which significantly enhanced the inhibitory impact of ZGS II on cell migration. CONCLUSION: Our study findings reveal that ZGS II efficiently suppresses both metastasis and tumor growth in OS by a novel mechanism that entails the inhibition of the CBX4-regulated Wnt/ß-catenin pathway. These outcomes highlight the promising potential of ZGS II as a therapeutic agent for managing metastatic OS, thus justifying the need for additional clinical investigations.

Study of the mechanism of Shexiang Baoxin pill-mediated angiogenesis in acute myocardial infarction.

AIM: The Shexiang Baoxin pill (SBP) is a commonly used drug for the treatment of coronary artery disease in China. More recently, some studies have found that it improved coronary microvascular function. This study aimed to explore the possible mechanism by which the SBP promotes angiogenesis after acute myocardial infarction (AMI). METHODS: A rabbit model of acute myocardial infarction was established by ligating the left anterior descending coronary artery with silk thread, and the limb lead electrocardiogram was recorded to determine the success of the model. The rabbits were divided into a control group (SBP + normal rabbit group), a sham operation group, a saline + AMI group and an SBP + AMI group. There were 10 rabbits in each group. The animals were sacrificed and myocardial tissue was collected seven days after the operation. Haematoxylin-eosin staining was used to observe the histological changes in the rabbit myocardium in each group. The degree of acute myocardial infarction was observed with picric acid staining, which was used to detect the expression of vascular endothelial growth factor (VEGF), silent information regulator 1 (SIRT1), Beclin1 and mTOR protein in the myocardial tissue of each group. Immunofluorescence CD31-labelled microvascular density (MVD) was used to observe the vascular regeneration of the rabbits in each group. RESULTS: Compared with the normal saline + AMI group, the myocardial infarction area of the SBP + AMI group decreased and CD31 immunofluorescence-labelled MVD increased. Compared with the control and sham operation groups, the expression of VEGF, Beclin1 and mTOR in the normal saline + AMI group and the SBP + AMI group increased, while the expression of SIRT1 decreased. Compared with the normal saline + AMI group and the SBP + AMI group, the positive expression of VEGF, Beclin1, mTOR and SIRT1 in the SBP + AMI group was significantly increased. CONCLUSION: Autophagy was enhanced after acute myocardial infarction. SBP may affect angiogenesis through the SIRT1/mTOR signalling pathway after acute myocardial infarction to inhibit ventricular remodelling and a decline in cardiac function.

Syringin alleviates hepatic fibrosis by enhancing autophagic flux and attenuating ER stress-TRIB3/SMAD3 in diabetic mice.

Type 2 diabetes mellitus (T2DM) is a key risk factor for the developing of metabolic liver injury and easily evolving to advanced fibrosis. Syringin (SYR), isolated from Acanthopanax senticosus, has anti-inflammatory, anti-oxidant, and anti-apoptotic properties. However, its hepatoprotective effects and mechanisms in T2DM-induced liver fibrosis remain unclear. Here, we investigated whether syringin (SYR) could serve as a therapeutic agent for liver fibrosis and its mechanism in high-fat diet (HFD)/streptozotocin (STZ)-induced type 2 diabetic mice. C57BL/6 mice were induced with T2DM via HFD and STZ injection and treated with different doses of SYR. Serum lipid parameters and liver function indicators were measured, and hepatic histology and fibrosis were examined. The mechanism of SYR was explored through molecular analyses Results demonstrated SYR improved oral glucose tolerance, decreased the levels of ALT, AST, and AKP, and reduced hepatic lipid deposition in diabetic mice. Moreover, SYR ameliorated epithelial-to-mesenchymal transition to reverse hepatic fibrosis via suppressing TRIB3-SMAD3 interaction to restrain nuclear localization of SMAD3. Strikingly, SYR reversed hyperglycemia-induced deficiency in autophagic flux by regulation of Raptor/mTORC1, triggering nuclear translocation of TFEB to improve autophagosome-lysosomal fusion. In brief, SYR potentially ameliorates hepatic injury and fibrosis by enhancing autophagic flux and inhibing TRIB3 activation in diabetic mice.

Aucubin supplementation alleviate diabetes induced-disruption of blood-testis barrier and testicular damage via stabilizing cell junction integrity.

Disruption of blood-testis barrier (BTB) was a crucial pathological feature of diabetes induced-testicular injury at early phase. Aucubin (AU), a main active component in Eucommiae Cortex, has drawn attention for its benefits against male reproductive system disease. The current study was aimed at investigating the protective role of AU and exploring the underlying mechanism in diabetic model. A murine model of type 2 diabetes mellitus (T2DM) was induced by high-fat diet (HFD) combined with streptozocin (STZ). Testicular weight index and morphology, sperm quality, integrity of BTB and protein levels were analyzed. The underlying mechanism of the protective effect of AU was further explored in Sertoli cells (SCs) cultured with high glucose (HG). Our results showed AU inhibited testicular structural destruction, restored disruption of BTB and improved abnormal spermatogenic function in diabetic mice. Consistent with in vivo results, HG induced decreased transcellular resistance and increased permeability in SCs monolayers, while AU exposure reverses this trend. Meanwhile, reduced expression of Zonula occludin-1(ZO-1) and Connexin43(Cx43) in testicular tissue diabetic mice and HG-induced SCs was prominently reversed via AU treatment. Mechanistic studies suggested a high affinity interaction between AU and c-Src protein was identified based on molecular docking, and the activation of c-Src was significantly inhibited in AU treatment. Furthermore, AU significantly increased the expression of Cx43 and ZO-1 proteins HG-induced SCs, which can be further enhanced in gene-silenced c-Src cells to some extent. Our results suggested that AU ameliorated disruption of BTB and spermatogenesis dysfunction in diabetic mice via inactivating c-Src to stabilize cell junction integrity.

Aucubin ameliorates liver fibrosis and hepatic stellate cells activation in diabetic mice via inhibiting ER stress-mediated IRE1α/TXNIP/NLRP3 inflammasome through NOX4/ROS pathway.

Type 2 diabetes (T2DM) is closely associated with hepatic injury, which could promote/exacerbate hepatic inflammation, steatosis, and accelerate liver fibrosis progression. Aucubin (AU), as an active ingredient isolated from Eucommia ulmoides, exists a nutritional value in hepatoprotective effect and diabetic complications. However, whether it possesses more outstanding features on improving liver injury in diabetic conditions and the underlying mechanism is unclear. Our research investigated the treatment of AU on liver fibrosis and potential mechanisms on high-fat diet/streptozotocin-induced diabetic mice and high glucose (HG)&TGF-ß1-induced LX-2 cells. Results showed that AU restored hepatic function without affecting blood sugar levels in diabetic mice. Meanwhile, the enhanced levels of total cholesterol, triglycerides, and LDL-c were reversed in hepatic tissue after AU treatment. Histomorphology assays including H&E, Masson, PAS, Oil red and Sirius red staining showed that AU treatment reduced liver swelling, steatosis and fibrosis. Mechanistic studies showed that AU alleviated NLRP3 inflammasome activation and inflammatory responses via inhibiting ER stress-mediated IRE1α/TXNIP signaling pathway, which could postpone the development of T2DM induced hepatic fibrosis. In addition, the ROS generation and the up-regulated expression of NADHP oxidase 4 (NOX4) in the liver tissue were suppressed by AU treatment. Moreover, in vitro model, NOX4 activation was prominently enhanced and AU treatment blocked HG&TGF-ß1-induced NOX4 derived superoxide generation and thereby ameliorating hepatic stellate cell activation, which can be abrogated in the overexpression of NOX4 LX-2 cells. In addition, inhibition effects on ER stress-mediated IRE1α/TXNIP/NLRP3 inflammasome by AU treatment also were abolished in the overexpression of NOX4 LX-2 cells. Meanwhile, molecular docking results indicated that AU and NOX4 protein have a higher affinity. Taken together, AU might be a potential nutraceutical or therapeutic drug to ameliorate hepatic impairment and fibrosis in T2DM.

Complanatoside A targeting NOX4 blocks renal fibrosis in diabetic mice by suppressing NLRP3 inflammasome activation and autophagy.

BACKGROUND: Diabetic nephropathy (DN) is an important cause of end-stage renal disease. Complanatoside A (CA), an active component from Semen Astragali Complanati, has been reported to be a potential candidate for the treatment of kidney diseases. However, the underlying mechanisms and protective effects of CA in DN remain unclear. PURPOSE: In this paper, the effects and the mechanism of CA against ameliorating DN were investigated in vivo and in vitro. STUDY DESIGN: Here, a high-fat diet/streptozotocin-induced diabetic model and TGF-ß1-induced HK-2 cells were used to explore the protective effects and mechanisms of CA on DN in vivo and in vitro. METHODS: Major biochemical indexes, Histopathological morphology, and Immunohistochemistry have explored the therapeutic effect of CA on DN. Subsequently, TGF-ß1-induced HK-2 cells were utilized to investigate the anti-renal fibrosis effect of CA. Finally, the mechanism of CA against renal fibrosis was studied via western blotting, immunofluorescence, transfection, and molecular docking. RESULTS: The results showed that CA attenuated glomerular hypertrophy, collagen matrix deposition, and tubular interstitial fibrosis in diabetic mice. Moreover, the activation of TGF-ß1-inducible epithelial-mesenchymal transition (EMT) was hindered by CA treatment in HK-2 cells. Mechanistically, the data suggested that upregulated NOX4 during diabetes and TGF-ß1 in HK-2 cells was prominently diminished after CA treatment. Furthermore, CA exposure inhibited NLRP3 inflammasome activation and downstream inflammation gene expression such as IL-18 and IL-1ß in vivo and vitro. These findings indicated that CA obstructed the EMT to protect renal tubular epithelial cells against fibrosis via blocking NLRP3 activation, which was associated with inhibiting NOX4. Besides, the markedly raised autophagy levels in the diabetic model characterized by increasing LC3II/LC3I and Beclin1 were reversed after CA treatment, which is also a pivotal mechanism against renal fibrosis. More importantly, specific NOX4 overexpressed in HK-2 cells abolished that CA exposure blocked TGF-ß1-induced-EMT, ROS generation, NLRP3, and autophagy activation. Meanwhile, the inhibition of cell migration, ROS generation, autophagy, and renal inflammation after CA treatment was more pronounced in NOX4-deficient HK-2 cells. CONCLUSION: Our findings provided evidence that CA might be a potential therapeutic agent for DN by ameliorating NLRP3 inflammasome and autophagy activation via targeting NOX4 inhibition.

Restirring and Reheating Effects on Microstructural Evolution of Al-Zn-Mg-Cu Alloy during Underwater Friction Stir Additive Manufacturing.

Friction stir additive manufacturing (FSAM) can be potentially used for fabricating high-performance components owing to its advantages of solid-state processing. However, the inhomogeneous microstructures and mechanical properties of the build attributed to the complex process involving restirring and reheating deserve attention. This study is based on the previous research of the underwater FSAMed 7A04 aluminum alloy and adopts a quasi in situ experimental method, i.e., after each pass of the underwater FSAM, samples were taken from the build for microstructural observation to investigate the restirring and reheating effects on microstructural evolution during the underwater FSAM. Fine-grain microstructures were formed in the stir zone during the single-pass underwater FSAM. After restirring, the grain size at the bottom of the overlapping region decreased from 1.97 to 0.87 µm, the recrystallization degree reduced from 74.0% to 29.8%, and the initial random texture transformed into a strong shear texture composed of the C {110}<11¯0>. After reheating, static recrystallization occurred in the regions close to the new additive zones, increasing the grain size and recrystallization degree. This study not only revealed the microstructural evolution during the underwater FSAM but also provided a guideline for further optimization of the mechanical properties of the Al−Zn−Mg−Cu alloy build.

Effect of Post-Fabricated Aging on Microstructure and Mechanical Properties in Underwater Friction Stir Additive Manufacturing of Al-Zn-Mg-Cu Alloy.

The fabricated Al-Zn-Mg-Cu alloy build has low mechanical properties due to the dissolution of strengthening precipitates back into the matrix during friction stir additive manufacturing (FSAM). Post-fabricated aging was considered an effective approach to improve the mechanical performance of the build. In this study, various post-fabricated aging treatments were applied in the underwater FSAM of Al-7.5 Zn-1.85 Mg-1.3 Cu-0.135 Zr alloy. The effect of the post-fabricated aging on the microstructure, microhardness, and local tensile properties of the build was investigated. The results indicated that over-aging occurred in the low hardness zone (LHZ) of the build after artificial aging at 120 °C for 24 h as the high density of grain boundaries, subgrain boundaries, dislocations, and Al3Zr particles facilitated the precipitation. Low-temperature aging treatment can effectively avoid the over-aging problem. After aging at 100 °C for 48 h, the average microhardness value of the build reached 178 HV; the yield strength of the LHZ and high hardness zone (HHZ) was 453 MPa and 463 MPa, respectively; and the ultimate tensile strength of the LHZ and HHZ increased to 504 MPa and 523 MPa, respectively.

Interface Characteristics and Mechanical Properties of Ultrasonic-Assisted Friction Stir Lap Welded 7075-T6 Aluminium Alloy.

In this work, friction stir lap welding (FSLW) and ultrasonic-assisted friction stir lap welding (UAFSLW) was applied to 6-mm-thick 7075-T6 alloy sheets using three welding tools with the same process parameters. The joint formation, microstructural characteristics, and mechanical properties of the resulting lap joints were then investigated. The results showed that ultrasonic vibration significantly promoted the flow of metal at the interface, enlarged the size of the stirred zone (SZ), and reduced the angle between the hook defect and the interface. During lap shear testing, the FSLW and UAFSLW joints fractured in a similar manner. The fracture modes included tensile fracture, shear fracture, and a mixture of both. Cold lap and hook defects may have served as crack-initiation zones within the joint. Under configuration A (i.e., upper sheet on the retreating side (RS)), all joints failed in the shear-fracture mode. The effective lap width (ELW) of the joint welded using tool T2 was the greatest. This resulted in a higher shear fracture strength. The maximum shear fracture strength of the UAFSLW joint was 663.1 N/mm. Under configuration B (i.e., upper sheet on the advancing side (AS)), the shear fracture strength was greatly affected by the fracture mode. The highest shear fracture strength of the UAFSLW joint, 543.7 N/mm, was welded by tool T3. Thus, under otherwise identical conditions, UAFSLW joints can withstand a greater fracture shear strength than FSLW joints, as ultrasonic vibration helps to mix the material at the interface, thus, enlarging the SZ and diminishing the cold lap defects.

Fatigue Behaviour of 7N01-T4 Aluminium Alloy Welded by Ultrasonic-Assisted Friction Stir Welding.

This study investigates the effects of axial ultrasonic vibration on the microstructure evolution, residual stresses distribution and fatigue fracture behaviour of a 7N01-T4 joint, and demonstrates that ultrasonic vibration can significantly promote the flow of plasticised metals, expand the stirred zone (SZ) width and refine the grain size. The longitudinal residual stresses of the joints are dominant, and the peak longitudinal residual stresses of the thermo-mechanically affected zone (TMAZ) on the advancing side (AS) (TMAZ-AS) in the ultrasonic-assisted friction stir welding (UAFSW) joint are 31.5 MPa lower than those in the friction stir welding (FSW) joint. Compared to that of FSW joints, the fatigue strength of UAFSW joints increases by 20 MPa at 107 cycles (stress ratio of R = 0.1). At high-stress levels, crack initiation occurs at the TMAZ-AS, and is mainly attributed to high residual stresses and second-phase particles. At low-stress levels, fatigue cracks are likely to initiate in the transition zone (TZ).