MN as a prominent subtype of CKD, is a pathologically defined renal glomerular disorder characterized by the presence of immune complexes on the outer surface of the basement membrane[16, 17]. This autoimmune disease is typified by the deposition of immunoglobulin G (IgG), relevant antigens, and complement components, including the membrane attack complex (MAC)[18, 19]. The immunological conflict results in significant protein loss in the urine, known as proteinuria, primarily due to the disruption of podocyte structure caused by immune complex deposition and MAC formation[19]. Recent research indicates a significant rise in the prevalence of IMN in China[20]. Therefore, actively seeking a treatment for MN, especially for IMN, is our clinical challenge and the direction of our research.
Network pharmacology analyzed the intersection of the active components of SQ and the disease targets of IMN, as depicted in Fig. 2 of the Wayne diagram, yielded a total of 195 common targets(Figure. 2). By identifying the overlapping targets between the active components of SQ and the gene targets of MN, it was postulated that SQ may possess therapeutic potential through its intervention on these shared targets and thus made the SQ-active ingredient-MN target network and PPI diagram(Figure. 3–4). Furthermore, GO and KEGG analyses suggested that SQ primarily engages in enzyme binding, signaling receptor binding, kinase binding, response to lipid, regulation of cell death, cell proliferation, apoptotic process, extracellular space, etc, and disease of the most closely related signaling pathways containing Lipid and atherosclerosis, HIF-1 signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, Apoptosis, mTOR signaling and so on(Figure. 5).
Podocytes, the smallest and most fundamental cell of the glomerulus[21, 22], were utilized to establish the cell model of MN through the use of PAN. Podocyte function is dependent on actin cytoskeleton regulation within the foot processes, structures that link podocytes to the glomerular basement membrane. Actin cytoskeleton dynamics in podocyte foot processes are complex and regulated by multiple proteins and other factors[23]. Changes in the actin cytoskeleton within a cell are necessary for maintenance of cell shape, cell motility and intracellular transport[24]. Our previous study demonstrated that SQ offers protection against podocyte cytoskeletal proteins, including CD2AP and α-actinin4[15, 25, 26]. In this study, upon further examination of the podocytes' cytoskeletal protein, it was observed that PAN induces impairment in the F-actin protein, resulting in polymerization breakdown. However, it was found that SQ has the ability to restore the integrity of the skeletal cytoskeleton proteins. The effectiveness of this protective effect and its dependence on drug concentration are illustrated in Figure. 8. This investigation revealed that both SQ and CsA possess the ability to restore F-actin expression and enhance polymerization. Notably, the effectiveness of SQ in this regard is contingent upon the concentration of the drug.
Phosphoinositide 3-kinase (PI3K), AKT serine/threonine kinase (Akt), and mammalian target of rapamycin (mTOR) are kinases that mediate cellular signaling pathways involved in the regulation of various cellular processes, including the cell cycle, cell survival, cytoskeleton rearrangements, metabolism, and protein synthesis[27], ectopic or too intense activation of this signaling is a causal event in diseases characterized by uncontrolled cellular expansion[28, 29]. PI3K plays a crucial role in controlling diverse cellular processes such as growth, survival, metabolism, apoptosis, and autophagy[30]. AKT serves as a primary effector of PI3K, upon activation, AKT phosphorylates tuberous sclerosis complex 2, which disrupts the formation of inhibitory TSC1/TSC2 heterodimers, subsequently activating TOR complex 1, this complex, consisting of mTOR, performs distinct functions within the cell[31]. mTOR is a serine/threonine protein kinase that forms the catalytic subunit of two distinct protein complexes, known as mTOR Complex 1 (mTORC1) and 2 (mTORC2), the two complexes exert their activity towards distinct substrates and, as a consequence, regulate different cellular functions[32]. Our network pharmacology demonstrates that SQ for IMN may be involved in the pathway. In vitro experiments, we have confirmed this inference by observing that PAN intervention in MPC5 activates the PI3K/AKT/mTOR signaling pathway, leading to a significant increase in AKT/mTOR phosphorylation levels(Figure. 9). However, we have observed that various concentrations of SQ exhibit remarkable effects in reducing the expression of p-AKT and p-mTOR, as well as inhibiting the activation of this signaling pathway's phosphorylation. These findings may potentially induce subsequent alterations in downstream apoptosis and autophagic activity.
Apoptosis is characterised by distinct morphological alterations in cellular structure, accompanied by enzyme-dependent biochemical mechanisms[33]. Bcl-2/Bax and cleaved caspase-3 are crucial factors in the advancement of cellular apoptosis. The Bcl-2 family significantly influences the facilitation or inhibition of the intrinsic apoptotic pathway, which is initiated by mitochondrial dysfunction[34, 35, 36]. Certain investigations propose that kidney-related apoptosis is mediated by Bcl2 proteins and could potentially be controlled through systemic intervention aimed at impeding conformational bax activation and/or reduction in Bcl2 levels induced by renal stress[37]. Caspase-dependent apoptosis is responsible for approximately 90% of cellular turnover in homeostatic conditions[38]. According to the structure and the roles of caspase-3 in various cellular mechanisms, mainly in apoptosis, this enzyme was specifically considered as a therapeutic target to combat apoptosis-related diseases[39]. Cleaved caspase3, a downstream protein of the Caspase family, serves as the final product of apoptosis[40, 41, 42, 43]. In our study, depicted in Figure. 10, it is evident that PAN-induced cells exhibited suppressed Bcl2 expression and heightened expression of Bax and cleaved Caspase-3, indicating an increase in apoptotic activity. Conversely, SQ was found to activate Bcl2 expression, decrease Bax and cleaved Caspase-3 expression, and decelerate cellular apoptosis.
These findings imply that certain active components within the drug composition of SQ possess the capability to inhibit apoptosis and regulate certain signaling pathway and facilitate cytoskeleton structure repair. Consequently, these results support the alignment between our SQ and network pharmacology, as the in vitro experiments substantiated the conjecture made by network pharmacology.
We through network pharmacology found that SQ and IMN have the same therapeutic targets, and dig out the potential mechanism of SQ treatment of IMN, earlier studies have shown that SQ can activate autophagy activity, improve cytoskeletal protein expression, has the effect of protecting damaged podocytes. Our study has yielded novel findings, indicating that SQ exerts a regulatory effect on cell apoptosis and has significant feedback implications for the associated signaling pathways(Figure. 11). These findings were validated through in vitro studies and molecular docking technology, which confirmed the strong binding affinity of the active components in SQ with the disease targets(Figure. 6, Supplementary Table 2).