AS is a chronic inflammatory disease that can be caused by endothelial damage and lipid deposition, leading to inflammatory reactions, thrombosis, thickening of blood vessel walls, and arteriosclerosis. Inflammatory response is a driving factor for AS and runs through the entire process. Endothelial cell damage and cellular dysfunction are classic markers and predictive factors for the occurrence of AS [5,32]. When endothelial cells are injured or dysfunctional, inflammatory cytokines and cell adhesion molecules are secreted in large quantities. The secreted chemokines and other effector proteins lead to the aggregation of a large number of inflammatory cells, the enhancement of inflammatory reaction of vascular wall, the destruction of the integrity of tunica intima, and the aggravation of the pathological process of AS [33–34]. In this study, we used ox-LDL to induce inflammatory damage in HUVECs. The previous research team searched for literature and combined it with CCK8 to select the optimal concentrations of ox-LDL and BBR for intervention in HUVECs, which were 50mg/mL and 20µmol/L, respectively. When the vascular endothelium is damaged and the cell membrane ruptures, inflammatory cells can secrete IL-18 and IL-1β and a large number of inflammatory factors such as LDH are released into the extracellular space. This study found that the secretion of LDH in the supernatant of HUVECs suggests an increase, and LDH is one of the markers of cell membrane rupture, and its release to some extent reflects the degree of cell damage [4]. At the same time, ELISA was used to detect the protein content of inflammatory factors IL-18 and IL-1β in the supernatant, and the results showed a significant increase, indicating the presence of cell damage in HUVECs. This is consistent with the results of previous literature suggesting cell inflammation damage. Firstly, it was determined that the construction of a cell inflammation damage model was successful.
Endothelial pyroptosis is one of the main ways of endothelial cell injury, and NLRP3 inflammatory body dependent Caspase-1 is the classic way of pyroptosis [35].The accumulation of ox-LDL is one of the ways to trigger endothelial pyroptosis. When ox-LDL accumulates in the vascular wall, it triggers the activation of NLRP3 inflammasome, and the expression levels of NLRP3, Caspase-1 and related inflammatory factors are significantly increased, through NLRP3/Caspase-1/IL-1β Signal pathways promote endothelial cell activation and dysfunction [36,37]. In arterial plaque, pyroptosis caused by NLRP3 inflammasome is highly correlated with plaque rupture and vasculitis, and pyroptosis related molecules, NLRP3, ASC, Caspase-1, and GSDMD also show a trend of high expression in unstable plaque [38].
In this study, scanning electron microscope was used to find that after ox LDL stimulated HUVEC for 24 hours, cells swelled, membrane ruptured, accompanied by a large number of bubble like projections and pore formation, and pseudopodia was obvious, which was consistent with the typical morphological changes of pyroptosis. After stimulating HUVECs with ox-LDL, we found an increase in NLRP3 levels, indicating activation of NLRP3 inflammasomes. Activated Caspase-1 is composed of its subunits p10 and p20 heterotetramer, which are both effective components of Caspase-1. The formation of its subunits p10 and p20 not only reflects the activation level of Caspase-1, but also reflects the degree of pyroptosis [39]. Therefore, we used RT-qPCR and WB to detect Caspase-1 and Caspase-1 p10 respectively, and the results were higher than those of the normal group, suggesting that Caspase-1 was activated, and the expression level of GSDMD was up-regulated. GSDMD is an executive protein of pyroptosis, and a marker of pyroptosis [40]. In addition, activated Caspase-1 can induce IL-1β and IL-18 precursors to become active IL-1β and IL-18, allowing inflammatory substances to be released into the extracellular space through membrane pores [41], while explaining the results of inflammatory factors in the previous experiment. Ox-LDL can induce the pyroptosis of HUVECs, which has been verified in this experiment.
Berberine, as a traditional Chinese herbal medicine, has powerful effects in anti-inflammatory, lipid-lowering, improving vascular endothelial function, inhibiting inflammatory cell infiltration and smooth muscle proliferation, migration, and stabilizing plaques [2,42]. It has been found that berberine can treat digestive system, respiratory system, nervous system and other related diseases by inhibiting the classic pyroptosis pathway mediated by NLRP3 inflammasome [43,44]. However, the regulatory mechanism of berberine on NLRP3 inflammasome mediated pyroptosis is still unclear. We demonstrated whether BBR could inhibit ox-LDL induced pyroptosis of HUVECs. Before ox-LDL induced endothelial pyroptosis, HUVECs were pretreated with 20µmol/L BBR 1 hour in advance. Observation of cell morphology under scanning electron microscopy showed that the cell surface of the drug group was slightly rough. After BBR treatment, the pores and folds on the cell membrane surface were significantly reduced compared to the model group. BBR can alleviate endothelial cell damage and inhibit the pyroptosis of HUVECs induced by ox-LDL. The WB and RT qPCR results suggest that BBR can reduce the expression of NLRP3 mediated pyroptosis protein molecules, and downregulate the relative expression of NLRP3, Caspase-1, GSDMD mRNA, while IL-18 and IL-1 β The secretion of LDH in the cell supernatant was also reduced. We can conclude that BBR can inhibit NLRP3 inflammasome mediated endothelial pyroptosis, and its mechanism may be that BBR can reduce endothelial cell damage by downregulating NLRP3/Caspase-1/GSDMD pathway, intervening in inflammatory cascade reaction, reducing the production of IL-18, IL-1β, LDH and other downstream products.
TXNIP is the second signal activated by NLRP3 and can directly bind to NLRP3. When TXNIP is activated, it immediately separates from TRX and acts directly on NLRP3 [45]. After the activation of NLRP3 inflammasomes, the expression of Caspase-1 and GSDMD proteins increased, and the release of inflammatory factors IL-18 and IL-1β increased. After gene knockout of TXNIP, the activation of NLRP3 inflammasomes was affected, and downstream molecules were significantly reduced [46]. Multiple research results have shown that many drugs, such as Tangshen Formula and Taurine, are involved in regulating TXNIP to achieve the goal of treating related diseases [47,48]. However, there are few studies on the regulatory effect of BBR on TXNIP, especially in the AS field. Therefore, the research team studied the regulatory mechanism of berberine on TXNIP, and further elaborated the mechanism of BBR's effect on NLRP3 inflammatory body mediated endothelial pyroptosis by regulating the level of TXNIP.
We infected HUVECs with Lentivirus TXNIP to construct a TXNIP overexpression system. We found that after overexpression of TXNIP, the expression levels of NLRP3, Caspase-1 and GSDMD were significantly increased, the levels of IL-18 and IL-1β inflammatory factors were increased, and the content of LDH in the supernatant was increased. Therefore, overexpression of TXNIP could aggravate NLRP3 mediated pyroptosis. After overexpression of TXNIP, we pretreated HUVECs with BBR for 1 hour, and then continued to interfere with HUVECs with ox LDL for 24 hours. The results showed that the expression levels of NLRP3, Caspase-1, and GSDMD were inhibited, and the levels of downstream products IL-18 and IL-1β were reduced, and the LDH release was reduced, indicating that BBR can offset the pyroptosis effect caused by overexpression of TXNIP to a certain extent, indicating that BBR can inhibit endothelial pyroptosis by inhibiting the overexpression of TXNIP. It can be seen that TXNIP may be the target of BBR, and its mechanism may be through down-regulation of TXNIP/NLRP3/GSDMD signaling pathway to inhibit ox LDL induced endothelial pyroptosis, thus playing a role in alleviating endothelial cell damage and delaying the occurrence and development of AS.
The mechanism of pyroptosis and AS is complex, and the research is not thorough. How NLRP3 inflammatory bodies drive AS needs to be further explored.The characteristic of berberine action is multiple pathways and targets, and the mechanism is relatively complex. Although the research group has done some research at the cell level, the cell type is single, and the pyroptosis pathway is limited, which cannot represent the in vivo experiment, and needs further systematic improvement. In this study, the mechanism of berberine inhibiting pyroptosis has not been verified in clinical practice, and there is a lack of relevant clinical data. Therefore, more in vivo studies and clinical experiments are needed to clarify the molecular pathway and specific mechanism of berberine on pyroptosis, so as to provide a basis for the development of AS target inhibitors.