Platelets are not only involved in primary hemostasis but also in the formation of thrombus induced by inflammation. Platelet activators include not only thrombin and ADP but also molecules involved in inflammation (64). Platelets as non-nucleated cells also contain members of the NF-κB family and their corresponding signaling molecules, which are involved in platelet activation and secondary feedback loops (56). Activated platelets express or secrete pro-inflammatory and pro-coagulant substances on their surfaces, such as adhesion molecules, growth factors, cytokines, and the fibrinolytic inhibitor PAI-1, inducing surface aggregation of coagulation factors (65).

The injury of the vein wall or vein endothelial cells caused by various factors is one of the factors that cause DVT. Studies have confirmed that NF-κB signaling molecules exist in endothelial cells (63). When injured, endothelial cells are activated, which can inhibit the expression of thrombomodulin (TM) and activate the expression of endothelial tissue factor (TF), resulting in the activation of adhesion molecules such as P-selectin and clotting factor vWF (66), and the endothelial cells change from anticoagulant, anti-inflammatory, and vasodilator functions to proinflammatory and pre-thrombotic states.

Inflammatory cells represented by neutrophils and monocytes were able to rapidly aggregate and adhere to the venous endothelium (67). Among them, neutrophils can activate coagulation factors XII, initiate endogenous coagulation, and also form neutrophil extracellular traps (NETs) after apoptosis. NETs, as part of the body’s innate immunity, are an extracellular network of fibers made up of disaggregated chromatin (DNA fibers and histones) released by neutrophils and more than 30 granule proteins with antimicrobial properties (68, 69), providing a supporting basis for thrombosis through their fiber network structure (70). They interact with platelets to further stimulate platelet aggregation, activate thrombin, and accelerate the DVT process (71, 72) ( Figure 1 ).

DVT group and miR-181b overexpression and inhibition of rat models were constructed by producing a deep-vein thrombosis model and injecting normal saline, miR-181b mimics, and inhibitors into the tail vein. The changes of NF-κB (P65) in the venous endothelium of rats in each group were analyzed, and the expression level of NF-κB (P65) in the venous endothelium of rats in the Normal group was used as a reference. The results showed that the expression of NF-κB (P65) in the venous endothelium of rats in the 181b-i group was the highest, followed by the DVT group, and the 181b-m group was lower than the DVT group but higher than the Normal group; moreover, the difference was statistically significant (P<0.05). These results indicate that the expression of NF-κB (P65) is increased in the rat vena cava DVT model, and miR-181b can inhibit the expression of NF-κB (P65). By comparing the length and wet weight of the thrombus in each model group, no thrombus formation was observed in the Normal group. Overexpression of miR-181b can shorten the length of the thrombus and lighten the wet weight after thrombus formation in rats; inhibition of miR-181b expression can lengthen the length of thrombus and increase the wet weight after thrombus formation. These results suggest that miR-181b can reduce the formation of DVT by inhibiting the NF-κB signaling pathway to a certain extent (78).

Up-regulating the expression of miR-150 inhibited thrombosis in DVT rats. The effect of miR-150 on inflammation was studied in vivo and in vitro. The rats were injected with LV-NC, and it was found that the administration of LV-miR-150 inhibited the platelet aggregation inhibition rate and TXB2 content in the rat model and significantly inhibited thrombosis. Transfected into in vivo skin cells, PAI-1, TNF-α, IL-6, and IL-8 levels were significantly reduced, indicating that miR-150 alleviated inflammation and inhibited apoptosis of vascular endothelial cells. The expression level of NF-κB p50 in vascular endothelial cells transfected with miR-150 mimics was significantly decreased, while the expression level of NF-κB p50 was significantly increased by miR-150 inhibitors. These results suggest that miR-150 may have a negative regulatory effect on NF-κB p50. Therefore, overexpression of miR-150 can be used as a potential therapeutic target for future DVT (79).

This study found that by overexpression of miRNA-141, the expression of TLR4 and its signaling pathway-related proteins NF-κB, Rac1, and IL-1β in vascular tissues of thrombotic rats was significantly down-regulated, and by restoring the expression of TLR4 and NF-κB, the expression of Rac1 and IL-1β was restored at the same time, and multiple related indexes of miRNA-141 on thrombus were significantly reversed (80). Activated TLR4 induces late activation of NF-κB, which significantly increases TNF-α expression and causes widespread inflammation (53). Furthermore, TNF-α stimulates the TNF receptor (TNFR) and induces phosphorylation of IκB kinase (IKK), which in turn enhances NF-κB activity (81). NF-κB is the end point of the TLR4/NF-κB pathway and the regulatory hub of an inflammatory response, and its activation can enhance the inflammatory response and promote the formation of a thrombus. This may be a key mechanism by which miRNA-141 inhibits thrombus formation by regulating the TLR4/NF-κB signaling pathway ( Table 1 ).

Qihong Tongluo prescription is mainly composed of Astragalus and safflower. Astragalus has a variety of biological functions, including potent immunomodulatory, antioxidant, anti-inflammatory, and antitumor activities (82). Isoflavones, saponins, and polysaccharides are three types of beneficial compounds for their pharmacological activity and therapeutic efficacy (82–84). Astragalus polysaccharides decreased the expression of IL-1β, IL-6, TNF-α, and INF-γ by regulating the toll-like receptor 4 (TLR4)/NF-κB signaling pathway (85, 86). In addition, safflower can inhibit platelet activation, adhesion, and aggregation (87, 88). In summary, we believe that Qihong Tongluo prescription’s inhibition of thrombosis is the result of the joint action of its active components.

The high incidence of thrombotic events is one of the clinical features of coronavirus disease (COVID-19) due to the high inflammatory response caused by the virus. Gegen Qinlian pill (GQP) is a traditional Chinese medicine, which can inhibit toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) signaling (89–91), has good anti-inflammatory activity, has a good effect on the treatment of COVID-19, and has shown anti-thrombotic potential. In our study, GQP treatment significantly reduced the expression of TNF-α, NLRP3, and NF-κB, reduced lung, liver, and tail thrombosis, and increased tail blood flow in mice (92). This at least partially supports the hypothesis that GQP can inhibit inflammation-induced thrombosis by inhibiting NF-κB/NLRP3 signaling.

HLJJD is a famous prescription in China, and its main compounds have been studied as baicalin and berberine (93). Baicalin, a flavonoid compound extracted from the root of Scutellaria baicalensis, has significant anti-inflammatory and antibacterial effects, scavenging oxygen free radicals and anti-allergic reactions (94, 95). Baicalin can inhibit the NF-κB signaling pathway, reduce the expression level of the p-NF-κB p65 protein, and reduce inflammatory response (96, 97). Berberine is an isoquinoline alkaloid isolated from Coptis chinensis, a Chinese medicinal plant, and has significant anti-inflammatory effects (98, 99). For the infection-induced tissue injury model, the gene and protein expression levels of TNF-α, TLRs, and NF-κB p65 were significantly reduced in the berberine treatment group (100–102). TLR4 is a pattern recognition receptor, and activated TLR4 induces late activation of NF-κB, which significantly increases TNF-α expression and causes widespread inflammation (53). TNF-α stimulates the phosphorylation of IκB kinase (IKK), which in turn enhances the activity of NF-κB (81). As the endpoint of the TLR4/NF-κB pathway and the regulatory hub of the inflammatory response, the activation of NF-κB can enhance the inflammatory response and promote the formation of thrombosis.

Through intravenous injection of the effective components of Huanglian Jidutang into the thrombus model, the results showed that the contents of IL-1β, IL-6, and TNF-α and the expression levels of TLR4, NF-κB, NLRP3, and Caspase-1 were decreased (103), and they also showed significantly reduced thrombus dry weight, Block platelet aggregation, and adhesion induced by collagen (104). In conclusion, the inhibitory effect of the active components of Huanglian Jiedu Decoction on the NF-κB pathway is the cause of alleviating inflammation and reducing thrombosis.

LSW is a classic proprietary Chinese medicine with anti-inflammatory and analgesic effects (105). Its main components are cow gallstone, musk secretion, toad secretion, pearl shell, realgar, and borneol (106). In PR8-infected cells, LSW significantly down-regulated the expression levels of IL-1β, TNF-α, IL-6, and IFN-γ. In mice infected with PR8, LSW reduced the secretion of TNF-α, IL-1β, IL-6, and IFN-γ in lung tissue, significantly improving survival. In addition, LSW significantly reduced the expression levels of TLR4, phosphor-NF-κB p65, and phosphor-iκBα (107). It is suggested that LSW exerts anti-inflammatory effects by regulating the TLR4/NF-κB signaling pathway.

Rhein is widely found in a variety of Chinese herbs, including Rhubarb palmatum, aloe curacao, cassia stenophyllum, and polygonum multiflorum. It has antioxidant, antiviral, anti-inflammatory, anti-tumor, and immunomodulatory activities (108). In mice infected with PR8, rhein significantly improved survival and reduced the lung index and lung expression levels of IL-1β, IL-6, IL-8, and TNF-α. In addition, rhein significantly reduced the protein levels of TLR2, TLR3, and TLR4 and the phosphorylation of NF-κB p65 in PR8-infected cells. The addition of TLR4 and NF-κB activators can antagonize the inhibitory effect of rhein on viral replication (109), so the anti-inflammatory effect of rhein may be related to inhibiting the activation of the TLRs/NF-κB signaling pathway.

H. cordatum Thunb. is an important plant medicine with antiviral, antibacterial, anti-inflammatory, and antioxidant activities (110–112). Flavonoids, one of the effective components of this phytomedicine, can reduce lung and intestinal damage in mice, inhibit the over-release of tumor necrosis factor-α, IL-1, IL-8, and MCP-1 in the lung tissue of infected mice, and inhibit the up-regulated expression of TLR and NF-κB p65 proteins (113). HCP may play an anti-inflammatory role by inhibiting the activation of the TLR/NF-κB signaling pathway ( Table 2 ).

There are studies that Xiaoshuantongmai decoction can mediate microRNA-181b intervention in deep-vein thrombosis. According to the equivalent dose ratio of kg body weight of human and animal, the drug dose was converted, and based on the deep-vein thrombosis model, normal saline and Xiaoshuantongmai decoction were administered. The results showed that the expression level of miR-181b in the venous endothelium of the Xiaoshuantongmai Tang group was the highest, followed by the blank control group and the sham operation group, and the lowest was in the model control group, with a statistical difference. It was proved that Xiaoshuantongmai decoction can up-regulate the expression of miR-181b in the venous endothelium of DVT model rats (78).

Acute pulmonary thromboembolism (APE) is a disorder of pulmonary circulation caused by a blockage of the pulmonary artery. Extensive inflammatory responses have been demonstrated in the lung tissue of APE rats, accompanied by significantly elevated levels of tumor necrosis factor-α, interleukin-1-β, and IL-8 (114, 115). High levels of NF-κB were also observed in rats after APE induction (116, 117). Aspirin and salicylate have been reported to inhibit NF-κB equally (118, 119). Healthy SD rats were randomly divided into a control group, sham operation group, APE model group, and aspirin low-dose, medium-dose, and high-dose groups. After APE induction for 6, 24, and 72 h, rats in the low-, medium-, and high-dose aspirin groups were given daily doses of aspirin at 150, 300, and 600 mg/kg, respectively, for 3 consecutive days. The other groups were given the same amount of normal saline. In the APE model group, thrombus formation, alveolar wall injury, pulmonary hemorrhage, and inflammatory cell infiltration occurred at all time points. After aspirin treatment, pathological changes such as pulmonary hemorrhage and inflammatory cell infiltration were reduced. Compared with the APE model group, the expression of the NF-κB protein measured by Western blotting was significantly decreased in other groups at each time point (P<0.05, P<0.001). The highest expression of the NF-κB protein was observed in the APE model group, and NF-κB protein expression decreased gradually in a dose-dependent manner in rats receiving aspirin (120). In summary, aspirin can significantly inhibit the NF-κB pathway in a dose-dependent manner to reduce inflammation and alleviate lung injury after APE.

Clinically, ticagrelor and clopidogrel (antiplatelet coagulants) are often combined with PCI for acute coronary syndromes (ACS) (121). They cure ACS12 adenosine diphosphate (ADP) receptors by targeting the platelet P2Y to inhibit platelet aggregation and reduce thrombosis (122). In this study, human umbilical vein endothelial cells (HUVECs) were cultured with ticagrelor or clopidogrel and given lipopolysaccharide (LPS) and CD14. Human umbilical vein endothelial cells (HUVECs) were cultured with ticagrelor or clopidogrel and given lipopolysaccharide (LPS) and CD14. Ticagrelor and clopidogrel reduce the expression of TNF-α, IL-1, IL-6, IL-8, and IL-2, inhibit p65 phosphorylation and IKB-α degradation, and significantly reduce the amount of nuclear translocation p65 (123, 124). These findings suggest that ticagrelor and clopidogrel inhibit the production of inflammatory cytokines by inhibiting the NF-KB pathway.

PAR-1 can be activated by thrombin to regulate platelet aggregation and endothelial permeability, so it is clinically used as a target for anti-platelet drugs to prevent thrombosis (125–127). Vorapaxar is a representative drug (128). IR induction was performed in rat lung models by perfusion in vitro. Male rats were treated with the specific PAR-1 antagonist vorapaxar or the control agent with 40 min of ischemia and 60 min of reperfusion. In vitro, mouse lung epithelial cells (MLE-12) were treated with vorapaxar and subjected to hypoxic reoxidation (HR). We found that vorapaxar reduced the production of thrombin, inflammatory factors, cytokine-induced neutrophil chemokine-1, interleukin-6, and tumor necrosis factor-α, pulmonary edema and neutrophil infiltration, and it alleviated lung cell apoptosis and down-regulated the nuclear factor-κB (NF-κB) pathway. It also blocked HR-induced NF-κB activation and the production of inflammatory chemokines in MLE12 cells. The results suggest that vorapaxar acts by blocking PAR-1 expression and modulating the NF-κB pathway (129) ( Table 3 ).