To our knowledge, this is the first research using synovial metabonomic approach to explore the potential applications of HL in treating synovial diseases. According to the pathway analysis of MetaboAnalyst, HL mainly affected two key pathways, that is, arachidonic acid metabolism and glycerophospholipid metabolism. Previous studies have shown that both pathways are closely associated with some synovial diseases, such as osteoarthritis and rheumatoid arthritis, which are involved in the pathogenesis of both diseases [13–17]. Previous proteomics study shows that HL also affects 4 key protein pathways (ribosome, RNA transport, hematopoietic cell lineage, and mRNA surveillance pathway), which may participate in the degenerative synovial diseases [2]. Therefore, combined with the pathway analysis of both studies, we find that HL may have a therapeutic advantage for the treatment of osteoarthritis (one of the degenerative diseases in synovium). The speculation is consistent with the validation test results of previous research [2].
Combined with the previous proteomics and current metabonomics studies, we find that both protein and metabolite biolabels of HL may affect the platelet aggregation and apoptosis regulation processes in the synovium [2]. In addition, the present experiment also showed that HL can regulate the expression of some endogenous metabolites, which are involved in inflammation, angiogenesis, and carcinogenesis processes (Fig. 4). In the following sections, we briefly discuss the biochemical processes of these potential biolabels, which may be helpful to explore the therapeutic potentials of HL for synovial diseases.
The aggregation of platelets in the synovium is significant for the treatment of synovial diseases, which can release the growth factors and promote synovial tissue repair and regeneration [18, 19]. Our previous proteomics study has demonstrated that HL can significantly increase the levels of Gp1bb, Itga2b, and Itgb3 in physiological situation, which indicates that HL may promote platelet aggregation in the synovium [2]. In this respect, the data from the current experiment complemented our previous research. Arachidonic acid is the precursor of prostaglandin E2, an increase in whose level may facilitate prostaglandin E2 production. As shown in Fig. 5b, the levels of prostaglandin E2 are increased significantly in HL-treated groups, which can potentiate platelet aggregation [20]. However, prostaglandin E1 may produce an opposite effect on prostaglandin E2 in this process, which inhibits platelet aggregation [21]. HL reduced the content of 8,11,14-eicosatrienoic acid in synovial tissue. The metabolite can be converted into prostaglandin E1, whose levels were increased significantly in HL-treated groups (Fig. 5a). From these, we known that HL may promote the conversion of 8,11,14-eicosatrienoic acid to prostaglandin E1. In addition, the up-regulation of arachidonic acid by HL can also induce prostaglandin E1 production [22]. Therefore, we speculate that the up-regulation of prostaglandin E1 may be one of the important molecular mechanisms by which HL inhibits platelet aggregation in synovial membrane of osteoarthritis model [2]. Combining the above description, through its regulation of prostaglandin E1/ prostaglandin E2, HL may selectively promote or inhibit platelet aggregation in synovial membrane under different situations. This may also explain our previous research results showing the dual effects of HL on synovial platelet aggregation in the joints of healthy rats and osteoarthritis rats [2].
Abundant mononuclear cells infiltration and overexpression of inflammatory factors are seen in synovial diseases including osteoarthritis and rheumatoid arthritis [23, 24]. The changes in the levels of vanilpyruvic acid, 8,11,14-eicosatrienoic acid, arachidonic acid, and prostaglandin A1 may participate in the mechanism of HL against synovial inflammation. Vanilpyruvic acid is a catecholamine metabolite, an increase in whose level may mean the accumulation of catecholamine in synovial tissue. One previous study has shown that catecholamine-producing cells are present in inflamed synovial tissue of osteoarthritis and rheumatoid arthritis, and produced catecholamines has strong anti-inflammatory activities [25]. Prostaglandin E1, converted from 8,11,14-eicosatrienoic acid, may modify the inflammatory response and suppress arthritis [26, 27]. However, prostaglandin E2 (one of the metabolites of arachidonic acid) may antagonize the effects of prostaglandin E1, which is an inflammatory mediator and triggers the inflammatory response in the synovium [28]. Additionally, synovial NF-κB plays a crucial role in synovial inflammation [29]. Prostaglandin A1, a potent inhibitor of NF-κB, also has anti-inflammatory effects [30]. From these data, HL may also have a bidirectional effect on synovial inflammation.
Apoptosis is one of the inducing factors of synovial diseases [31, 32]. The regulation of prostaglandin A1, 9E,11E-octadecadienoic acid, PC(o-18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), PC(22:2(13Z,16Z)/14:0), and PC(14:0/20:2(11Z,14Z)) by HL may show the possibility of inhibiting synovial apoptosis. In addition to its anti-inflammatory activity, prostaglandin A1 also exerts anti-apoptotic effects via inhibiting the activity of NF-κB [33, 34]. 9E,11E-Octadecadienoic acid is a conjugated linoleic acid isomer, which can induce apoptosis [35]. Additionally, the phosphatidylcholines have pro-apoptotic potentials as well [36], the dysregulation of which in synovial tissue is associated with osteoarthritis [37]. Suppression of these four metabolites by HL might enhance the inhibition of prostaglandin A1on apoptosis. However, on the contrary, HL can significantly increase the contents of two phosphatidylcholines (PC(22:1(13Z)/14:0) and PC(14:0/20:3(5Z,8Z,11Z))), which might lead to apoptosis [36]. In addition, phosphatidylcholine can donate its phosphocholine to ceramide for the synthesis of sphingomyelin, abnormal metabolism of which may affect the level of sphingomyelin to some extent [36]. SM(d18:0/22:3(10Z,13Z,16Z)) is a sphingomyelin, the activation of whose pathway is also involved in the apoptosis process [38]. From these point of view, HL may produce the bidirectional effects on apoptosis, which was in line with the results of previous proteomics study [2]. Besides protein biolabels [2], endogenous metabolites may also be used to characterize the apoptosis regulation potentials of HL.
LysoPC(18:2(9Z,12Z)) and LysoPC(16:0) are the lysophospholipids, which have a role in lipid signaling by acting on lysophospholipid receptors. Lysophospholipids can promote angiogenesis [39]. Synovial angiogenesis is involved in the pathogenesis of various synovial diseases, such as osteoarthritis and rheumatoid arthritis [40]. Antiangiogenic drugs used in synovial diseases are available [41]. Additionally, lysophospholipids also have stimulatory roles in cancer progression and stimulate the proliferation/survival of cancer cells [39]. Therefore, the inhibitory effects of HL on these two lysophospholipids might be conducive to the inhibition of synovial angiogenesis and carcinogenesis.
In summary, through the regulation of prostaglandin E1 and E2, HL exerts the dual effects on platelet aggregation in the synovium. This may be helpful for the protection of HL from synovial lesions under different situations. Additionally, from the analysis at the level of metabolite, HL is suitable for treating synovial diseases, especially osteoarthritis. The treatment may be associated with the platelet aggregation, apoptosis, inflammation, angiogenesis, and carcinogenesis processes, which may be applied to update the information characterizing the treatment of HL on synovial diseases.