Liver fibrosis is a pathological process characterized by abnormal deposition of extracellular matrix (ECM), which is an important factor contributing to the increasing global burden of disease [1]. Due to viral / parasitic infection, inflammation, mechanical injury, autoimmune diseases and other factors, non parenchymal cells in the liver, namely hepatic stellate cells (HSCs), are activated, transformed into myofibroblasts, secrete a large amount of ECM and deposit in the liver interstitium, forming scars [2]. . As an intermediate link from chronic liver disease to liver failure / liver cancer, how to reverse liver fibrosis is a hot topic of great research significance.
Ligustrum lucidum is a plant of OleaceaeLigustrum lucidum Ait.'s dried and mature fruit, also known as "Holly seed", has a very high medicinal value [4]. Shennong herbal classic once recorded that "Ligustrum lucidum is solid, tastes bitter and flat, mainly enriches the middle, soothes the five internal organs, nourishes the spirit, and eliminates all diseases" [5]. Ligustrum lucidum contains a variety of active ingredients, such as triterpenoids: oleanolic acid, ursolic acid, acetyl oleanolic acid, etc; Iridoids: Ligustrum lucidum glycosides, tertileZhenLigustrine glycosides, ligustrine glycosidesOleuropicric acidEt al; Flavonoids: apigenin, quercetin, etc; Phenylethanol glycosides: salidroside, verbascoside, etc., have the effects of regulating liver protection, immunity, anticancer, antitumor, etc. [6]. In addition, Ligustrum lucidum also contains some polysaccharides, fatty acids, etc. [7].
Based on the related research of transforming growth factor - β (tgf- β) / Smad, reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) / reactive oxygen species, phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt), p38 mitogen activated protein kinase (MAPK), Janus kinase (JAK) / signal transducer and activator of transcription (STAT), nuclear factor - κ B (nf- κ b) and other signaling pathways, this paper reviews the anti hepatic fibrosis mechanism of Ligustrum lucidum, so as to provide reference for the clinical application of Ligustrum lucidum.
1 liver fibrosis
When chronic liver disease develops to the later stage, hepatocytes are severely damaged, inflammatory cells infiltrate and secrete a large number of inflammatory factors, leading to HSC activation and proliferation. Under normal conditions, HSCs exist in the sinusoidal space of the liver in a resting state [2,8], and participate in the regulation of ECM production and degradation, as well as liver sinusoidal blood flow. However, when the liver is damaged, tissue resident macrophages scattered in the sinusoids of the liver, namely Kupffer cells, activate and promote HSC activation under the joint action of a variety of cytokines. Because HSCs express tgf- β 1 receptors and can also secrete tgf- β 1, a positive feedback regulatory loop is formed in this case, resulting in the sustained activation of HSCs.
. Under normal circumstances, the synthesis and degradation of ECM are regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), and are in a dynamic balance. However, when liver fibrosis occurs, this balance is disrupted, ECM synthesis increases and degradation decreases, resulting in excessive synthesis of ECM, which further causes liver fibrosis [9].
2 anti hepatic fibrosis mechanism of active components of Ligustrum lucidum
2.1 regulatory mechanism of tgf- β /smadsignaling pathway
. The tgf- β / smadsignaling pathway mainly consists of three parts: tgf- β ligands located outside the cell, tgf- β receptors on the cell membrane surface, and Smad proteins that transmit signals inside the cell [11]. Smad Protein plays a role downstream of tgf- β /smadsignaling pathway, directly participating in and affecting the occurrence of liver fibrosis [12].
There are two forms of tgf- β /smad: basal state and activated state [10,13]. When in the basal state, tgf- β ligands are isolated by ECM or other substances and exist stably in the form of complexes, making it unable to bind to receptors and play a role. However, when activated, tgf- β ligands dissociate from the corresponding complexes and directly bind to serine / threonine kinases on the cell membrane, namely, type I receptors (tgf- β R-I) and t β R-II, or bind to the above two receptors under the action of other auxiliary molecules. Afterwards, t β R-II acts on the juxtamembrane regulatory region of t β R-I to phosphorylate it, and then causes receptor regulated Smad (r-smad) to undergo phosphorylation reaction. Finally, phosphorylated r-smad forms a complex with Smad3 to enter the nucleus and regulates the expression of corresponding genes [10].
Flavonoids have a unique protective effect on the liver and can alleviate the occurrence of liver fibrosis [14-15]. Apigenin is the main flavonoid in Ligustrum lucidum seed, which also exists in citrus fruits, parsley, celery leaves and other plants [16]. Apigenin can down regulate the expression of TGF - β 1, Smad3 and p-smad3 in animal models of liver fibrosis induced by CCl4 and bile duct ligation, indicating that apigenin can inhibit TGF - β 1 / Smad3 pathway; By measuring the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, and hematoxylin eosin and Masson staining, it was found that apigenin could significantly improve the arrangement disorder of hepatocytes, and the proliferation of fibrous tissue and inflammatory cell infiltration were significantly reduced. It can be seen that apigenin has a certain therapeutic effect on liver fibrosis, which may be related to the inhibition of tgf- β 1/smad3 pathway [17].
In addition, luteolin, quercetin, Douglas fir and other flavonoids in Ligustrum lucidum can also play an anti fibrotic role through this pathway. Luteolin, a natural tetrahydroxyflavonoid, widely exists in chrysanthemum, pepper, onion and other plants. It has a wide range of pharmacological effects, including anti-tumor, antioxidant and so on [18]. By exploring the relevant mechanisms of luteolin affecting HSC proliferation and activation, it was found that luteolin can significantly inhibit the phosphorylation of Smad2 and Smad3, indicating that luteolin can inhibit tgf- β 1/smad3 pathway and has the potential of anti liver fibrosis [19]. Quercetin is also a flavonoid that widely exists in a variety of plants, with antioxidant, anti-inflammatory and other functions [20]. Similarly, quercetin could also significantly reduce the levels of tgf- β 1, p-Smad2 and p-smad3 in serum; Immunohistochemical staining showed that the expression levels of tgf- β 1, p-Smad2 and p-smad3 in liver tissue were significantly decreased after quercetin treatment, suggesting that quercetin has an inhibitory effect on tgf- β 1/smad3 pathway [21].
In addition, by detecting AST and ALT in the liver of mouse model of liver fibrosis, it was found that quercetin could reduce the levels of AST and alt, and alleviate the development of liver fibrosis. The above results suggest that quercetin can inhibit liver fibrosis through tgf- β 1/smad3 pathway [21]. Douglasine exists in many plants such as Larix olgensis, Astragalus membranaceus, onion and so on. It has four phenolic hydroxyl groups and one hydroxyl group. Due to this special chemical structure, it has become the most potent natural antioxidant, with a variety of pharmacological effects such as anti oxidative stress and liver protection [22]. TGF-β1、α-SMAThe mRNA and protein levels of Smad3 were significantly down regulated, and the protein levels of Smad3 were also down regulated in 40 and 80 mg/kg dose groups [23], indicating that saikosaponin can inhibit the activation of HSCs by inhibiting tgf- β 1/smad3signaling pathway, thus playing an anti hepatic fibrosis role.
Phenylethanol glycosides are a class of natural glycosides. So far, 18 phenylethanol glycosides have been isolated from Ligustrum lucidum, such as salidroside and echinacoside [7]. Among them, echinacoside is composed of glycosyl, phenylallyl, and phenylethanol groups, which is abundant in the rhizomes of Echinacea plants and has liver protection, anti-tumor, anti-inflammatory and other effects [24]. Studies have found that echinacoside can down regulate the expression and phosphorylation levels of Smad2 and Smad3, and up regulate the expression of Smad7, suggesting that echinacoside has the potential function of regulating HSC activation by inhibiting tgf- β /smadsignaling pathway [25].
Salidroside, as one of the main pharmacodynamic components in Ligustrum lucidum, has a wide range of pharmacological effects, including liver protection, anti-tumor, anti oxidative stress and other functions [26]. Salidroside can inhibit the tgf- β /smadsignaling pathway by downregulating the expression of tgf- β 1 in HSCs, affect the activation and autophagy of HSCs, lead to the decline of ECM synthesis, and promote the recovery of imbalanced MMP2 and TIMP1 [9]. Yue Xueliang [27] studied the effect of salidroside on CCl4 induced mouse liver fibrosis model and found that it can effectively alleviate liver fibrosis. Compared with the model group, the expression of tgf- β and Smad3 in liver tissue of salidroside 20 and 40 mg/kg groups were significantly down regulated, suggesting that salidroside may inhibit liver fibrosis through tgf- β /smad3signaling pathway.
Ligustrum lucidum also contains some iridoid terpenoids and triterpenoids, which may play a potential role in anti liver fibrosis through tgf- β /smadsignaling pathway. Ligustrin is an active component of iridoid substances with multiple pharmacological effects in Ligustrum lucidum [28]. In vivo experiments showed that ligustrin could reduce the liver index and down regulate the expression of ALT and AST in liver fibrosis mice, indicating that ligustrin could effectively improve the degree of liver injury in CCl4 induced liver fibrosis. α-SMA， And the expression of t β R-I, t β R-II, p-Smad2, p-smad3 and Smad4. These findings suggest that ligustrin may alleviate liver fibrosis through tgf- β / smadsignaling pathway [29]. . Studies have shown that Potentilla acid can not only significantly inhibit the activation of HSCs, but also significantly down regulate the expression of Smad4, suggesting that Potentilla acid may regulate the activation of HSCs through tgf- β /smadsignaling pathway [31].
2.2 regulation mechanism of nox4/ reactive oxygen species signaling pathway
NOX4 is a member of NOx. ; NOx in non phagocytic cells can also produce reactive oxygen species, but its role is mainly to regulate apoptosis, proliferation, etc. [34]. In the process of liver injury, NOX4 promotes the synthesis of reactive oxygen species by inducing the expression of p22phox subunit, which in turn activates tyrosine protein kinase and triggers oxidative stress response [35]; High concentration of reactive oxygen species in hepatocytes can inhibit caspases and induce apoptosis[36]. In addition, NOX4 is also involved in the proliferation and apoptosis of HSCs and plays an important role in the migration of myofibroblasts [37].
. Studies have found that ursolic acid can inhibit the expression of NOX4 in HSCs and alleviate the oxidative stress response of liver tissue. Moreover, ursolic acid can also inhibit HSC activation and hepatocyte apoptosis [39]. It may be achieved by inhibiting the activation of NOx caused by the pro fibrotic factor angiotensin II (AngII), and then inhibiting the proliferation of HSCs and the synthesis of type I collagen [40-41]. These findings suggest that ursolic acid can reverse the occurrence of liver fibrosis through nox4/ reactive oxygen species signaling pathway.
Hydroxytyrosol is a phenylethanol glycoside compound and one of the effective ingredients in Ligustrum lucidum seed. It is a natural polyphenol compound in the form of esters in Oleaceae plants and has antioxidant activity [42]. .
2.3 regulatory mechanism of pi3k/aktsignaling pathway
PI3K is an important molecule located upstream of the pi3k/aktsignaling pathway, which can phosphorylate phosphatidylinositol-4,5-bisphosphate (PIP2) to PIP3 after activation [44]. In phosphoinositide dependent protein kinase（
Under the action of phosphoinositide dependent protein kinase (PDK), PIP3 recruits Akt to the plasma membrane and phosphorylates it, thereby activating akt[45], positively regulating the cell cycle, promoting the activation and proliferation of HSCs and the synthesis of collagen, thus leading to liver fibrosis [46].
Ursolic acid and Potentilla acid in Ligustrum lucidum can exert anti liver fibrosis function through pi3k/ aktsignaling pathway. The study found that the expression of pi3k/aktsignaling pathway related proteins (PI3K, p-Akt, p-p38 and MAPK) was significantly increased in rat hepatic stellate HSC-T6 cells stimulated by AngII, indicating that AngII can activate pi3k/aktsignaling pathway. Compared with AngII group, the expression of pi3k/ aktsignaling pathway related proteins and the level of type I collagen mRNA in ursolic acid + AngII group were significantly decreased, and the activation and proliferation of HSCs were also significantly decreased, suggesting that ursolic acid can inhibit the activation and proliferation of HSCs through pi3k/aktsignaling pathway, reduce the synthesis of type I collagen, and alleviate liver fibrosis [40]. Similar to ursolic acid, Potentilla acid can also significantly down regulate the expression levels of p-pi3k, p-Akt, p-mTOR and phosphorylated focal adhesion kinase related proteins of pi3k/akt/ mammalian target of rapamycin (mTOR) signaling pathway in HSCs, suggesting that Potentilla acid can regulate liver fibrosis through pi3k/akt/mtorsignaling pathway [47].
Apigenin, quercetin and Douglas fir, the flavonoids in Ligustrum lucidum, can also exert anti liver fibrosis function through pi3k/aktsignaling pathway [21,23,48]. Among them, apigenin can alleviate the degree of liver injury and pathological changes in mice with liver fibrosis, and also has a significant inhibitory effect on epithelial mesenchymal transition. Further study found that apigenin could significantly inhibit the expression levels of p-pdk1 and p-Akt, suggesting that the anti fibrotic effect of apigenin may be achieved by inhibiting the pdk1/aktsignaling pathway [48]. Similarly, quercetin could effectively reduce the expression of p-pi3k, p-Akt in serum and p-Akt in liver tissue; In mouse liver fibrosis model, quercetin can inhibit HSC activation and promote ECM degradation, suggesting that the inhibitory effect of quercetin on liver fibrosis may be closely related to pi3k/akt pathway [21].
2.4 regulatory mechanism of p38 mapksignaling pathway
MAPK is a protein kinase distributed in the cytoplasm. As an important member of the MAPK family [49], it has the ability to double phosphorylate serine and tyrosine. It can participate in signal transduction by binding membrane receptors and regulating intracellular factors, and finally activate in the form of conserved tertiary kinase cascade and participate in the occurrence of liver fibrosis and other diseases [50]. . Moreover, the higher the degree of HSC activation, the higher the level of p-p38 MAPK; After blocking the p38 mapksignaling pathway, the degree of HSC activation was significantly reduced [19,51].
Betulin in Ligustrum lucidum is a triterpenoid compound, which has anti AIDS, anti-tumor and other activities. . Another triterpenoid in Ligustrum lucidum also has a similar effect: in HSC-T6 cells, ursolic acid can inhibit leptin induced p-p38 MAPK level, and its inhibitory effect is similar to that of p38 MAPK inhibitor SB203580 [53], suggesting that ursolic acid can negatively regulate p38 mapksignaling pathway, and then play an anti liver fibrosis role.
Oleuropein is a polyphenol compound with a natural schizoiridoid glycoside backbone in Ligustrum lucidum, which has anticancer, antioxidant, anti-inflammatory and other activities [54]. Using a mouse model of liver fibrosis, it was found that oleuropein can alleviate liver pathological injury, and compared with the control group, the p38 phosphorylation level and the protein expression of transcription factor ETS domain containing protein (Elk-3) in liver tissue of oleuropein group were significantly down regulated [55], indicating that oleuropein may participate in the regulation of liver fibrosis through p38 mapk/elk-3signaling pathway.
. In addition to tgf- β /smad and pi3k/aktsignaling pathway, quercetin can also regulate liver fibrosis through p38 mapksignaling pathway [57].
2.5 regulation mechanism of jak/statsignaling pathway
JAK is a kind of protein kinase mainly distributed on the surface of cell membrane, which participates in cytokine receptor-mediated signal transduction and is upstream of jak/ statsignaling pathway. Its family members include Jak1, JAK2, JAK3, Tyk2, etc. [58]. Stat is a kind of cytosolic protein with signal transduction and transcriptional activation functions. As a substrate of JAK, stat plays a signal transduction function and can transmit signals from upstream to nucleus to regulate the expression of corresponding genes. . Jak/statsignaling pathway plays an important role in the occurrence of liver fibrosis [60]. Liver fibrosis related molecules, such as leptin, interferon alpha, interleukin-4 (IL-4), etc., will activate the intracellular jak/ statsignaling pathway after binding to specific receptors on the HSC cell membrane, gradually transduce the signal into the nucleus, and regulate the expression of related target genes [60].
Leptin is an important factor causing liver fibrosis, which can activate jak2/stat3signaling pathway. However, after treatment with ursolic acid, the expression levels of p-jak2 and p-STAT3 in leptin activated HSCs will be significantly decreased, and the inhibitory effect of the two is similar to that of JAK inhibitor AG490 [61], suggesting that ursolic acid can inhibit leptin induced activation of jak2/ stat3signaling pathway and has potential anti liver fibrosis function. Similar to ursolic acid, luteolin can also significantly reduce the level of p-STAT3 in a dose-dependent manner, suggesting that its anti hepatic fibrosis function can be achieved by inhibiting statsignaling pathway [62].
2.6 regulatory mechanism of nf- κ bsignaling pathway
Nf- κ B can be expressed in normal or diseased state of the body, participate in a variety of transcriptional regulation, and play an important role in the occurrence of liver fibrosis. Rel protein family is a class of proteins related to the structure of nf- κ B, including P50, p52, p65, RelB and c-Rel, which can enter the nucleus and bind to the corresponding target genes to play a regulatory role [63]. In the process of liver fibrosis, Kupffer cells synthesize various inflammatory factors through the nf- κ bsignaling pathway, constantly activating HSCs, while inhibiting the apoptosis of activated HSCs, and promoting the further development of liver fibrosis [64].
In Ligustrum lucidum, some triterpenoids can regulate the occurrence of liver fibrosis through nf- κ bsignaling pathway, including ursolic acid, Potentilla acid, betulin and betulinic acid. Among them, ursolic acid can inhibit the activation of NOx and down regulate the activity of nf- κ B, thereby reducing the proliferation and transformation of HSC cells and the synthesis of ECM, and slowing down the process of liver fibrosis [65]. Unlike ursolic acid, Potentilla acid inhibits the nf- κ bsignaling pathway, promotes the apoptosis of HSCs, and inhibits the production and deposition of ECM by downregulating the phosphorylation levels of nf- κ bsignaling pathway related proteins p-p65, P-I κ b- α, and p-ikk α / β [66-67]. Similarly, betulin and betulinic acid can also down regulate the levels of nf- κ B and P-I κ B α and inhibit the activation of HSCs, indicating that betulin and betulinic acid can also play a role in the occurrence of liver fibrosis by inhibiting nf- κ bsignaling pathway [52].
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3 conclusion and Prospect
Since the implementation of the "anti drug prohibition order", traditional Chinese medicine has attracted widespread attention due to its advantages of no obvious adverse reactions, wide sources, easy access, and wide pharmacological effects. Its substitution for antibiotics has become a trend. Based on the signaling pathway that plays an important role in the occurrence of liver fibrosis, including tgf- β /smad, nox4/ reactive oxygen species, pi3k/akt, p38 MAPK, jak/stat and nf- κ B, this paper summarizes the anti liver fibrosis mechanism of the active ingredients of Ligustrum lucidum (Fig. 1). The active ingredients in Ligustrum lucidum, such as ursolic acid, apigenin, quercetin and Douglas fir, can inhibit the activation and proliferation of HSCs and the formation of myofibroblasts, regulate hscapoptosis, maintain the balance between MMP and TIMP, reduce ECM synthesis, and play an anti liver fibrosis function through one or more of the above signaling pathways.
Some active ingredients in Fructus Ligustri Lucidi can participate in the regulation of liver fibrosis through a variety of signaling pathways, for example, anthocyanin can play an anti fibrotic role through pi3k/ akt/mtor and tgf- β /smadsignaling pathway, salidroside can play an anti fibrotic role through tgf- β /smad and nf- κ bsignaling pathway, but how these signaling pathways work in coordination with each other, and whether there is a competitive relationship between different active ingredients acting through the same signaling pathway, are the contents that need to be further studied in the follow-up.
Moreover, for the pathological model of liver fibrosis, the commonly used modeling methods are mainly divided into four categories: chemical drug injection, special diet feeding, genetic modification, and surgery [69]. Among them, CCl4 subcutaneous injection method is widely used because of its advantages of convenience, low cost and easy repetition. However, rodents are sensitive to the toxicity of CCl4, and it is very easy to cause animal death due to excessive dose in the process of modeling. It is necessary to consider the dosage, ratio, frequency and time of administration according to animal species, body weight and other factors. At present, CCl4 subcutaneous injection is mostly used in the study of the anti liver fibrosis effect of traditional Chinese medicine monomer or compound, and the etiology is relatively single, but the etiology of liver fibrosis is diverse in clinic. Therefore, when analyzing the anti liver fibrosis mechanism of Ligustrum lucidum and other Chinese herbs, whether it is appropriate to use CCl4 liver fibrosis model needs further discussion. In addition, there are still some unresolved substances in Ligustrum lucidum. Whether these substances have synergistic or antagonistic effects with known substances still needs further study.
References (omitted)
Come Source: ye Ziyu, hexuedong, zhengyongjun, liwanjing, Zeng QingQin, Li ; Jing, Zheng Yadong, Yang Yongchun, zhaojinguo, songhouhui. Research progress on the mechanism of anti liver fibrosis of active components of Ligustrum lucidum [J] . Chinese herbal medicine, 2024, 55 (4): 1389-1396