Peng Yue,Su Aolei,Zhao Tiejian,Zheng Yang,Shang Zhihao

1.Guangxi University of Chinese Medicine,Nanning 530200，China;2.Medical Basic Department of Guangxi Medical College,Nanning 530021,China

Abstract Hepatic fibrosis is a reversible pathological phenomenon in the early and middle stages,but but no satisfactory intervention drugs have been available so far.Recent studies have suggested that microcirculation disturbance of liver is one of the important pathogenesis of chronic liver disease,the improvement of microcirculation is beneficial to the recovery of liver function and the delay of liver fibrosis.Hepatic stellate cells are the core cells of hepatic fibrosis,and also the most critical cells that affect the microcirculation of the liver.While TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs which are based on the action of hepatic stellate cells are two pathways that have very important influence on the inflammatory response of liver,the proliferation and apoptosis of hepatic stellate cells,and the secretion of fibrogenic cytokines.It was found that Plumbapin,the active ingredient of Guangxi specialty ethnic medicine,has the definite effect of promoting blood circulation and removing blood stasis and antihepatic fibrosis,but its mechanism is not clear.In this study,the research progress of the above problems was reviewed,and further research ideas were derived as follows:the pharmacological effect of Plumbapin on anti-hepatic fibrosis,promoting blood circulation and removing stasis was based on the influence of TLR4/MyD88/NF-κB and MAPKs signal pathway.

Keywords Plumbapin;Anti-hepatic fibrosis;Promoting blood circulation and removing blood stasis;TLR4 signal pathway;Animal model

There are more than 30 million patients with chronic liver disease in China,and liver fibrosis is the necessary stage for them to develop to cirrhosis.Liver fibrosis is a kind of chronic liver disease that causes fatty change,inflammation and necrosis of liver cells,and excessive hyperplasia and abnormal deposition of extracellular matrix components in the necrotic area.There are many and complex causes of liver fibrosis,almost any factors that can cause chronic liver damage can lead to liver fibrosis,such as chronic hepatitis B,chronic hepatitis C,fatty liver hepatitis(including alcoholic or non-alcoholic),autoimmune liver disease,schistosomiasis liver disease,drug-induced liver disease and some congenital metabolic diseases[1].Liver fibrosis is a typical pathological feature of chronic liver disease,25%-40% of which can develop into the end-stage cirrhosis of fibrosis.Liver fibrosis and cirrhosis have become one of the main reasons that affect the quality of life and medical expenses of patients with liver disease[2].It has been reported that there are more than 30 million chronic hepatitis B patients in China,and the annual incidence of cirrhosis is 2.1%.The cancer rate of cirrhosis was 17% in 5 years and 40% in 10 years[3].The researchers have realized that liver fibrosis is a reversible pathological phenomenon in the early and middle stage.In June of 2011,Professor Friedman proposed in the Special Meeting of Liver Fibrosis Held by the European Association for the Study of the Liver that the fact that the treatment of hepatitis B and hepatitis C can resist fibrosis proves the reversibility of liver fibrosis,while compared with pulmonary fibrosis,cardiac fibrosis and scleroderma,liver fibrosis is more easily reversed[4].As early as the 1970s,Professor Hans Popper,the founder of modern Hepatology,stated that whoever can prevent or delay liver fibrosis will be able to cure most liver diseases.Therefore,it is very important to prevent and interfere in the pathological process of liver fibrosis as early as possible,delay its development and even reverse it,so as to improve the quality of life and prognosis of patients.But so far,no satisfactory means have been found to solve this major global health problem[5].

1 Hepatic Stellate Cells and Hepatic Fibrosis

Hepatic stellate cells(HSC)accounts for 5%-15% of the total cells in normal liver tissue.The results showed that although many cells in the liver can synthesize ECM,hepatic stellate cells are the main source of ECM in chronic liver disease[6];similar evidences have been obtained in the models of liver fibrosis induced by carbon tetrachloride(CCl4),iron overload and biliary tract obstruction[7];it was found that the activated hepatic stellate cells increase significantly in liver lesions and their surrounding sites,directly dominating the development and progression of liver fibrosis by various etiologies.The proliferative and activated HSC can synthesize and secrete a large amount of ECM,so the activation of HSC is the key link of hepatic fibrosis[8].When the liver is constantly stimulated by chronic injury,the hepatic stellate cells will transform from the normal relatively static phenotype to the activated phenotype,and some functional changes will occur,such as the increase of cell proliferation rate,the decrease of cell apoptosis,mobility,contractility,chemotaxis,the variety and quantity of combined ECM will change[9],and a series of fibrogenic cytokines will be synthesized;leptin is one of the most important mitogenic factors,which can promote the division,proliferation and activation of HSC.

2 TLR4 Signal Pathway and Hepatic Fibrosis

Toll-like receptors(TLRs)are a kind of natural immune receptor,which are mainly distributed on the surface of immune cells;they can directly recognize and combine with some pathogens or their products,which can trigger a series of signal transduction and lead to the release of inflammatory mediators,so it plays an important role in natural immune defense.TLR4 is the first TLR-related protein discovered by human,lipopolysaccharide(LPS)which is the main ingredient of cell wall of Gram-negative bacteria is its main ligand[10].Recent studies have found that TLR4 expressed in various kinds of liver cells can regulate the natural immune response of liver cells,participate in the pathophysiological process of liver,and play an important role in the occurrence and development of liver diseases[11].HSC is the main cell in the liver that directly promotes the formation of hepatic fibrosis through lipopolysaccharide/TLR4 signal transduction[12];through two important signal pathways of TLR4/MyD88/NF-κB and MARKs,HSC can directly enhance the inflammatory response of the liver,affect the proliferation and apoptosis of HSC cells,the secretion and synthesis of collagen,and promote the secretion of profibrogenic cytokines,thus promoting the formation of hepatic fibrosis.

The activation of TLR4 signaling pathway by LPS is mainly through the myeloid differentiation factor 88(MyD88)-dependent signaling pathway[13].The specific steps are as follows(Fig.1):the poly-merized TLR4 forms a complex through the binding of MyD88-like adaptor protein with MyD88,which can interact with IL-2 receptor-related kinase(IRAK)and lead to its phosphorylation,the phosphorylated IRAK can bind to tumor necrosis factor receptor-related factor 6(TRAF6).After binding,TRAF6 can mediate signal transduction through two ways as follows:(i)Activating the IKK complex of nuclear transcription factor κB(NF-κB)inhibitor.Under the action of the complex,IκB is phosphorylated and recognized by ubiquitin ligase complex and degraded,thus NF-κB is released and transferred into the cell nucleus to induce specific gene expression.NF-κB is the most important transcription factor in the inflammatory response[14],which can lead to the release of IL-8,monocyte chemoattractant protein(MCP)1 and intercellular adhesion mol-ecule(ICAM)1 and other pro-inflammatory factors,enhance the inflammatory response of the liver to form an"inflammatory cascade",and ultimately promote the formation of hepatic fibrosis.Tang et al.[15]found that the expression of HSC collagen is related to NF-κB,which inhibits the expression and activity of NF-κB,and signi-ficantly inhibits the expression of collagen.In recent years,it has also been found that NF-κB has anti-apoptotic effect,some apoptotic stimulation can activate NF-κB,and activated NF-κB can block TNF-α-mediated apoptosis[16].In conclusion,NF-κB is an important fibrogenic factor,which can enhance the inflammatory response of liver,regulate the proliferation and apoptosis of HSC,promote the synthesis and secretion of collagen.(ii)Activating TGF-β-activated kinase 1(TAK-1).TAK-1 activates mitogen-activated protein kinase-6(MKK6)by phosphorylation,and further activates mitogen-activated protein kinase(MAPKs)pathway by phosphorylation.This pathway is an important system for eukaryotic cells to regulate and mediate intracellular signal transduction,which is involved in animal cell proliferation,apoptosis,differentiation,transformation and other biological reactions,it mainly includes extracellular signal-regulated kinase(ERK),c-jun-N-terminal kinase(JNK)and P38-MARK signal pathway;through the phosphorylation cascade effect of such key enzymes as RAF,MEK and ERK,these three pathways can activate induced transcription factors,such as Elk-1,c-fos and c-jun,and then regulate the transcription of downstream genes,resulting in a series of cell biological effects,such as cell proliferation,apoptosis and synthesis of cytokines and collagen[17].Liver studies have found that ERK1/2 pathway is involved in the proliferation of hepatic stellate cells,the secretion of ECM and the expression of a variety of fibrogenic cytokines[18];while JNK pathway after activating can enhance the cell activation of HSC,promote cell proliferation and increase its gene expression[19];P38-MAPK pathway can affect the process of hepatic fibrosis by regulating the balance of hepatic inflammatory response,interfering with HSC cell cycle,inducing apoptosis,regulating the effects of TGF-β1 and leptin on HSC,and regulating the secretion of cytokines[20].It was found that CCl4can induce the increase of the expression of ERK1,JUK2 and P38 genes,which are the key signal molecules of MAPK signaling pathway,and the increase of phosphorylated ERK1,JUK2 and P38 protein level in liver tissue of rats with hepatic fibrosis[21].All of the above results suggested that all the key signaling molecules in MAPKs signaling pathway may be the new targets of hepatic fibrosis treatment;by changing the activity of these pathways,the proliferation and apoptosis of HSC cells,the secretion and synthesis of collagen,and the level of pro-fibrogenic cytokines and other core links in the development of liver fibrosis diseases can be directly affected.

Fig.1 Schematic diagram of activation process of TLR4/MyD88/NF-κB,MAPKs signal pathway

3 Study on the Relationship Between Blood Stasis and Hepatic Fibrosis in Traditional Chinese Medicine

As for hepatic fibrosis,the theory of traditional Chinese medicine held that hepatic fibrosis has the characteristics of blood stasis,hypersplenism or mass in the abdomen,which belongs to the category of hypochondriac pain,jaundice,accumulation and abdominal mass[22].Peng Bo[23]proposed the treatment of stasis and advocated dark stasis first,and thought that the treatment of hepatic fibrosis should be based on dispersing blood stasis in the whole process.Liu Chenghai et al.[24]made a statistical analysis of the clinical data of 38 papers and more than 3 800 case s about the treatment of hepatic fibrosis by traditional Chinese medicine in recent years,and found that stagnation of qi and blood stasis,and Qi deficiency and blood stasis are the main TCM syndromes of hepatic fibrosis.Liver stores blood,controls conveyance and dispersion,and is Yin in substance and Yang in function;stagnation of Qi and deficiency of Qi can hinder the ascending and descending functional activities of visceral qi and blood transport and running of liver,which becomes the physiological basis for the formation of blood stasis.Blood stasis is an important pathological product in the course of chronic liver disease,after the formation of it,it becomes the cause of further aggravating the process of liver fibrosis.In modern medical research,it has been believed that there will be a series of chain reactions related to changes in vasoactive factors (TGF-β1,VEGF,etc.),ECM synthesis and secretion,and enzyme activity(uPA/tPA and MMPs,etc.),structural damage and dysfunction of HSEC,etc.,resulting in changes in liver morphology and hemorheology,which are consistent with the characteristics of"blood stasis syndrome"in traditional Chinese medicine.The results of Blood Research Institute of the Chinese Academy of Medical Sciences showed that the essence of blood stasis is the hyperplasia and degeneration of fibrous connective tissue and microcirculation disturbance[25].Because blood stasis syndrome is one of the essence of most liver fibrosis diseases,promoting blood circulation by removing blood stasis therapy has become an important means and entry point of TCM treatment and control of liver fibrosis.

4 Establishment of Animal Model of Liver Fibrosis Disease with Blood Stasis Syndrome of Integrated Traditional Chinese and Western Medicine

The causes of liver fibrosis are various and complex,almost any physical and chemical factors that can cause chronic liver damage and TCM etiology and pathogenesis can lead to liver fibrosis.In the study of anti-hepatic fibrosis of traditional Chinese and western medicine,no matter which kinds of animal liver fibrosis induced by single factor is used as the model,due to the limitations of the modeling method and the relative single pathogenicity,it is difficult to completely and accurately duplicate the complex fibrosis lesions of human liver(of course,it can’t duplicate the complicated blood stasis syndrome of traditional Chinese medicine),which limits the applicability and research value of these models.Moreover,in the traditional single factor modeling method,some of the modeling cycle is too long,some of the mortality is too high,some of the fibrosis formation is less stable[26].It is necessary to study and establish an improved animal model with combined induction of complex factors and high replication of pathological changes,so as to provide a good research platform for the research on the prevention and treatment of liver fibrosis by combining traditional Chinese and western medicine.

4.1 Hepatic fibrosis model of blood stasis type The establishment of the bloodstasis liver fibrosis model required by the research of traditional Chinese medicine for promoting blood circulation and removing blood stasis and preventing liver fibrosis should be prepared on the basis of the theory of blood stasis syndrome of traditional Chinese medicine and the theory of activating blood circulation and removing blood stasis.There are two kinds of blood stasis models in the past,one is the animal model of traditional Chinese medicine that simulates the causes of blood stasis,such as trauma,cold coagulation,qi stagnation,qi deficiency,Yin deficiency and Yang deficiency,etc.,the other is to simulate the pathological morphological changes or pathophysiological functions of blood vessels or blood in western medicine,such as vascular obstruction, microcirculation disturbance,hemodynamic disorder and so on.Peng Yue et al.[27]have established an animal model of liver fibrosis of blood stasis type,which is induced by multiple factors,such as dimethylnitrosamine(DMN)and injection of norepinephrine(NE)as well as bovine serum albumin(BSA),intragastric administration of ethanol solution,high-fat and low-protein feeding,etc.;this model has obvious advantage in accuracy,and it can reproduce the syndrome of blood stasis similar to human liver fibrosis.At the end of the modeling,the rats have obvious syndromes of blood stasis in traditional Chinese medicine,such as ecchymoses of the tongue,varicose veins under the tongue,darkening of the eyeballs,ecchymoses of the tail,weight loss,dull hair and easy to fall off;pathological examination showed pathological changes of fibrosis of western medicine,such as proliferation of liver fibrous tissue,formation of pseudolobule,expansion of hepatic sinusoid and blood stasis,etc..This showed that the liver fibrosis model of blood stasis syndrome induced by complex factors has been established,and its pathological indexes and syndrome observation results are consistent with liver fibrosis and blood stasis syndrome of traditional Chinese medicine,which provides a powerful model support for the study of the mechanism of promoting blood circulation and removing blood stasis of national drugs.

4.2 Animal model of liver fibrosis induced by carbon tetrachloride(CCl4)At present,the animal model of liver fibrosis induced by carbon tetrachloride(CCl4)is mostly used to study the mechanism of liver fibrosis,the correlation between serum markers and histopathology,and the screening of anti-fibrosis drugs.The conventional method is to induce liver fibrosis by repeated small doses of CCl4,and give CCl40.5-2.0 mL/kg twice a week,which can lead to liver fibrosis within a few weeks.However,the hepatotoxicity of CCl4is rapid and severe,and the mortality rate of animals is very high,moreover,the obtained model lacks the simulation of alcoholic liver damage.In recent years,researchers have improved the method:CCl4and ethanol are combined for modeling,and the concentrations of CCl4and ethanol are gradually increased,so that the rats can tolerate the induced drugs and reduce the mortality of the animals in the process of modeling,the success rate of the obtained liver fibrosis model is higher(the pseudolobule rate is 86.6%,far higher than 66.7% of the traditional CCl4model),and the mortality is significantly reduced(only 10% died)[28].

5 Studies on the Anti-hepatic Fibrosis and Anti-blood Stasis Syndrome of Plumbagin

Plumbago zeylanica L.is mainly distributed in tropical and subtropical areas,and is mainly distributed in Guangxi,Guangdong and Yunnan of China;it is widely distributed and rich in resources in Guangxi,and is a special medicinal plant in Guangxi.It is acrid,bitter and astringent in taste and poisonous,and has the functions of dispelling wind,removing blood stasis and detoxification[29].In Zhuang Medicine of Guangxi,it is called"Dian Bang"or"Luo Duan",its leaves are used to treat traumatic injury,ulcerative carbuncle and hepatosplenomegaly;in Yao Medicine of Guangxi,it is called"Meng Lao Hu"or"San Qian San";in Dai Medicine of Yunnan,it is called"Bi Bi Pie",its dry root can treat rheumatism pain,hyperosteogeny,pain in liver area,hepatosplenomegaly and other diseases with definite effect.Modern medical research showed[30]that Plumbago zeylanica L.has the effect of treating hepatitis,cirrhosis,bruises and sprains,and it has obvious inhibitory effect on the growth of liver cancer cells[31].The experimental study of Fuzheng Huayu Jiedu Prescription,which is mainly composed of Plumbago zeylanica L.,showed that it can improve the speed of hepatic blood flow,inhibit the activation of HSC,interfere with the formation of the basement membrane of the hepatic sinusoid,and has obvious effects of anti-fibrosis,promoting blood circulation and removing blood stasis[32].No matter from the records of folk application of various nationalities,the efficacy of single or compound clinical application,or from the results of modern scientific experimental research,the pharmacological effect of Plumbago zeylanica L.is effective in the treatment of liver fibrosis.

The results showed that the main active ingredient of anti-hepatic fibrosis drug of Plumbago zeylanica L.is Plumbagin which has the effect of promoting blood circulation and removing blood stasis.According to the determination,the content of Plumbagin in the roots of Plumbago zeylanica L.is consistent with its effect of promoting blood circulation and removing blood stasis[33]. It was reported that Plumbagin has the effects of anti-tumor,anti-inflammatory,anti-fibrosis,anti-anaphylaxis,anti-hyperglycemia and so on[34].Plumbagin is most widely distri-buted in the liver of rats and has the effect of antihepatic fibrosis,its mechanism may be related to p-Akt expression in PI3/Akt signal transduction pathway in the process of down-regulating liver fibrosis[35].In conclusion,Plumbagin is a natural antifibrotic and anti-tumor natural drug with great potential and application prospect.

In the studies of the anti-liver fibrosis and anti-blood stasis syndrome of Plumbagin,the former researchers found the following results:(i)The decoction of Plumbago zeylanica L.can significantly inhibit the acute liver damage of CCl4and lipid peroxidation of liver tissue[36];the extract of Plumbago zeylanica L.has the effect of reducing the fibroplasia in the liver tissue of experimental hepatic fibrosis mice.(ii)The serum containing Plumbago zeylanica L.can obviously inhibit the proliferation of HSC-T6 strain and promote its apoptosis,and reduce the expression and deposition area of type I and type III collagen[37].(iii)Taking hepatic stellate cell strain(HSC-LX2)as the research object,the target sites and molecular mechanism of the inhibitory effect of Plumbagin on HSC-LX2were explored from the aspects of cell proliferation,apoptosis,collagen synthesis and secretion,and the expression of fibrogenic cytokines,the results showed that the proliferation rate of HSC-LX2cells was significantly inhibited by Plumbagin,which blocked the cells from entering the proliferation cycle and promoted the apoptosis of HSC-LX2cells[38];it was also observed that Plumbagin could reduce the synthesis and secretion of α-SMA,type I and type III collagen of HSC-LX2cells,reduce the expression level of TGF-β1,VEGF and other fibrogenic factors,and at the same time increase the expression of MMP1,MMP13[39].(iv)The intervention effects of Plumbagin on the ultrastructural changes of hepatic sinusoidal endothelial cells(HSEC)and the change of secretory function of HSEC cells,and the microvascular lesions and microcirculation disorders in liver in hepatic fibrosis animal model and cell model were studied.The results showed that a variety of cytokines(such as ET-1,VEGF,CTGF,etc.)can have an impact on the structure and function of hepatic fibrosis HSEC,causing the formation of basement membrane and the loss of fenestration of HSEC,leading to hepatic microcirculation disorders.While Plumbagin can make the liver fibrosis model group disappear or a large number of reduced hepatic sinuses window reopen,and the continuous basement membrane under the endothelium disappears,which shows that the drug can obviously inhibit the phenomenon of hepatic sinuses capillary,and improve the role of liver microcirculation[40].This result is very consistent with the modern western medicine’s explanation of"promoting blood circulation and removing blood stasis",which confirms the effect of Plumbagin on promoting blood circulation and removing blood stasis,and proves that it is reasonable and feasible to further research and develop the mechanism of the effect of Plumbagin on promoting blood circulation and removing blood stasis.

However,what are the pathways and mechanisms through which the above cellular biological effects of Plumbagin can produce the effects of anti-liver fibrosis,promoting blood circulation and removing blood stasis?Many literatures indicated that TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs,which are activated by lipopolysaccharide,are based on HSC cells and have a very important influence on liver inflammatory response,proliferation and apoptosis of hepatic stellate cells and secretion level of fibrogenic cytokines.Thus it can be inferred that whether the effect of Plumbagin on inhibiting HSC cell proliferation,promoting apoptosis,reducing collagen secretion,and changing the secretion volume of pro-fibrogenic cytokines are caused by TLR4/MyD88/NF-κB and MAPKs signaling pathway?

6 Conclusions and Prospects

Based on the summary of the mechanism of liver fibrosis,the mechanism of promoting blood circulation and removing stasis,the core status of HSC,and the pharmacological research progress of Plumbagin,it has been confirmed that Plumbagin which is a national drug has the definite anti-hepatic fibrosis effects of inhibiting the proliferation of hepatic stellate cells,promoting their apoptosis,reducing the synthesis and secretion of liver collagen,promoting fibrinolysis,and reducing the secretion of fibrogenic cytokines,etc.;it has been further observed that the drug can inhibit the capillarization of hepatic sinuses and improve the microcirculation of liver,which further suggest that the drug has the effect of promoting blood circulation and removing blood stasis.Therefore,further study can be carried out and a hypothesis is put forward that the effects of Plumbagin on anti-hepatic fibrosis,promoting blood circulation and removing blood stasis(inhibiting HSC pro-liferation,promoting apoptosis,reducing collagen synthesis and secretion,changing the secretion of fibrogenic cytokines,inhibiting the phenomenon of hepatic sinus capillarization,improving liver microcirculation,etc.)are produced by affecting TLR4/MyD88/NF-κB and MAPKs signaling pathway.

The idea of further research is as follows:three groups of research models can be established:the rat model of hepatic fibrosis with blood stasis syndrome(TCM model),the modified CCl4animal model(western medicine model)and the activated HSC-LX2(cell model);TLR4/MyD88/NF-κB and TLR4/MyD88/MAPKs are selected to study,which have important effects on liver inflammation,hepatic stellate cell proliferation and apoptosis,and the secretion level of pro-fibrosis factors;the transcription level and expression level of such six most critical signaling molecules as TLR4,MyD88,NF-κB,ERK,JUK and P38-MARK,as well as the specific location of their expression in tissues and cells are observed.The research contents can be set as follows:(i)The intervention of Plumbagin on TLR4/MyD88/NF-κB and MAPKs channels of two groups of animal models will be observed;the mechanism and specific target of anti-hepatic fibrosis,promoting blood circulation and removing blood stasis of Plumbagin will be explored.(ii)The intervention of Plumbagin on TLR4/MyD88/NF-κB and MAPKs channels in HSC-LX2cell model will be observed;the research results of cell model,combined with the results of two groups of animal models,can be interpreted from the two levels of tissue and cell,which can more comprehensively and accurately explain the specific effects of the drug on TLR4/MyD88/NF-κB,MAPKs channels.These can provide more scientific basis for further development and utilization of Plumbagin,which is a special national drug in Guangxi,in the treatment of liver fibrosis.(iii)The differences and similarities of TLR4/MyD88/NF-κB and MAPKs channel activation in Chinese and western model animals without drug intervention will be observed;two groups of models with different guiding theories and different inducements of traditional Chinese medicine and western medicine can get two groups of data,which can be interpreted separately and explained comparatively,these can provide a new target site for the treatment of liver fibrosis in traditional Chinese and western medicine more comprehensively,more accurately and more pertinently,and give enlightenment.