hepatitis is caused by many factors including alcohol , viruses , drugs , immune injury , and idiopathic factors and has remained a serious human health problem throughout the world [ 1 , 2 ] . the features of hepatitis are the release of inflammatory cytokines , the elevation of aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) , and hepatocyte apoptosis and necrosis .
although many treatments are currently used in the clinic , the therapeutic effect is not ideal .
concanavalin a ( cona ) has the ability to stimulate the activation of t lymphocytes , mostly cd4 t - cells , leading to immune hepatitis in mice [ 3 , 4 ] .
natural killer ( nk ) t - cells and macrophages are also closely associated with the progression of liver injury caused by cona .
therefore , cona - induced hepatitis is an ideal model to investigate the mechanisms and treatments of t - cell - dependent hepatotoxicity .
injection of cona increases the proinflammatory cytokines il-1 , il-6 , ifn- , and tnf- [ 69 ] .
research has shown that there is a close relationship between high cytokine levels and early liver injury .
moreover , il-1 is a central cytokine associated with liver injury [ 11 , 12 ] .
several signaling pathways have been associated with the underlying mechanisms of cona - induced hepatitis .
studies have shown that cona - induced immune hepatitis was significantly attenuated with the inhibition of the phosphorylation of jnk [ 1316 ] .
apoptosis or programmed cell death is associated with liver injury caused by cona [ 15 , 17 ] .
bcl-2 family members , including bcl-2 , bcl - xl , bax , and bad , play key roles in the apoptotic pathway .
bax and bad represent proapoptotic proteins , while bcl-2 and bcl - xl represent antiapoptotic proteins .
autophagy , first described by ashford and porter , is characterized by the formation of autophagosomes and autolysosomes and is an intracellular degradation system that focuses on defective organelles .
autophagy plays important roles in various biological processes , including innate immunity , inflammatory responses , and adaptive immunity . however , autophagy is also called type ii programmed cell death and is intimately associated with eukaryotic cell death and apoptosis . therefore , we consider that autophagy is a double - edged sword .
microtubule - associated protein 1 light chain 3 ( lc3 ) and beclin-1 are widely considered as markers of autophagy .
shikonin , a natural product extracted from lithospermum erythrorhizon , has many biological functions , including antibacterial , antioxidant , anti - inflammatory , and antitumor activities .
recently , shikonin was shown to play an important role in regulating the process of inflammation , exerting strong anti - inflammatory effects .
lee and colleagues found that shikonin could effectively inhibit allergic airway inflammation in a model of asthma and suppress bone marrow - derived dendritic cell ( bm - dc ) maturation in vitro .
shikonin exerted anti - inflammatory effects by interfering with the degradation of i-b and then suppressed the activation of nf-b , as described by andjar and colleagues .
xiong et al . also found that shikonin could reduce the release of proinflammatory cytokines in cerulein - induced acute pancreatitis in mice .
however , the mechanism of action of shikonin in a model of cona - induced autoimmune hepatitis remains unclear .
the present study investigated the underlying mechanism of action of shikonin in cona - induced autoimmune hepatitis .
we hypothesized that shikonin could reduce the level of il-1 upregulated in cona - induced hepatitis and ameliorate liver injury , as measured by serum hepatic enzymes , proinflammatory cytokines , and histological changes , which may be partly associated with the c - jun n - terminal kinase ( jnk)/p - jnk pathway .
shikonin , dimethyl sulfoxide ( dmso ) , and cona were purchased from sigma - aldrich ( st . louis , mo , usa ) .
antibodies used in the study were from cell signaling technology ( danvers , ma , usa ) , including il-1 , tnf- , ifn- , lc3 , beclin-1 , caspase 9 , bax , bcl-2 , total jnk , p - jnk , and p62 .
the pcr kit was purchased from takara ( takara biotechnology , dalian , china ) .
the microplate test kits for ast and ast were purchased from nanjing jiancheng bioengineering institute ( jiancheng biotech , china ) .
male balb / c mice ( 68 weeks old , 23  2  g ) were purchased from shanghai laboratory animal co. , ltd .
the mice were housed in a clean environment at 24  2c and an alternating 12  h light and dark cycle .
the study was approved by the animal care and use committee of shanghai tongji university .
a total of 48 mice were randomly divided into four groups as follows : a normal control group treated with saline solution , a dmso group treated with 2% dmso , and two shikonin groups treated with shikonin at doses of 7.5  mg / kg and 12.5  mg / kg . shikonin was dissolved in 2% dmso .
the serum and liver tissues were gathered and used to analyze data immediately , including liver enzymes , the levels of cytokines , and pathological changes .
cona was dissolved in normal saline solution at a concentration of 2.5  mg / ml and injected at 20  mg / kg via tail vein to induce acute hepatitis according to previous study .
shikonin was diluted with 2% dmso and injected intraperitoneally 2  h prior to cona challenge .
a total of 96 mice were treated by tail intravenous injection of cona 2  h before administrating shikonin .
the mice were randomly divided into four groups:(1)normal control ( n = 24 ) : mice were injected with saline solution only.(2)cona group ( n = 24 ) : mice were injected with 20  mg / kg cona via the tail vein.(3)low dose group ( n = 24 ) : mice were intraperitoneally injected with 7.5  mg / kg shikonin 2  h before cona challenge.(4)high dose group ( n = 24 ) : mice were intraperitoneally injected with 12.5  mg / kg shikonin before cona challenge . normal control ( n = 24 )
: mice were injected with 20  mg / kg cona via the tail vein .
low dose group ( n = 24 ) : mice were intraperitoneally injected with 7.5  mg / kg shikonin 2  h before cona challenge .
high dose group ( n = 24 ) : mice were intraperitoneally injected with 12.5  mg / kg shikonin before cona challenge .
based on a previous study , blood was collected at three time points 3 , 6 , and 24  h rapidly after the mice were sacrificed .
after blood collection , the serum was separated by centrifugation at 2000  rpm at 4c for 10  min and used to detect liver function and cytokine levels .
the levels of alt and ast were measured with an automated chemical analyzer ( olympus au1000 , japan ) .
il-1 , tnf- , and ifn- were measured by enzyme - linked immunosorbent assay ( elisa ) kits ( r&d systems , usa ) according to the manufacturer 's protocols .
the middle portion of the left liver lobe was cut and fixed in 4% paraformaldehyde for at least 24  h. after fixation , the specimen was embedded in paraffin ; sections were cut at a thickness of 5  m and stained with hematoxylin and eosin ( h&e ) . the inflammatory level and tissue damage
prepared paraffin - embedded liver sections ( 5  m ) were heated at 60c for 1  h and then dewaxed and rehydrated by using xylene and different concentrations of alcohol . to recover the antigens ,
the paraffin - embedded sections were treated with an antigen - retrieval technique , including heating in a water bath at 95c for 10  min , and then covered in hydrogen peroxide solution ( 3% ) for 20  min at 37c to block the activity of endogenous peroxidase .
the nonspecific binding sites were blocked with 5% bovine serum albumin ( bsa ) at 37c for 20  min and then incubated for 10  min at room temperature .
the liver specimens were incubated overnight with il-1 ( 1  :  100 ) , tnf- ( 1  :  100 ) , ifn- ( 1  :  100 ) , bax ( 1  :  100 ) , bcl-2 ( 1  :  100 ) , p - jnk ( 1  :  100 ) , and lc3i / ii ( 1  :  500 ) . the next day , the liver sections were incubated with a secondary antibody , and a diaminobenzidine kit was used to analyze antibody binding . finally , the slices were observed under a light microscope .
the ratios of brown staining areas and total areas were calculated using image - pro plus software 6.0 .
after recovery from 80c storage , liver tissues were rapidly ground in liquid nitrogen and then lysed with ripa lysis buffer supplemented with protease inhibitors ( pi ) and phenylmethanesulfonyl fluoride ( pmsf ) .
the protein concentration was detected with the bicinchoninic acid ( bca ) protein assay ( kaiji , china ) .
equivalent amounts of total protein ( 120  g ) were boiled and mixed with 5  sds - page sample loading buffer .
the proteins were separated by using different concentrations of sodium dodecyl sulfate ( sds ) polyacrylamide gels and then transferred to polyvinylidene difluoride ( pvdf ) membranes .
nonspecific binding was blocked with 5% nonfat milk ( diluted in pbs ) for 1  h and incubated overnight at 4c with primary antibodies : -actin ( 1  :  1000 ) , il-1 ( 1  :  200 ) , tnf- ( 1  :  200 ) , ifn- ( 1  :  200 ) , bcl-2 ( 1  :  500 ) , bax ( 1  :  500 ) , caspase 9 ( 1  :  500 ) , beclin-1 ( 1  :  500 ) , lc3 ( 1  :  1000 ) , p62 ( 1  :  500 ) , total jnk ( 1  :  1000 ) , and p - jnk ( 1  :  500 ) .
membranes were washed with pbst three times for 10  min and then incubated with a secondary goat anti - rabbit or anti - mouse antibody ( 1  :  2000 ) for 1  h at 37c . finally , the membranes were washed with pbst three times for 10  min and then scanned using the odyssey two - color infrared laser imaging system .
total rna was extracted from frozen liver tissues and transcribed into cdna using the reverse transcription kit ( takara biotechnology , china ) , according to the manufacturer 's protocols .
sybr green quantitative rt - pcr was performed to detect target gene expression using a 7900ht fast real - time pcr system ( applied biosystems , ca , usa ) , according to the protocols for sybr premix ex taq ( takara biotechnology ( dalian ) co. , ltd . ,
the fresh liver tissues were fixed in 4% paraformaldehyde for 1  h. then the fixed liver tissues were washed three times by pbs for 15  min before they were dehydrated overnight in 30% sucrose at 4c .
then the sections were infiltrated with oct ( sakura , usa ) for 2  h and stored at 80c .
5  m liver sections were cut by a freezing microtome . the prepared sections ( 5  m ) dried at room temperature for 10  min ; then the sections were washed three times by pbs for 5  min each time .
and then the prepared sections were incubated with ros fluorescent probe - dhe ( 60  m , diluted by pbs ) for 90  min .
after incubation , the sections were washed three times by pbs for 5  min each time .
one - way analysis of variance ( anova ) was used to analyze the datum of q - pcr , elisa , ros scavenging activity , the levels of ast and alt , the gray value of western blotting , and the areas of inflammation and necrosis in immunohistochemistry . in all comparisons , p values
to determine whether the drug and the solvent affected liver function , we examined the effects of shikonin and 2% dmso on liver enzymes and the release of cytokines .
as shown in figure 1(a ) , serum ast and alt levels did not differ between the 2% dmso and shikonin groups and the control group .
the serum il-1 , tnf- , and ifn- levels were comparable between the four groups .  
cona can activate t - cells to induce autoimmune hepatitis . to detect the effect of shikonin on cona - induced hepatitis
, mice were pretreated with shikonin 2  h before cona injection . based on the experimental design ,
the serum and liver tissues of mice were collected at three time points : 3 , 6 , and 24  h.   figure 2(a ) shows that the serum ast and alt levels were significantly increased at the three time points , with a peak at 6  h after cona injection . however , pretreatment with shikonin clearly reduced the serum levels of liver enzymes and the high dose was more effective .
as shown in figure 2(b ) , abundant necrotic areas were observed in the cona group , while the shikonin groups showed minor liver injury at the three time points , indicating that pretreatment with shikonin dramatically decreased liver necrosis .
image - pro plus software showed statistically significant differences between the groups . taken together , these results indicated that pretreatment with shikonin effectively reduced cona - induced liver injury in mice .
the development of autoimmune hepatitis is closely associated with the release of proinflammatory cytokines , including il-1 , tnf- , and ifn-. the levels of il-1 , tnf- , and ifn- were determined by elisa and found to be significantly increased in the cona group , showing a peak at 6  h after cona induction ( figure 3(a ) ) .
shikonin pretreatment dramatically reduced the levels of these proinflammatory cytokines , particularly at 6  h. to confirm our results , we used real - time pcr to detect the mrna expression of il-1 , tnf- , and ifn- at each time point .
the mrna expression of these cytokines was decreased by shikonin pretreatment compared with the cona group , especially in the high dose group ( figure 3(b ) ) .
the protein expression of il-1 , tnf- , and ifn- was significantly increased in the cona group at all the three time points and reached peak at 6  h ( figure 3(c ) ) , consistent with the mrna expression .
however , the protein expression of these cytokines decreased in both shikonin pretreatment groups , and shikonin administered at 12.5  mg / kg was more effective , indicating that the effects of shikonin on cona - induced hepatitis were dose - dependent .
immunohistochemical staining was used to determine the expression of the inflammatory cytokines in the normal control , cona , and cona + shikonin groups ( figure 3(d ) ) .
these results provided strong evidence that pretreatment with shikonin could decrease the release of inflammatory cytokines such as il-1 , tnf- , and ifn- in autoimmune hepatitis caused by cona .
we investigated the expression of bcl-2 , bax , caspase 9 , lc3 , beclin-1 , and p62 .
we used real - time pcr and western blot technologies to detect the expression of apoptosis and autophagy markers at the mrna and protein levels , as shown in figures 4(a ) and 4(b ) .
bax and caspase 9 , the proapoptotic proteins , were significantly upregulated in the cona group and downregulated in the shikonin pretreatment groups at all the three time points .
bcl-2 , an antiapoptotic marker , was downregulated in the cona group and upregulated in the shikonin treatment groups .
the expression of lc3 , beclin-1 , and p62 was inhibited with increasing drug doses , with the highest level in the cona group .  
figure 4(c ) shows that the results of immunohistochemistry were consistent with those of western blotting . in conclusion
, these results provided strong evidence that shikonin can attenuate hepatocyte apoptosis and autophagy and protect the liver tissue from pathological damage in cona - induced autoimmune hepatitis .
we showed that shikonin can attenuate autoimmune hepatitis caused by cona through the inhibition of the release of proinflammatory cytokines , such as il-1 , tnf- , and ifn-. however , the underlying mechanism remains unclear .
evidence indicates that the jnk / p - jnk pathway is closely associated with inflammatory responses and the progression of apoptosis and autophagy .
therefore , we examined whether shikonin could protect liver tissues by suppressing the jnk / p - jnk pathway in cona - induced hepatitis .
western blot analysis was used to determine the protein levels of total jnk and the phosphorylation of jnk .
however , the level of phosphorylated jnk was significantly increased in the cona - treated group and clearly decreased in the shikonin - pretreated groups at all the three time points ( figure 5(a ) ) .
immunohistochemical staining was used to detect the expression of p - jnk , as shown in figure 5(b ) .
the results of western blotting and immunohistochemical staining suggested that shikonin treatment can ameliorate liver injury in cona - induced hepatitis in part through the jnk / p - jnk pathway .
previous studies have shown that il-1 plays a crucial role in cona - induced hepatitis and was suggested to play a role in the activation of jnk .  
figure 5 shows that the protein level of il-1 was consistent with the changes of p - jnk .
autoimmune hepatitis is an inflammatory disease and its incidence has been increasing , which has resulted in an important global burden and poses a serious risk to human lives .
however , there are no effective treatment measures , underscoring the need to identify effective therapy options .
shikonin , a promising anti - inflammatory drug , has attracted the attention of scientists worldwide .
several studies show that the release of inflammatory cytokines is involved in autoimmune hepatitis , including il-1 , tnf- , ifn- , and il-6 , giving rise to the development of liver injury [ 7 , 26 ] .
il-1 , which is secreted by activated macrophages , plays a significant role in the necrosis of liver tissues .
several studies have shown that shikonin exerts anti - inflammatory effects by downregulating the expression of the inflammatory cytokine il-1 in animal models [ 24 , 28 ] . however , the mechanism of shikonin in concanavalin a - induced autoimmune hepatitis remains largely uncertain and requires further investigation . here , we established a model of cona - induced acute hepatitis to investigate the mechanism underlying the anti - inflammatory effect of shikonin .
our results showed that shikonin pretreatment has a protective effect on cona - induced liver injury , as shown by serum liver enzymes levels , the release of inflammatory cytokines , and pathological changes .
the levels of serum ast and alt and the range of necrosis of liver tissues on biopsy were markedly reduced by both doses of shikonin ( 7.5  mg / kg and 12.5  mg / kg ) , particularly in the high dose group .
our results showed that pretreatment with shikonin suppressed the release of the inflammatory cytokines tnf- and ifn- and most significantly il-1. therefore , we examined shikonin could prevent liver tissue from injury caused by cona by inhibiting the expression of il-1 and the phosphorylation of jnk [ 29 , 30 ] .
jnk , one of the members of the family of mitogen - activated protein kinases ( mapks ) , is activated by many types of factors , such as immune responses , cell stress , and especially the inflammatory cytokine il-1 [ 31 , 32 ] .
the active form of jnk is p - jnk , which is closely associated with apoptosis induced by il-1 .
reported that the apoptosis of pancreatic beta - cells induced by il-1 occurs mainly via the activation of the jnk pathway .
revealed that the cell - permeable peptide inhibitor of jnk ( jnki1 ) could effectively protect beta - cells from death induced by il-1 .
kim et al . found that the jnk pathway is closely associated with il-1 production in alzheimer 's disease .
cona administration induced the phosphorylation of jnk to generate p - jnk , which was translocated from the cytoplasm to the mitochondrial membrane or cell nucleus causing liver injury .
found that ellagic acid ( ea ) pretreatment significantly attenuated liver injury in cona - induced hepatitis through the phosphorylation of jnk .
a recent study showed that astaxanthin protects liver tissues in a model of cona - induced immune hepatitis via the jnk / p - jnk pathway .
we examined whether shikonin protected liver tissues in cona - induced hepatitis via the inhibition of the jnk / p - jnk signaling pathway .
our results showed that cona enhanced the expression of p - jnk , and this was significantly decreased by pretreatment with shikonin , indicating that shikonin attenuated liver injury caused by cona by reducing the phosphorylation of jnk . to investigate
how shikonin reduced liver injury by modulating the phosphorylation of jnk , we detected the expression of bcl-2 , bax , and caspase 9 .
bcl-2 and bax belong to the bcl-2 family , which is an antiapoptotic protein , while bax represents a proapoptotic protein , and the balance of bcl-2 and bax determines cell survival or cell apoptosis   ( figure 6 ) . the phosphorylation of jnk results in the inactivation and phosphorylation of bcl-2 , leading to the release of cytochrome c and the activation of caspase - mediated apoptosis .
our results showed that pretreatment with shikonin could increase the expression of bcl-2 and reduce the expression of bax and caspase 9 through the effect of p - jnk .
these results indicated that shikonin could attenuate hepatic cell apoptosis in cona - induced hepatitis through the jnk / p - jnk pathway .
recent studies showed that bcl-2 is a central regulator of autophagy and apoptosis and functions by interacting with beclin-1 .
when bcl-2 is inactivated by the effect of p - jnk , it causes the displacement of bcl-2 from beclin-1 , thereby triggering autophagy .
the induction of autophagy results in the formation of autophagosomes , which can generate several markers of autophagy , including lc3 , beclin-1 , and p62 .
the results of pcr and western blotting demonstrated that shikonin pretreatment decreased the expression of lc3 and beclin-1 and increased the expression of p62 compared with the cona - treated group .
cona - induced hepatitis involves many complex and multifactorial mechanisms and these mechanisms need to be investigated further
. moreover , additional studies are needed to examine the protective effects of shikonin on liver injury .
 
 objective . shikonin possesses anti - inflammatory effects . however , its function in concanavalin a - induced acute liver injury remains uncertain . 
 the aim of the present study was to investigate the functions of shikonin and its mechanism of protection on cona - induced acute liver injury . 
 materials and methods . 
 balb / c mice were exposed to cona ( 20  mg / kg ) via tail vein injection to establish acute liver injury ; shikonin ( 7.5  mg / kg and 12.5  mg / kg ) was intraperitoneally administered 2  h before the cona injection . 
 the serum liver enzyme levels and the inflammatory cytokine levels were determined at 3 , 6 , and 24  h after cona injection . results . 
 after the injection of cona , inflammatory cytokines il-1 , tnf- , and ifn- were significantly increased . 
 shikonin significantly ameliorated liver injury and histopathological changes and suppressed the release of inflammatory cytokines . 
 the expressions of bcl-2 and bax were markedly affected by shikonin pretreatment . 
 lc3 , beclin-1 , and p - jnk expression levels were decreased in the shikonin - pretreated groups compared with the cona - treated groups . 
 shikonin attenuated cona - induced liver injury by reducing apoptosis and autophagy through the inhibition of the jnk pathway . conclusion . 
 our results indicated that shikonin pretreatment attenuates cona - induced acute liver injury by inhibiting apoptosis and autophagy through the suppression of the jnk pathway . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion