nonalcoholic fatty liver disease ( nafld ) refers to the accumulation of hepatic steatosis not due to excess alcohol consumption . it is estimated that approximately 20%40% of the people in various countries have excess fat accumulation in the liver . nafld encompasses a spectrum of disorders ranging from hepatic steatosis to nonalcoholic steatohepatitis ( nash ) , cirrhosis , or hepatocellular carcinoma . despite its severity and high prevalence many factors including lipid metabolic disturbance , insulin resistance ( ir ) , oxidative stress , and mitochondrial dysfunction are known to cause the occurrence and aggregation of nafld . according to the prevailing theory of nafld pathogenesis , ir and lipid dysregulation are the  first hit  toward nafld , which may trigger following  hits  spontaneously such as oxidative stress , inflammation , necrosis , apoptosis , and fibrosis . therefore , targeting steatosis and ir in the early stage of nafld is extremely important for the retardation of nafld in both basic research and clinical trials . although the initial accumulation of fat in the liver can occur through several mechanisms , most previously done research implicates ir as the key mechanism in primary nafld , leading to hepatic steatosis . ir is associated with an increase in peripheral lipolysis , uptake of fatty acids by the liver , and synthesis of triglycerides , which promote the accumulation of hepatic fat . nowadays , the methods for treating nafld involve rational diet , exercise , and medicines including metformin , statins , and fibrates . however , these drugs have some adverse effects or contraindications , and no consensus exists on the most effective drug therapies . recently , emerging evidence suggests the possible use of herbal derivatives in treating nafld , such as green tea , garlic , resveratrol , and coffee   in both preclinical studies and clinical trials . the therapeutic hepatoprotective activity of ethanolic extracts of edible lotus leaves was studied , indicating that the hepatoprotective activity of lotus leaf extract was comparable with that of a standard treatment comprising silymarin , a known hepatoprotective drug . np is the germ of the lotus seeds , which tastes bitter and generally can be removed . a previous research demonstrated the protective effect of total alkaloid and alkaloid compounds including liensinine , isoliensinine , or neferine from np against oxidative stress induced by tertiary - butyl hydroperoxide , suggesting their involvement in the cytoprotective effect on oxidative stress in liver . furthermore , total alkaloid and alkaloid compounds from np displayed a significant cytoprotective effect against hepatic steatosis ( data not reported ) . therefore , the aim of the present study was to investigate the effects of npa on ameliorating ir in high - fat diet- ( hfd- ) induced nonalcoholic fatty liver disease in mice . and the possible mechanisms were also studied . hfd , composed of 5% lactalbumin , 10% cholesterol , 20% lard oil , and 1% yolk powder , was obtained from research diets , inc . serum blood glucose , free fatty acid , alanine amino transferase ( alt ) and aspartate aminotransferase ( ast ) detection kits , and bicinchoninic acid protein assay kit were obtained from nanjing jiancheng institute of biotechnology ( nanjing , china ) . phosphorylated insulin receptor substrate 1 ( irs-1 ) and c - jun n - terminal kinase ( jnk ) antibody were purchased from sigma chemicals co. the insulin enzyme immunoassay kit was purchased from puer biotechnology co. , ltd . enzyme - linked immunosorbent assay ( elisa ) kits for tumor necrosis factor alpha ( tnf- ) and interleukin-6 ( il-6 ) were supplied by glory science co. ltd . then , the extraction was centrifuged at 1500  g for 15  min , and the supernatant was collected . next , 5% hcl solution was used to immerse the extract , followed by the addition of naoh to regulate ph to 9 - 10 . the crude sample was then extracted with chloroform using liquid - liquid separation . the collected soluble fraction from chloroform the alkaloid composition of npa was also reported in previous studies [ 13 , 14 ] , which indicated that 3 alkaloid components of liensinine , isoliensinine , and neferine make up the majority in npa . male wistar rats ( 180200  g ) were provided by the experimental animal center of fujian university of traditional chinese medicine ( fuzhou , china ) . the animals were housed in a room with a controlled temperature ( 21  3c ) , relative humidity ( 65%70% ) , and 12 h light / dark cycles . after adaptation for 1 week , the rats were used for the experiment approved by the ethical committee and the laboratory animal center of fujian university of traditional chinese medicine . group i ( normal control ) was given distilled water ( 10  ml / kg body weight ) and normal feeding . groups ii  vi were administered with hfd feeding for 8 weeks to induce nafld in the animal . among them , group ii was designed as the model control group . groups iii  v were administered with npa ( 200 , 400 , and 600  mg / kg , resp . at the end of the experiment , blood samples of all rats were obtained via the abdominal vein under anesthesia after an overnight fasting . liver sample from each rat was collected for molecular measurement and stored at 80c for the assays . serum was obtained from blood after centrifugation ( 2500  g , 10  min ) . the serum blood glucose , free fatty acid , alt , and ast levels of rats were detected using corresponding detection kits according to the manufacturer 's protocols . fasting serum insulin was determined using elisa kit for insulin following the manufacturer 's protocol . using an elisa reader , the homeostasis model assessment of ir ( homa - ir ) was calculated as [ fasting blood glucose ( mmol / l )  fasting serum insulin ( mu / l)/22.5 ] . an elisa kit for tnf- or interleukin-6 ( il-6 ) was employed for estimating the serum levels of these parameters . western blotting assay for detecting phosphorylation of jnk or irs-1 in liver tissue was determined according to the method described by li et al .   with some modification . then , it was denatured , resolved with sodium sulfate poly acrylamide gel electrophoresis , and transferred onto a polyvinylidene difluoride membrane ( pvdf ) . after blocking , the membranes were individually incubated overnight with mouse anti - jnk ( 1  :  700 dilution ) , mouse anti - p - jnk ( 1  :  700 dilution ) , mouse anti - irs-1 ( 1  :  700 dilution ) , or mouse anti - p - irs-1 ( 1  :  700 dilution ) , respectively , followed by washing and incubation with secondary antibodies to show the resulting bands . the data were analyzed using one - way analysis of variance coupled with duncan post hoc multiple range test at 95% confidence level using spss ( version 16.0 , chicago , usa ) . before the start of the experiment , the average body weight of each group was similar . as showed in table 1 , after feeding with hfd for 8 weeks , the body weights of rats in the nafld group significantly increased compared with the control group , indicating that hfd had a significant influence on body weight . however , after oral administration of npa ( 400 and 600  mg / kg ) for 4 weeks , the body weight of rats significantly reduced compared with the nafld group . therefore , the consumption of npa could significantly reduce the body weight of nafld rats . to correlate hepatic injury with nafld , serum alt and ast measurements were conducted in each group of rats . as shown in figure 1 , the progression of nafld significantly induced the higher levels of both serum alt and ast compared with the control rats . however , the levels of alt and ast significantly decreased in a dose - dependent manner after npa treatment , and the hepatoprotective effects produced by npa at the dose of 600  mg / kg reach the highest as well as the positive control group . therefore , npa treatment potently alleviated hepatic injury by reducing the levels of aminotransferases . as noted , ir and glucose metabolism dysfunction are typical clinical symptoms of nafld and metabolic syndrome . eight weeks of daily ingestion of hcd contributed to the insulin resistance of the model group , which exhibited a significant increase in fasting blood glucose ( fbg ) compared with the control group . however , after npa exposure , the concentrations of fbg in blood significantly decreased in a dose - dependent manner compared with the model group . the fasting serum insulin level increased significantly in association with the elevated fbg in the model group ; however , npa intake at higher dose significantly decreased the fasting insulin level in groups iv - v compared with the nafld group . a high - fat , cholesterol - rich diet induced a significant increase of ir in the nafld model group animals compared with the control group , as indicated by the high homa - ir . npa administration in groups iv - v could decrease their homa - ir level compared with the model group , reaching the control range ( table 2 ) . our results showed that the nafld model rats could exhibit a much higher level of free fatty acid ( fft ) compared with the control group ( figure 2 ) . however , treating the nafld model with npa at a concentration of 400 or 600  mg / kg could significantly inhibit the fft level in serum , suggesting its significant effect on alleviating steatosis in rats . rats fed with hfd for 8 weeks showed a significant increase in serum tnf- and il-6 levels compared with the rats in the control group . treatment with npa ( 400 or 600  mg / kg ) significantly decreased these high levels compared with the control group . notably , npa at a higher dose could decrease the serum il-6 level to a much greater extent , which was comparable with the positive control group ( figures 3(a ) and 3(b ) ) . the results showed that jnk phosphorylation significantly increased in the nafld rats compared with the normal group . thus , increased jnk phosphorylation might explain the increased ir ( figure 4(a ) ) . it is well established that the insulin signaling pathway is impaired in obese objects as a result of decreased irs phosphorylation . in the present nafld model , western blot results showed that hepatic tissue levels of phosphorylated irs-1 were inhibited by the induction of nafld . however , cotreatment with npa completely restored the levels of these proteins ( figure 4(c ) ) , indicating the basis of improving ir in nafld . the aim of the present study was to evaluate the hepatoprotective effect of npa by ameliorating ir in a model of hfd - induced nafld . hfd is well known to increase body weight and induce ir , in addition to an inflammatory and oxidative burden in rodent models . in the present study , high - fat , the aforementioned findings confirmed that 8 weeks of hfd feeding in rats resulted in increments in body weight , adiposity index , systemic ir , and inflammatory markers . hfd feeding could also increase serum ffa , causing the overloads of ffa in hepatocytes and promoting lipogenesis . nafld is defined histologically as the presence of lipid droplets in more than 5% of hepatocytes , not caused by excessive alcohol consumption , drugs , or viruses . ir represents the main link among obesity , metabolic syndrome , and liver disease with fat accumulation , that is , nafld . the present research investigated the effect of npa on the metabolic alterations in the glucose level and ir in hfd - induced nafld . feeding the rats with hfd resulted in dramatic increases in the fbg , ffa , and ir . it is well known that infusions of triglyceride emulsions to rapidly increase ffa levels would cause a decrease in insulin sensitivity . therefore , controlling ir , as well as associated glucose metabolism and lipid accumulation , is the first but most important step toward the therapy of nafld . the adipocytes are no longer considered passive cells that store excessive triglycerides but are instead considered active cells that regulate the energy balance and secrete the proinflammatory cytokines il-6 and tnf- . tnf- is an important mediator of ir due to its ability to influence the tyrosine kinase activity of the insulin receptor . in the present study , this suggested that multiple immunomodulatory factors could contribute to the chronic inflammatory condition and hepatocytes injury observed in nafld . it is assumed that inflammatory factors may be mediators of ir because the plasma levels of both markers are correlated negatively with insulin sensitivity . excessive fat accumulation in hepatocytes , regardless of its cause , tends to induce the activation of nuclear factor-b , which is the key regulator of inflammation . subsequently , the expression of inflammatory cytokines tnf- and il-6 may lead to infiltration of neutrophils and inflammation in liver injury . impaired irs-1 phosphorylation is responsible for reduced insulin signaling and impaired downstream pi3k / akt signal transduction . it is well established that the insulin signaling pathway is impaired in obese patients as a result of decreased irs phosphorylation . some authors suggest that lipolysis induced by tnf- and il-6 , resulting in the inhibition of irs , may represent a major mechanism . thus , it could be considered that the restoration of irs-1 might be beneficial for the amelioration of nafld . the present results showed that insulin hypersecretion was prevalent in the group of nafld model rats , which significantly decreased on administering npa . considering the survival - promoting role of irs-1 in several hepatic diseases , particularly nafld , the restoration of this pathway would definitely contribute to the improvement in ir . lipid peroxidation products can activate transcription factor nf - kb in patients with nafld , a key transcription factor that regulates the expression of several proinflammatory cytokines and hepatic inflammation . in fact , it is now generally established that jnk1 is central to obesity - induced ir , although jnk2 might also play a contributing role . the present results suggested that jnk phosphorylation might be responsible for aggravating ir , as nafld group advanced to more severe form . hence , inhibition of jnk phosphorylation by npa adds to its modulation effect on ir during napld progression . in conclusion , the present results supported the view that the pathogenesis of nafld was complex with inducing inflammation and ir , as well as increasing serum glucose and free fatty acid level . moreover , cotreatment with npa could regulate ir through restoring phosphorylation of irs-1 and alleviating phosphorylation of jnk in liver tissue , suggesting its involvement in modulating ir by protein phosphorylation during nafld progression . further investigations are needed to ensure the safety , benefits , and utility of npa in patients with nafld .
this study aimed to investigate the effects of total alkaloids from nelumbinis plumula ( npa ) on insulin resistance ( ir ) of high - fat diet- ( hfd- ) induced nonalcoholic fatty liver disease ( nafld ) . 
 rats were fed with hfd for 8 weeks to induce nafld . 
 then , the effect of npa on ameliorating ir in hfd - induced nafld was evaluated . 
 fasting serum insulin was determined using an enzyme - linked immunosorbent assay ( elisa ) kit for insulin following the manufacturer 's protocol . 
 some inflammatory cytokines such as tumor necrosis factor alpha ( tnf- ) and interleukin-6 ( il-6 ) were determined using elisa kits to assess the inflammatory burden in rats . 
 the results showed that hfd could induce a significant increase in blood glucose and ir in rats . 
 however , rats treated with npa ( 400 or 600  mg / kg ) showed improved ir and reduction in serum inflammatory cytokines tnf- and il-6 . 
 further investigation indicated that npa could inhibit ir by restoring the insulin receptor substrate-1 ( irs-1 ) and suppressing the expression of c - jun n - terminal kinase ( jnk ) phosphorylation . 
 the present results supported the view that the pathogenesis of nafld was complex with inflammation , together with increasing serum glucose and ir . also , jnk and irs phosphorylation were suggested for their involvement in the modulating of ir during nafld progression . 
 therefore , npa may serve as a potential natural remedy against ir in nafld .