Serum HGF ,PCIII and PLT are Noninvasive Markers for the diagnosis of nonalcoholic fatty liver disease

Background/Aims: It was suggested that serum HGF,PCIII and PLT play important roles in nonalcoholic fatty liver disease (NAFLD). Thus, we aimed to evaluate their clinical utility in the diagnosis of patients with suspected NAFLD. Methods:300 Patients with NAFLD were compared to 102 matched controls.All were subjected to history taking, anthropometric measurements, and abdominal ultrasonography, as well as laboratory assessments of liver functions, fasting lipid profile, GLU, serum PLT, HGF and PCIII. Results:The levels of HGF,PCIII and PLT were higher in NAFLD cases than controls ,and with progressive increases as the severity of fatty liver increasedP<0.05.HGF,PCIII and PLT were correlated with various clinical parameters and severity of NAFLDP<0.05.The optimal cut-off values for HGF in diagnosis of mild, moderate and severe fatty liver were 14.1pg/ml(AUROC 0.753,P0.004), 15.4pg/ml(AUROC 0.836, P < 0.001), 17.7pg/ml(AUROC 0.903, P < 0.001). PCIII had no value in differentiate mild from moderate fatty liver ,but its ability to diagnose severe fatty liver was significant. A cut-off value for PCIII to diagnose severe fatty liver was 7.9ng/L(AUROC 0.773).The optimal cut-off values for PLT in the diagnosis of mild, moderate and severe fatty liver were 194×10^9/L(AUROC 0.732), 195×10^9/L(AUROC 0.765), 200×10^9/L(AUROC 0.925), respectively with P < 0.001. When three indicators were tested together,the AUROC(95%CI)curve for diagnose NAFLD was 0.881(sensitivity0.760,specificity 0.873)(P<0.001) . Conclusion:.Combined detection of serum HGF, PCIII and PLT may be an effective non-invasive method for diagnosing NAFLD. group from mild NAFLD group,moderate NAFLD group and normal group, but It showed no significant difference in mild NAFLD group and moderate NAFLD group.Even more,PLT can distinguish normal group from all NAFLD group, but It showed no significant difference in mild NAFLD group and moderate NAFLD group,so as moderate NAFLD group and severe group.

SUBJECT AREAS 6% to 37%, with a reported pooled overall global prevalence of 25% [2],As the global epidemic of NAFLD, the clinical and economic burden of NAFLD will become enormous.Although Liver biopsy is the gold standard for the diagnosis, grading, and staging in patients with NAFLD, but it has its own limitations, such as the risk of sampling error, high rates of inter-and intraobserver differences, risk of complications, and the high cost and so on [3]. Thus, it is important to develop efficient noninvasive diagnostic methods for NAFLD and numerous noninvasive diagnostic method have been developed worldwide.
Hepatocyte growth factor (HGF) is a multifunctional pleiotropic cytokine,is not only an anti-apoptotic and anti-fibrotic factor in the liver, but also a adipokine [4].HGF is initially produced as a biologically inactive, single-chain precursor form,After proteolytic cleavage,it converts to the active form to The development of hepatic fibrosis is an important event in the progression of liver disease. Serum type III procollagen peptide (PIIIP), a degradation product of the type III collagen precursor and has been shown to be a negative prognostic factor in chronic hepatitis [8].Serum PIIINP levels increased significantly along with the increasing severity of liver inflammation[9].Procollagen-III peptide also could identify adipose tissue(AT)-associated inflammation in type 2 diabetes with or without nonalcoholic liver disease,Higher PIIINP levels correlated with greater BMI and visceral AT area and were associated with systemic signatures of AT-associated inflammation[10].
The platelet is the primary cell for hemostasis and tissue repair.Many studies have shown that PLT plays a role in the inflammatory response after hepatic damage [11,12].Recent clinical and experimental evidences also support that platelets play several roles in the progression of malignancies and inversely, cancer can also influence platelet count and activity [13].Sangbin Han et al. 's findings suggested that platelets play an important role in HCC cells metastasis and preoperative platelet count was independently correlated with post-transplant HCC recurrence [14].
Thus, the current study attempted to evaluate the clinical utility of serum HGF,PIIIP and PLT levels in the diagnosis of suspected NAFLD.

Patients and methods
A total of 300 patients diagnosed with NAFLD and 102 sex-and age-matched healthy volunteers without NAFLD were included in this study from Novemble 2016 to Novemble 2018. All patients were diagnosed with NAFLD based on ultrasound imaging made under fasting conditions.The grade of fatty liver was categorized as follows: (1) mild steatosis: increased echogenicity of the liver as compared with the renal cortex or spleen; (2) moderate steatosis: obscured hepatic and portal vein walls, and (3) severe steatosis: impaired visibility of the diaphragm [15].Subjects with a history of excessive alcohol consumption (weekly consumption of ≥140 g for men or ≥70 g for women), other liver diseases such as chronic hepatitis, or use of drugs known to be associated with the development of fatty liver, body weight reduction or thyroid disorders were excluded [16].
Physical examination of each patient included body weight, height,calculation of body mass index (BMI) .Blood samples were collected after 12 hours of fasting,the serum was obtained after centrifugation at 3000 RPM for 15min and Stored in a -80℃ low temperature refrigerator.Analyses

Statistical analysis
A statistical analysis was performed using Medcalc 19.0. The measurement data conforming to the normal distribution were expressed by mean ± standard deviation (SD).T-test was used for comparison between two groups.Univariate anova was used for multigroup comparison and Lsd-t test was used for further pairwise comparison.
The measurement data of non-normal distribution were expressed by [M (p25-p75)], mann-whitneyu test was used for comparison between two groups, and kruskal-wallis H test was used for comparison between multiple groups.X 2 test was used to compare counting data between groups. The area under the ROC curve (AUC) was calculated, and its diagnostic validity was tested by Z test, and the optimal cut-off value (YI=Se+ sp-1) was determined by Jordan index.P<0.001 was considered statistically significant.Spearman rank correlation analysis was used to analyze the correlation between HGF, PCIII ,PLT and clinical parameters and severity of NAFLD (P<0.05). Table 1 showed the laboratory and clinical characteristics of NAFLD patients and the control group.Levels of BMI,GLU,ALT,AST,TG,ALP,GGT,GPRI(GGT/PLT), DBIL,IBIL,TBA,TP,HGF,PCIII and PLT were significantly higher in the NAFLD patients compared with the control group,ALB was significantly lower in the NAFLD patients compared with the control group,(all P<0.05).In terms of sex, age HDL and APRI(AST/PLT),no significant differences were found between the two groups. Table 2 showed the laboratory and clinical characteristics of varying degrees of NAFLD patients and the control group.levels of BMI,GLU,ALT,AST,TG,ALP,APRI, CHE,TG,TC,LDL,TBIL,DBIL,IBIL,TBA,HGF,PCIII and PLT were correlated With the severity of NAFLD (all P<0.05).In terms of sex, age,no significant differences were found in all groups.Moreover, the levels of HGF were significantly higher in all NAFLD group compared with Control group,But,It showed no significant difference in Mild NAFLD group and Moderate NAFLD group.In addition, PIIIP can distinguish severe NAFLD group from mild NAFLD group,moderate NAFLD group and normal group, but It showed no significant difference in mild NAFLD group and moderate NAFLD group.Even more,PLT can distinguish normal group from all NAFLD group, but It showed no significant difference in mild NAFLD group and moderate NAFLD group,so as moderate NAFLD group and severe group.

Discussion
NAFLD has become an emerging public health concern, given its remarkable growth in the worldwide over the recent decades [17]."Two hits" is the most accepted hypothesis in NAFLD.The "first hit" is closely related to insulin resistance, and the "second hit" is closely related to oxidative stress, lipid peroxidation, Immune disorders,inflammatory factors and so on [18]. NAFLD is considered to be the liver manifestation of metabolic syndrome and the prevalence of NAFLD is increasing in parallel with the global rise in obesity and type 2 diabetes mellitus (T2DM) [19].Therefore, in addition to the changes of HGF, PCIII and PLT in NAFLD, our study also focused on their relationship with BMI, liver function, blood lipid, blood glucose and other indicators.
In the liver,the major producer of HGF in the liver is hepatic stellate cell and it plays a crucial role in liver regeneration after hepatectomy or massive liver damage,protection against hepatocyte apoptosis and necrosis, and suppression of liver fibrosis progression [5].Our study also revealed significant differences between mild, moderate, and severe cases with respect to HGF, in that the more advanced the degree of fatty liver assessed by ultrasound, the higher the HGF level.That may be due to the fact that NAFLD is a chronic inflammatory state of the liver,at the initiation of the inflammatory response, IL-6 induces acute phase proteins, which are implicated in controlling the inflammation and in resetting homeostasis[20],these acute phase proteins include u-PA that promotes activation of pro-HGF to biologically active HGF [21].HGF-MET signaling can also modulate adaptive immune response by facilitating the migration of Langerhans cells and dendritic cells to draining lymph nodes [22],thus modulate inflammation in the liver.PIIINP is a collagen that reflect extracellular matrix collagen turnover [23]. In animal studies, a high-fat diet induced NASH mouse model exhibited elevated PIIINP [24]and Hamza et al. also reported that mean serum levels of PIIINP in children with NAFLD (assessed by ultrasonography) were higher than those in control children [25].Our results are consistent with previous studies and Elevated serum PIIINP has been previously been shown to be associated with elevated pro-inflammatory cytokines in liver disease [9].PLTs have a role in thrombosis formation and evidence pointing to association between PLT count and liver injury [26]. In a study, it was reported that patients with moderate-to-severe NAFLD had higher PLT counts than milder form of NAFLD [27]and PLT count has been included in some scoring systems such as NAFLD fibrosis score [28]. In addition, platelets play a pivotal role in both hepatic regeneration and fibrosis pathophysiology and Platelets contain HGF [29],Our study also found that both PLT and HGF in NAFLD increased with the increase in severity of NAFLD, which may be the protective mechanism of the liver in the inflammatory state.
In addtion,HGF,PCIII and PLT may represent links between obesity, inflammation, metabolism and cardiovascular risk factors [30][31][32].Previous studies have also shown that recombinant HGF treatment reduced intracellular lipid content, likely by accelerating lipid secretion in hepatocytes through activation of microsomal triglyceride transfer protein and apolipoprotein B [33].Besides, recombinant HGF treatment inhibited cholesterol overload-mediated hepatocyte lipotoxicity by suppressing production of reactive oxygen species [34]. HGF is also a potent suppressor of hepatic glucose production and output in a setting of insulin resistance and that it can potentially restore insulin responsiveness [35].Quilliot D. et al. [36]showed that circulating PIIINP correlated with markers of insulin resistance and signs of impaired ventricular dysfunction in non-diabetic obese subjects.Agarwal et al. [31]demonstrated that higher blood PIIINP levels were associated with greater carotid intimamedia thickness and impaired brachial artery reactivity. Fay WP.et al.showed PLT lifespan is known to be shorter in subjects with IR, and the interactions between inflammation and thrombosis provide apotential mechanism linking PLT count and MetS [37].MPV is one of the PLT function indices which reflects the PLT production rate and stimulation and It has been reported that MPV was found significantly increased in adult patients who have diabetes, impaired fasting glucose, and hypertension[19, 38] [39]. The current study also highlights significant correlations between HGF, PCIII,PLT and other components of the metabolic syndrome,which makes it possible for them to jointly detect NAFLD.As shown in our results, when HGF,PCIII and PLT were detected together,the AUROC curve for diagnose NAFLD was 0.881(sensitivity0.760,specificity 0.873).

Availability of data and materials
Raw data were generated at First Hospital of Shanxi Medical University. Derived data supporting the findings of this study are available from the corresponding author [Jinchun Liu] on request.

Ethics approval and consent to participate
This study was approved by the Ethics committee of the First Hospital of Shanxi Medical University, which complies with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals.All patients provided a written informed consent prior to inclusion in the study.

Competing interests
The authors declare that they have no conflict of interest.   Table 4 The relationship between HGF, PCIII and PLT and the degree of NAFLD   Figure 1 showed ROC curve analysis about the diagnostic performance of HGF in patients with different degrees of fatty liver disease. Figure 2 showed ROC curve analysis about the diagnostic performance of PCIII in patients with different degrees of fatty liver disease. Figure 3 showed ROC curve analysis about the diagnostic performance of PLT in patients with different degrees of fatty liver disease. Figure 4 showed the ROC curve analysis about the diagnostic performance of combined detection of HGF, PCIII and PLT in patients with fatty liver disease.