INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is a state defined by extreme fat accumulation in the form of triglycerides (steatosis) in the liver (more than 5% of liver cells histologically). It is considered nowadays as one of the most frequent causes of abnormal liver tests all over the world, and it ranges from simple steatosis to advanced fibrosis and cirrhosis. A subgroup of NAFLD patients were characterized by injury to the hepatocytes and inflammation in addition to excessive fat (steatohepatitis). The latter condition, nominated nonalcoholic steatohepatitis (NASH), is practically indistinguishable histologically from alcoholic steatohepatitis, and it is characterized by fatty infiltration of the liver with different degrees of inflammation, necrosis, and fibrosis, almost identical to those of alcoholic liver disease; without significant alcohol ingestion.1

Simple steatosis seen in NAFLD does not correspond to increased short-term morbidity or mortality, but advancement of this condition to NASH noticeably increases the risks of fibrosis, cirrhosis (cryptogenic cirrhosis), liver failure, and hepatocellular carcinoma (HCC). The prevalence of NAFLD ranges from 10 to 24% of the general population, while NASH affects about 3% of the lean population and nearly half of morbidly obese people.2

The term nonalcoholic steatohepatitis or NASH is expressing the clinical and pathological characters of NAFLD coupled with the pathological characters mostly present in alcoholic liver disease itself. This description is still proper as NAFLD can progress to cirrhosis with fat from simple steatosis, through NASH and fibrosis.3

Nonalcoholic steatohepatitis is a chronic liver disease, i.e., earning increasing significance due to its great prevalence all over the world, the difficulty in diagnosis with noninvasive diagnostic methods, and the risk of succession to advanced fibrosis and cirrhosis, all of which make NASH difficult for doctors. Although the natural history of NASH and the causes of disease progression remain mostly unknown, more than a few data suggest that in some patients the disease may follow an indolent course until it progresses to end-stage liver disease.4,5

Most patients have the clinical characteristics of insulin resistance syndrome, including obesity, hypertension, glucose intolerance, and typical dyslipidemia. Insulin resistance plays a role not only in obese individuals but also in lean nondiabetic patients with hepatic steatosis.6

The “two-hit” hypothesis is the leading theory for the pathogenesis of NASH. This model suggests that the initial event or first “hit” leads to steatosis, and the second “hit” leads to necroinflammation and fibrosis. Insulin resistance has been implicated as the first “hit” and various insults, such as oxidative stress, cytokine effects, and fatty acid toxicity are suspected as potential other hits that lead to hepatocellular injury.7 Tumor necrosis factor-alpha (TNF-α) in the liver can contribute to oxidative stress; furthermore, it may contribute to insulin resistance.8 Additionally, TNF-α also initiates fibrosis both by direct activation of hepatic stellate cells and by stimulating production of tumor growth factor (TGF)-beta, a potent profibrogenic cytokine.9

A complex relationship exists between endotoxins, stellate cell activation, and release of cytokines and chemokines due to increased intestinal bacterial overgrowth, increased gut permeability, and reduced endotoxin scavenging by the reticuloendothelial system. The endotoxin releases a battery of cytokines; interleukin (IL)-1, IL-6, and TNF-α from nonparenchymal cells.10 The general belief that intestinal bacterial overgrowth has a role in NAFLD is supported by the evidence that obesity and diabetes, which are the major risk factors for NASH, are also associated with intestinal dysmotility11 and small bowel bacterial overgrowth.12 Probiotics are traditionally defined as viable microorganisms that have beneficial effects in the prevention and treatment of specific pathologic conditions when they are ingested.13 There are many proposed mechanisms by which probiotics may protect the host from intestinal disorders, including production of inhibitory substances, blocking of adhesion sites, competition for nutrients, degradation of toxin receptor, and stimulation of immunity.13 Probiotics affect intestinal bacterial flora by increase of anaerobic bacteria and decrease of the population of potentially pathogenic microorganisms.14

This work aimed to evaluate the role of probiotics on the outcome of NASH in patients admitted to the Tropical Medicine Department, Faculty of Medicine, Zagazig University (inpatients and outpatients).

MATERIALS AND METHODS

This randomized controlled study was performed on 30 patients (17 males and 13 females), with body mass index (BMI) from 30 to 35 and average age of 44 with bright fatty liver in ultrasonography and raised alanine transaminase (ALT) and aspartate transaminase (AST) and positive liver biopsy findings. Patients were divided into group I (case group) that included 15 patients who received probiotics and group II that included 15 patients (control group) who did not receive probiotics and were consulting at Tropical Medicine Department, Faculty of Medicine, Zagazig University Hospitals between November 2014 and April 2016.

Inclusion Criteria

Patients with elevated liver enzymes and bright liver by ultrasonography were examined and investigated for selection of patients with NASH, which was proved by liver biopsy.

Exclusion Criteria

Patients with history of significant alcohol consumption more than 20 gm/day or under dietetic regimen; patients with positive hepatitis B and C virus markers; patients who were taking lipid-lowering medications, metformin, or thiazolidinediones; and patients with any other metabolic liver disease were excluded from the study. Kidney function tests were performed: For exclusion of cases with renal impairment. Compensated and decompensated cirrhotic patients; hepatic focal lesion; AST/ALT ratio >1 were also excluded.

All patients were subjected to the following: Full clinical history with stress on age and sex, change in appetite, dyspepsia, abdominal distension, right hypochondrial pain, and fatigue. Patients with history of medical diseases, e.g., diabetes mellitus, hypertension, bilharziasis, chronic viral hepatitis, pituitary disease, adrenal disease or pancreatic disease, history of surgical operation, e.g., gastric bypass or jejunoileal bypass were also excluded.

All patients were checked for parameters of general examinations, such as blood pressure, pulse, face complexion, hand and lower limb examination, body weight, height, and BMI that was defined by Kumar and Clark.15

Local examination of abdomen and liver was accomplished.

Laboratory assessment included complete blood count, hemoglobin level, white blood cells, and platelets counts. Parameters of liver function tests were also checked. Imaging of abdomen and the liver was done.

Probiotic Therapy

The patients were kept on probiotic supplementation. Acidophilus capsule (Lactobacillus acidophilus, which contains 2 billion viable organism, and mixture of rice flour, gelatin, and magnesium stearate) was given to patients 30 minutes before meal three times daily for 1 month duration (Acidophilus, Swanson Health Products, USA website, www.swansonvitamins.com).

Statistical Analysis

Data were checked, entered, and analyzed using Statistical Package for the Social Sciences version 18 for Windows. Data were expressed as mean ± standard deviation for quantitative variable, number and percentage for qualitative one. Chi-squared (χ2) or t-test and paired t-test were used when appropriate; p < 0.05 was considered significant; p < 0.001 was considered highly significant.

RESULTS AND DISCUSSION

Nonalcoholic fatty liver disease is probably the most common liver disorder in the world.16 It is generally believed that NAFLD affects 2.8 to 24% of the general population and affects adults and children.17

In addition, NAFLD is the consequence of excess triglyceride accumulation in hepatocytes in the absence of significant alcohol consumption.18 It includes a spectrum of hepatic changes from steatosis alone, to NASH, fibrosis, cirrhosis, and even HCC.19

Probiotics are traditionally defined as viable microorganisms that have beneficial effects in the prevention and treatment of specific pathologic conditions when they are ingested.13 There are many proposed mechanisms by which probiotics may protect the host from intestinal disorders, including production of inhibitory substances, blocking of adhesion sites, competition for nutrients, degradation of toxin receptor, and stimulation of immunity.13 Probiotics affect intestinal bacterial flora by increase of anaerobic bacteria and decrease of the population of potentially pathogenic microorganisms.14

This study is aimed to evaluate the role of probiotics on the outcome of NASH in humans.

This randomized controlled study was performed on 30 patients (17 males and 13 females), with BMI from 30 to 35 and average age of 44 with bright fatty liver in ultrasonography and raised ALT and AST and positive liver biopsy findings. Patients were divided into group I (case group) that included 15 patients who received probiotics and group II that included 15 patients (control group) who did not receive probiotics and were consulting at Tropical Medicine Department, Faculty of Medicine, Zagazig University Hospitals between November 2014 and April 2016.

In the present study of demographic, clinical, and abdominal ultrasound (U/S) findings before treatment for both groups, no significant difference was seen. Also this study showed no significant difference as regards complete blood count, liver function test, kidney function test, prothrombin time (PT), international normalized ratio (INR), lipid profile, and blood sugar. Due to all patients were complaining from nonalcoholic steatohepatitis then were divided randomly into two groups.

In the present study, patients were matched with control subject as regards posttreatment biochemical findings, which showed no significant difference (Tables 1 and 2). But there was a highly significant difference in ALT and significant difference in AST. Otherwise, other biochemical changes showed no significant difference between both groups This is in agreement with the results of Solga and Diehl,20 Loguercio et al,21 and Portincasa et al,22 which found similar changes in ALT and AST. These findings denote that the use of probiotics in these patients plays some role in improving the necroinflammatory status of the liver (Tables 3 to 6).

Table 1: Comparison between groups I and II as regards demographic, clinical, and U/S findings

VariablesGroup I (n = 15)Group II (n = 15)p-value
Age (years)44.20 ± 5.5144.33 ± 5.620.948
Gender                  
Male9 (60%)8 (53.3%)0.713
Female6 (40%)7 (46.7%)      
BMI (kg/m2)32.56 ± 1.1933.05 ± 1.270.284
Clinical presentation                  
Asymptomatic8 (53.3%)15 (100%)0.006
Dyspepsia4 (26.7%)0 (0%)0.100
Fatigue2 (13.3%)0 (0%)0.483
Right hypochondrial pain1 (6.7%)0 (0%)1.000
Clinical examination                  
Hepatomegaly4 (26.7%)4 (26.7%)1.000
Splenomegaly0 (0%)0 (0%)1.000
Ascites0 (0%)0 (0%)1.000
Liver in U/S                  
Average4 (26.7%)5 (33.3%)0.709
Enlarged11 (73.3%)10 (66.7%)      
Spleen in U/S                  
Average15 (100%)15 (100%)1.000
Splenomegaly0 (0%)0 (0%)      
Ascites in U/S                  
Absent15 (100%)15 (100%)1.000
Present0 (0%)0 (0%)      

Table 2: Comparison between groups I and II as regards laboratory findings before treatment

VariablesGroup I before (n = 15)Group II before (n = 15)p-value
Hemoglobin (g/dL)13.16 ± 0.8813.57 ± 0.840.208
WBCs (× 103/mm3)6.78 ± 1.727.36 ± 1.850.383
Platelet count (× 103/mm3)300.60 ± 78.33312.20 ± 75.560.683
Protein (g/dL)7.47 ± 0.777.45 ± 0.890.819
Albumin (g/dL)4.60 ± 0.444.64 ± 0.450.835
TSB (mg/dL)0.88 ± 0.380.86 ± 0.410.950
DSB (mg/dL)0.11 ± 0.040.12 ± 0.050.665
ALT (U/L)81.45 ± 23.3283.53 ± 12.010.962
AST (U/L)44.05 ± 14.6542.73 ± 9.950.967
PT (sec)15.19 ± 1.7215.86 ± 1.940.325
INR1.00 ± 0.091.04 ± 0.100.336
Serum creatinine (mg/dL)0.90 ± 0.291.05 ± 0.300.186
BUN (mg/dL)18.06 ± 6.8119.13 ± 7.460.686
Triglycerides (mg/dL)257.80 ± 55.02245.33 ± 70.780.755
Cholesterol (mg/dL)258.60 ± 31.70250.53 ± 45.120.771
LDL (mg/dL)158.53 ± 23.67154.73 ± 33.240.721
HDL (mg/dL)48.73 ± 4.8348.80 ± 5.000.971
FBS (mg/dL)104.86 ± 21.33107.06 ± 27.060.807
PBS (mg/dL)161.13 ± 53.37173.06 ± 73.910.819

WBC: While blood cell; TSB: Total serum bilirubin; DSB: Direct serum bilirubin; BUN: Blood urea nitrogen; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; FBS: Fasting blood sugar; PBS: Peripheral blood smear

Table 3: Comparison between groups I and II as regards laboratory findings after treatment

VariablesGroup I after (n = 15)Group II after (n = 15)  p-value
Hemoglobin (g/dL)13.20 ± 0.8513.67 ± 0.71  0.108
WBCs (× 103/mm3)6.79 ± 1.707.36 ± 1.67  0.366
Platelet count (×103/mm3)298.40 ± 76.37309.26 ± 70.02  0.688
Protein (g/dL)7.47 ± 0.777.42 ± 0.80  0.857
Albumin (g/dL)4.58 ± 0.454.66 ± 0.41  0.493
TSB (mg/dL)0.87 ± 0.370.88 ± 0.37  0.967
DSB (mg/dL)0.11 ± 0.040.12 ± 0.05  0.613
ALT (U/L)46.10 ± 20.7582.53 ± 10.41<0.001
AST (U/L)38.20 ± 11.5855.86 ± 5.11 0.03
Serum creatinine (mg/dL)0.89 ± 0.260.91 ± 0.20  0.142
BUN (mg/dL)17.73 ± 6.1318.46 ± 6.45  0.752

WBC: While blood cell; TSB: Total serum bilirubin; DSB: Direct serum bilirubin; BUN: Blood urea nitrogen

Table 4: Comparison between before and after treatment in group I as regards clinical and U/S findings

VariablesGroup Ip-value
Before treatment (n = 15)After treatment (n = 15)
Clinical presentation
Asymptomatic8 (53.3%)12 (80%)0.125
Dyspepsia4 (26.7%)1 (6.7%)0.250
Fatigue2 (13.3%)1 (6.7%)1.000
Right hypochondrial pain1 (6.7%)1 (6.7%)1.000
Clinical examination                  
Hepatomegaly4 (26.7%)4 (26.7%)1.000
Splenomegaly0 (0%)0 (0%)1.000
Ascites0 (0%)0 (0%)1.000
Liver in U/S                  
Average4 (26.7%)4 (33.3%)0.135
Enlarged11 (73.3%)11 (73.3%)      
Spleen in U/S                  
Average15 (100%)15 (100%)1.000
Splenomegaly0 (0%)0 (0%)      
Ascites in U/S                  
Absent15 (100%)15 (100%)1.000
Present0 (0%)0 (0%)

Table 5: Comparison between before and after treatment in group I as regards laboratory findings

VariablesGroup I p-value
Before treatment (n = 15)After treatment (n = 15)
Hemoglobin (g/dL)13.16 ± 0.8813.20 ± 0.85 0.334
WBCs (× 103/mm3)6.78 ± 1.726.79 ± 1.70 0.806
Platelet count (× 103/mm3)300.60 ± 78.33298.40 ± 76.37 0.021
Protein (g/dL)7.47 ± 0.777.47 ± 0.77 1.000
Albumin (g/dL)4.60 ± 0.444.58 ± 0.45 0.208
TSB (mg/dL)0.88 ± 0.380.87 ± 0.37 0.144
DSB (mg/dL)0.11 ± 0.040.11 ± 0.04 0.334
ALT (U/L)83.33 ± 10.9646.10 ± 20.75<0.001
AST (U/L)57.06 ± 7.8638.20 ± 11.58 0.03
Serum creatinine (mg/dL)0.90 ± 0.290.89 ± 0.26 0.414
BUN (mg/dL)18.06 ± 6.8117.73 ± 6.13 0.173

WBC: While blood cell; TSB: Total serum bilirubin; DSB: Direct serum bilirubin; BUN: Blood urea nitrogen

Table 6: Comparison between before and after treatment in group II as regards laboratory findings

VariablesGroup IIp-value
Before treatment (n = 15)After treatment (n = 15)
Hemoglobin (g/dL)13.57 ± 0.8413.67 ± 0.710.355
WBCs (× 103/mm3)7.36 ± 1.857.36 ± 1.670.937
Platelet count (× 103/mm3)312.20 ± 75.56309.26 ± 70.020.366
Protein (g/dL)7.45 ± 0.897.42 ± 0.800.362
Albumin (g/dL)4.64 ± 0.454.66 ± 0.410.519
TSB (mg/dL)0.86 ± 0.410.88 ± 0.370.340
DSB (mg/dL)0.12 ± 0.050.12 ± 0.050.582
ALT (U/L)83.53 ± 12.0182.53 ± 10.410.096
AST (U/L)58.73 ± 9.9555.86 ± 5.110.048
Serum creatinine (mg/dL)1.05 ± 0.301.07 ± 0.200.638
BUN (mg/dL)19.13 ± 7.4618.46 ± 6.450.329

WBC: While blood cell; TSB: Total serum bilirubin; DSB: Direct serum bilirubin; BUN: Blood urea nitrogen

This study showed improvement of dyspepsia in group I posttreatment; otherwise, there were no significant change in other symptoms and in abdominal U/S findings.

This study also showed significant decrease in ALT and AST in group I posttreatment in comparison to pretreatment; otherwise, there was no significant change in other laboratory findings, and these were in agreement with the findings of Loguercio et al21 and Wong et al.23

CONCLUSION

Probiotics treatment is effective, safe, well-tolerated, inexpensive, appropriate for long-term use, and optimally works at multiple levels to downregulate inflammatory mediators, and therefore, probiotics could be an option in the treatment of NASH.

Conflicts of interest

Source of support: Nil

Conflict of interest: None