| ObjectiveFirstly, patients with autoimmune hepatitis(AIH), patients with AIH liver cirrhosis and healthy controls were given oral lactulose and mannitol molecular probes, then their urine recovery rates were detected to evaluate the intestinal barrier function, in order to reveal the role of intestinal barrier function change, gut derived antigens such as lipopolysaccharide(LPS) in the occurrence and development of AIH. Further, the macrophage polarization of AIH patients liver tissues were explored, in order to explore and reveal the main function type of macrophages in the liver tissue of AIH patients.MethodFirst part:evaluation of intestinal barrier function in AIH patients5 cases of AIH patients, 5 cases of AIH liver cirrhosis patients and 10 cases of healthy controls were selected in Gastroenterology Department of Tianjin Medical University General Hospital between January 2015 and January 2016. The urine of all subjects were collected and lactulose and mannitol recovery were detected by high performance liquid chromatography(HPLC). The urine lactulose/mannitol ratio were calculated to evaluate intestinal barrier function. This study was approved by the hospital ethics committee(IRB2015-YX-002), all patients were informed consent.Second part:evaluation of liver macrophage polarization in AIH patients5 cases of AIH patients, 6 cases age and gender matched controls with other liver diseases and 5 cases of age and gender matched hepatic cyst patients were selected in Gastroenterology Department of Tianjin Medical University General Hospital between January 2015 and January 2016. Ultrasound guided liver biopsy were processed to get a 1.5cm long liver tissue. The activation and polarization of macrophages in all subjects' liver tissues were detected by immunohistochemistry and immunofluorescence staining. Further RT-PCR method was used to detect and verify the related surface markers and cytokines in the liver tissues. This study was approved by the hospital ethics committee(IRB2015-YX-002), all patients were informed consent.ResultFirst part: evaluation of intestinal barrier function in AIH patients(1) 5 patients with AIH and 5 patients with AIH liver cirrhosis were well tolerated, and no adverse reactions happened. One of 10 healthy controls(10%) appeared mild abdominal distension, two of 10(20%) appeared obvious abdominal distension, seven of 10(70%) appeared abdominal pain, diarrhea and other severe adverse reactions, three of 10 healthy volunteers(30%) collected no urine at the end.(2)Mannitol regression equations: y=1.096x+6.510; lactulose regression equations: y=1.032x+6.362. Among them, X is the common logarithm of standard concentration, y is the common logarithm of standard product peak area.(3)Only mannitol could be detected in healthy controls' urine samples, the mannitol recovery, mannitol recovery rate and L/M of healthy controls' urine samples were 92.35mg(14.60~209.4mg), 1.85%(0.29~4.19%) and 0.00(0.00~0.00)respectively. Compared to healthy controls, lactulose recovery?lactulose recovery rate and L/M of AIH group had no significant differences(all P>0.05), the mannitol recovery(362.22mg(176.67~515.36 mg)) and mannitol recovery rate(7.24%(3.53 ~ 10.31%)) had significant differences(P=0.023, 0.023); Compared to healthy controls, mannitol recovery(414.54mg(276.53 ~ 931.18mg)) ? mannitol recovery rate(8.29%(5.53 ~18.62%)) ? lactulose recovery(33.40mg(6.80 ~ 48.31mg)) ? lactulose recovery rate(0.37%(0.09~0.64%)) and L/M(0.05(0.02~0.10)) of AIH liver cirrhosis group were statistically higher(all P < 0.05); Compared to AIH patients, AIH patients with cirrhosis urine lactulose recovery(33.40 mg(6.80~48.31mg))?lactulose recovery rate(0.37%(0.09~0.64%))?L/M(0.05(0.02~0.10)) of AIH liver cirrhosis patients were statistically higher(all P < 0.05).Second part:evaluation of liver macrophage polarization in AIH patients(1) The number of macrophages in AIH patients increased: compared to fatty liver patients and hepatitis B virus patients, the absolute counts of CD68 positive cells in AIH patients increased significantly(P=0.034, 0.034), and there were no signficant differences between fatty liver patients and hepatitis B virus patients.(2)M2 type macrophages relatively low-grade expressed in AIH patients: compared to AIH patients and hepatitis B virus patients, the absolute counts of CD206 positive cells in fatty liver patients increased significantly(P=0.019, 0.046), and there were no signficant differences between AIH patients and hepatitis B virus patients. CD206 expression of fatty liver patients(8.08±1.63) were signicifantly higher than AIH patients(1.03±0.12) and hepatitis B virus patients(2.36±1.32), with significant statistical differences(all P <0.05), and CD206 expression of AIH patients and hepatitis B virus patients showed no significant differences(P>0.05); IL-10 expression of AIH patients(2.16±0.89) was significantly higher than that of patients with hepatitis B virus(0.93 ± 0.59), the difference was statistically significant(P<0.05), while AIH patients compared with fatty liver patients, hepatitis B virus patients compared with fatty liver patients, the relative expression of IL-10 showed no statistically significance(all P >0.05).(3) M1 type macrophages relatively over-expressed in AIH patients: there were CD68 and i NOS co-expressed M1 type macrophages in AIH patients and hepatitis B virus patients, and there were rare CD68 and i NOS co-expressed M1 type macrophages in fatty liver patients and liver cyst patients; TNF-? expression of AIH patients(13.29±7.55) and hepatitis B virus patients(12.39 ± 3.79) were significantly higher than patients with fatty liver patients(0.77±0.38), the differences were statistically significant(all P <0.05), and TNF- ? expression of AIH patients and hepatitis B virus patients showed no significant difference(P>0.05); i NOS expression of AIH patients(12.22±5.13) and hepatitis B virus patients(10.42±4.27) were significantly higher than that of patients with fatty liver(1.25±0.77), the differences were statistically significant(all P <0.05), and i NOS expression of AIH patients and hepatitis B virus patients showed no significant difference(P>0.05); IL-1?expression of AIH patients(5.90±3.01) and hepatitis B virus patients(12.19±3.71) were significantly higher than that of patients with fatty liver(1.76±1.42), the differences were statistically significant(all P<0.05), IL-1 ? expression of hepatitis B virus patients was higher than that of AIH patients(P<0.05).Conclusion(1) For the same dose of lactulose and mannitol, healthy controls showed a bad tolerance. We should pay much attention to explore a sensitive, well tolerated experimental dose. Healthy controls group had no intestinal barrier function changes,however, liver cirrhosis group's L/M ratio increased, intestinal barrier function changed. Compared with the former two groups, our research tended to prompt that AIH patients had no intestinal barrier function changes. However, we should optimize the method and further verify on the base of a large sample.(2) M1 macrophages over-expressed and played a major role in the inflammatory reaction and liver damage of AIH patients. |
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