Baseline Characteristics
All patients were Chinese. Table 1 shows the baseline characteristics at enrollment in the study. There were no significant differences in gender, age and creatinine in all groups. Nevertheless, the levels of alanine aminotransferase (ALT), total bilirubin, INR, AFP and MELD scores were significantly higher in ACLF group than that in the other group.
Complement components in plasma samples
The plasma concentrations of C1q were markedly reduced in HBV-ACLF than those in CHB group or in control group, but not the MBL and FB
We first examined the concentrations of the complement classical pathway component (C1q), the lectin pathway (MBL) and the alternative pathway (FB) in plasma samples from HBV-ACLF, CHB and control group. The concentrations of C1q were significantly lower in HBV-ACLF group than that in CHB group or in control group(Fig. 1A). The mean concentration of C1q was 177001 ng/ml in the HBV-ACLF group, but only 114640 ng/ml in CHB group and 50509 ng/ml in control group. However, There were no statistical significances of the plasma concentrations of MBL and FB in all groups(Fig. 1B-C), suggesting that the classical pathway plays the more important role in the inflammatory activities of HBV-ACLF. (Table 1)
The concentrations of complement components C3, C4, their degradation products and sC5b-9 in all groups
Complement C3 and its degradation product C3a were significantly higher in the control group than that in the HBV-ACLF group(Fig. 1D-E). The C3 is 15653 μg/ml in the control group, 8916 μg/ml in the CHB group and 6568 μg/ml in the HBV-ACLF group. C3 and its degradation product C3a were significantly higher in the control group than that in the HBV-ACLF group. The C4 is 1530 μg/ml in the control group, 1487 μg/ml in the CHB group and 1074 μg/ml in the HBV-ACLF group(Fig. 1F-H). The C3 and C3a level in ACLF survival group was higher than that in ACLF non-survival group, but without statistical difference. The concentrations of C1q, C3, C3a, C4, C4a and sC5b-9 were significantly higher in the control group than those in the HBV-ACLF group (3.5, 2.4, 2.1, 1.4, 1.3 and 6.0 fold, respectively). (Table 2)
The relationship between complement components and clinical parameter
The concentrations of C1q, MBL and FB were not correlated with the prognosis score
There are no statistical significance of the plasma concentrations of C1q, MBL and FB between the ACLF survival group and ACLF non-survival group. None of them were correlated with ALT, total bilirubin, albumin and HBV DNA. Only the C1q were observed to be positively correlated with AFP (P < 0.05). (Fig. 2A)
The correlation of C3, C4 and their degradation products levels with clinical parameter and prognosis score
C3 were observed to be positively correlated with AFP (P < 0.05). C3a was observed to be positively correlated with C3, r=4675, but P=0.0504. C3a and C4a were observed to be negatively correlated with creatinine (P < 0.05). C4 was observed to be negatively correlated with INR (P < 0.05). But none of them were correlated with ALT, total bilirubin, albumin and HBV DNA (Fig. 2B-E). C3, C3a and C4a were observed to be negatively correlated with MELDs (P < 0.05) (Fig. 3A-C). C3, C3a and C4 were observed to be negatively correlated with CLIF-C OFs (P < 0.05).
C3 activation in plasma in ACLF group
The complement system activation is mediated through the classical , the alternative and the lectin pathways. Each pathway converges at the C3 convertase level resulting in C3 cleavage. To further assess whether or not the C3 activation is involved in ACLF group, levels of C3 breakdown products in plasma samples were evaluated by Western blot. According to C3 level, we adjusted sample's volume to ensure the samples’ C3 concentration from two groups is the same for comparability. We repeated western blot test at least three times. Under the same conditions, C3 cleavage fragments iC3b were detected in both groups. Besides, and the level of iC3b was more in ACLF group than the one in CHB group. C3 cleavage fragments iC3b was detected in plasma from both ACLF group and CHB group. Prominent iC3b band was found in plasma samples in ACLF patients. (Fig. 4)