3.1 Patients Characteristics
A total of 50 participants were recruited in this study, including 18 LDSDS, 18 SGDHS, and 14 healthy subjects. Among the 18 LDSDS, 8 were males and 10 were females; among the 18 SGDHS, 12 were males and 6 were females; among the 14 healthy participants, there were 11 males and 3 females. There were significant differences in gender and age among the three groups (P<0.05). However, there was no significant difference in age and gender between the LDSDS and SGDHS(P>0.05). Serum aspartate aminotransferase (AST) levels, alanine aminotransferase (ALT) levels, total bilirubin (TBIL) levels, albumin (ALB) and HBV-DNA had no significant difference between the LDSDS and SGDHS(P>0.05) (Table 1).
Table 1 Patients characteristics.
Variables
|
LDSDS(n=18)
|
SGDHS(n=18)
|
HC(n=14)
|
P Value
|
Age(y) †
|
39.00±9.15
|
41.00±9.15
|
24.57±0.85
|
0.000
|
Female(%)‡
|
10(55.56%)
|
6(33.33%)
|
11(78.57%)
|
0.038
|
ALT(u/L) §
|
23.5(17.75~47.25)
|
25.5(20.25~36.23)
|
NA
|
0.696
|
AST(u/L) §
|
21.5(16~29.25)
|
25(20.75~28.25)
|
NA
|
0.323
|
TBIL(umol/L) §
|
13(10.38~14.55)
|
14(9.55~18.68)
|
NA
|
0.228
|
ALB(g/L) §
|
45(42.48~47.15)
|
44.85(42.18~47.2)
|
NA
|
0.729
|
HBV DNA(≥2000IU/ml,n(%)‡
|
7 (38.89%)
|
6 (33.33%)
|
NA
|
0.383
|
LDSDS, liver depression and spleen deficiency syndrome; SGDHS: spleen-gastric damp heat syndrome; HC: healthy controls; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; ALB, albumin; NA, not available; †Means ± SD; ‡Numbers and percentages; §Median and interquartile range.
3.2 Distribution of T lymphocytes in subjects
The results of flow cytometry showed that the CD8+T cell level was significantly decreased in LDSDS and SGDHS compared with the healthy controls (P<0.05), however, there were no significant difference observed (P>0.05) between LDSDS and SGDHS (Figure 2A). Significant decrease of CD4+T cells was observed only in the SGDHS, but not in LDSDS when compared with healthy controls (P<0.05). Moreover, the expression of CD4+T cell in LDSDS were increased compared with SGDHS (P<0.05) (Figure 2A). There were significant differences in Th17 cell expression among the three groups, and expression level of LDSDS was the highest(P<0.05) (Figure 2B). For Treg, both LDSDS and SGDHS exhibited significantly increased levels compared with the healthy controls (P<0.01), but there were no significant difference observed (P>0.05) between LDSDS and SGDHS (Figure 2C). In addition, the ratio of CD4+T to CD8+T cells and the ratio of Th17 to Treg cells reflect the immune balance of T cells. The specific percentage of each T lymphocyte was shown in Table 2. It can be seen that there was no significant difference in the ratio of CD4+T to CD8+T cells among the three groups. The ratio of Th17 to Treg cells of LDSDS and SGDHS was significantly higher than that of healthy controls(P<0.05)(Table 2). What has most interesting is that among the four T lymphocytes, only Th17 showed statistical differences not only between these two syndromes but also between the syndromes and healthy controls, suggesting that Th17 may serve as one of the potential biochemical indicators of the TCM-syndrome of CHB, which needs further exploration.
Table 2 Expression of T lymphocyte subsets (n=50, Mean ±SD)
T lymphocyte
|
LDSDS (%)
|
SGDHS (%)
|
HC (%)
|
CD4+T
|
33.64±5.08#
|
28.70±7.11**
|
36.58±4.76
|
CD8+T
|
25.64±6.66*
|
22.71±8.25**
|
32.07±6.27
|
CD4+T /CD8+T
|
1.38±0.36
|
1.37±0.34
|
1.18±0.30
|
Th17
|
13.01±5.11**#
|
9.12±3.74**
|
0.49±0.24
|
Treg
|
9.17±2.15**
|
10.35±1.86**
|
4.67±3.08
|
Th17/Treg
|
1.46±0.58**
|
0.89±0.39**
|
0.20±0.22
|
HC represents healthy controls. “*” represents statistically significant difference compared with the healthy control (P<0.05), “**” represents statistically significant difference compared with the healthy control (P<0.01); “#” represents statistically significant difference compared with SGDHS(P<0.05).
3.3 Analysis of HMGB1-PTEN pathway protein expression level
Through the Elisa, the HMGB1-PTEN proteins were quantified. The results showed the levels of HMGB1, PI3K, PDK1, and Akt in LDSDS and SGDHS were significantly higher than healthy controls (P<0.01) (Figure 3). Moreover, the expression levels of HMGB1, PI3K in LDSDS were even higher than that in SGDHS (P<0.01) (Figure 3). For PTEN, compared with healthy controls, it was significantly increased in LDSDS but significantly decreased in SGDHS(P<0.01), and exhibited a significant difference between the two syndromes(P<0.01) (Figure 3). Collectively, HMGB1, PTEN, and PI3K were highly expressed in LDSDS compared with the SGDHS(P<0.05) (Figure 3), indicating that the expression level of these three proteins may be related to the formation of TCM syndromes to some extent, but this need to be further studied in the following research.
3.4 Correlation analysis of HMGB1-PTEN pathway protein
To further elucidate the correlation strength among the HMGB1-PTEN pathway proteins, the protein-protein interaction networks between five proteins were constructed through the STRING database (https://www.string-db.org/). As shown in Figure 4, we found that HMGB1 was connected with PTEN, PI3K, PDK1 through Akt from the aspects of literature mining, experiments, databases, co-expression, gene neighborhood, gene fusion, co-occurrence, etc. The data demonstrated that the combined score of evidence suggesting a functional link in PI3K, PDK1, and Akt were above 0.8, and they had positive regulation. The combined scores of PTEN, PI3K, and Akt were above 0.9, but PTEN negatively regulates PI3K/Akt. Although the combined score of HMGB1 and Akt was 0.554, it can be seen from the number of lines in the figure, there was not only one way of connection between them.
The GeneMANIA database (http://genemania.org/) was used to further construct the network map related with HMGB1, PTEN, PI3K, PDK1, and Akt. As shown in Figure 5, there are many interaction modes such as physical interactions (67.64%), co-expression (13.50%), co-localization (6.17%), pathway-mediated (4.35%) and other aspects. Further analysis of the interaction mechanism revealed that HMGB1 is co-expressed with PDK1, which is associated with PI3K/Akt pathway, and can also co-express PTEN through CENPC, while PTEN directly negatively regulates PI3K/Akt pathway. That is to say, HMGB1 can regulate PI3K/Akt pathway by direct or indirect means. In the PI3K/Akt pathway, EGFR is the key target factor of the pathway and has a certain impact on mTOR signaling molecules that is the classic factor in liver disease research. These remind us that EGFR may be a key factor in HMGB1-PTEN protein network, which provides a target gene for further research.
3.5 Correlation analysis of HMGB1-PTEN pathway protein and immune cell levels
The proteins of HMGB1-PTEN pathway were, respectively, subjected to stepwise regression analysis on T lymphocyte subsets to construct a regression model. Next, take the LDSDS as an example: The adjusted R2 of all proteins were good fit. The results showed that it was highly consistent, mainly linearly correlated with CD4+T cells and Th17/Treg (Table 3). Similarly, in the SGDHS, the regression results showed that the HMGB1-PTEN pathway axis could significantly affect the differentiation of T lymphocytes and was highly consistent, all of which were significantly correlated with Treg cells. In healthy controls, the HMGB1-PTEN pathway proteins were significantly associated with CD4+T, CD8+T cells, and CD4+T/CD8+T. It can be seen that in the two syndromes, the immune cells affected by pathway regulation were different, in other words, linearly related to different lymphocytes, indicating that the physiological and pathological state of the body may affect the internal relationship between HMGB1-PTEN and immune cells.
Table 3 Multiple stepwise regression analysis of HMGB1-PTEN pathway and T lymphocytes in LDSDS
Pathway
|
CD4+T
|
CD8+T
|
CD4+T/CD8+T
|
Th17
|
Treg
|
Th17/Treg
|
HMGB1
|
10.855/0.983**
|
—
|
—
|
—
|
—
|
2.408/0.983*
|
PTEN
|
4.845/0.974**
|
—
|
—
|
—
|
—
|
—
|
PI3K
|
10.742/0.982**
|
—
|
—
|
—
|
—
|
2.390/0.982*
|
PDK1
|
10.824/0.983**
|
—
|
—
|
—
|
—
|
2.397/0.983*
|
Akt
|
10.808/0.983**
|
—
|
—
|
—
|
—
|
2.419/0.983*
|
The numbers on the left and right sides of each slash line represent the regression coefficient and coefficient of determination, respectively. *, **: p=0.05,0.01, respectively.