The aim of this study is to investigate traditional Chinese medicine syndrome (TCMS) patterns and their association with hepatitis B surface antigen (HBsAg) levels during the natural history of chronic hepatitis B virus infection (CHB). Patients were categorized according to the phase of CHB, as follows: immune tolerance (ITP); immune clearance (ICP); low or nonreplication (LRP); reactivation (RAP); hepatic cirrhosis (HC); and primary liver cancer (PLC). TCMS patterns were classified among the following types: spleen-kidney deficiency (SKD); liver-qi depression (LQD); damp-heat in liver-gallbladder (LGDH); liver-kidney deficiency (LKD); and blood stasis blocking collateral (BSBC). HBsAg levels and other serological indicators were quantified for all patients and their association with TCMS was statistically analyzed and determined. Two hundred and eighty-nine patients with CHB were included. During the natural history of CHB, TCMS patterns were statistically different among the different phases (
Due to high risk of developing acute or chronic hepatic failure and hepatocellular carcinoma (HCC), chronic hepatitis B virus (HBV) infection (CHB) remains a heavy burden and substantial challenge to global public health [
With advancement in understanding of CHB pathogenesis, the natural history of CHB has been recently classified into the following four phases: immune tolerance phase (ITP); immune clearance phase (ICP); low or nonreplication phase (LRP); and reactivation phase (RAP)[
Syndrome differentiation is the well-recognized essence of TCM, and as such identification of syndrome patterns is key to preventing and treating a disease by this approach [
As the first identified and main marker of HBV infection, hepatitis B surface antigen (HBsAg) is positively correlated with DNA and partly reflects the content of covalently closed circular DNA (cccDNA) [
In this study, we investigated TCMS patterns and their association with HBsAg levels during the complete natural history of CHB, including the four typical phases, HC, and PLC.
The study was reviewed and approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (Approval No. 2013:2-60). All patients voluntarily joined this study, and patients who accepted liver biopsy provided written informed consent.
This study was conducted in patients with CHB, from the Department of Integrative Chinese and Western Medicine at The Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). All patients were classified among the six groups, based on four typical phases (ITP, ICP, LRP, or RAP) and two late or advanced stages (HBV-related HC or PLC) in the course of CHB. The diagnostic criteria for the four typical phases were based on the 2012 European Association for the Study of the Liver [
Criteria of the classification of phases in the natural history of CHB.
| | | | |
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ITP | < ULN | + | > 107 | normal |
ICP | ≥ 2 × ULN | + | > 2000 | nonHC and nonPLC |
LRP | < ULN | - | < 2000 | normal |
RAP | ≥ 2 × ULN | - | > 2000 | nonHC and nonPLC |
HC | any | +/- | any | cirrhosis |
PLC | any | +/- | any | PLC |
Exclusion criteria for the study included (1) coinfection with hepatitis C virus, hepatitis D virus or human immunodeficiency virus, (2) coexistence of alcoholic, metabolic or autoimmune liver disease, (3) concomitant serious disease in heart, lung or kidney, (4) history of mental illness, (5) no signs of cirrhosis or PLC but having a history of taking anti-HBV medication, (6) diagnosis of nonHBV-associated liver cirrhosis, or (7) diagnosis of secondary liver cancer.
The following parameters were recorded for each patient: TCMS; demographical information; status and levels of serum liver biochemical markers and serum (qualitative) HBV markers and serum (quantitative) HBsAg, HBeAg, and HBV DNA levels.
Serum HBV markers were quantitatively measured by enzyme-linked immunosorbent assay (ELISA). Serum HBsAg titers were measured with a commercial detection kit (Abbott Architect assay; Abbott Diagnostics, Wiesbaden, Germany) following the manufacturer’s instructions. The dynamic range of the kit was 0.05-250 IU/mL. If titers were more than 250 IU/mL, the samples were diluted 1:500 or 1:1000 using the HBsAg Manual Diluent (Abbott Diagnostics). HBV DNA was qualitatively detected by fluorescence quantitative PCR assay (Da-An Gene, Sun Yat-Sen University, China).
TCMS of patients were differentiated among five types, with reference to the viral hepatitis diagnostic standards defined in December 1991 by the Internal Medicine Hepatopathy Committee of the Chinese Traditional Medicine Association [
Standards of TCMS were based on the Guiding Principles for Clinical Research on New Drugs of Traditional Chinese Medicine (published in 2003)[
Differentiating standards of the five patterns of TCMS.
| |
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LQD | Irritability, depression, fullness or pain in the hypochondrium, slight yellow coat on the tongue, taut pulse |
LKD | Dry eyes and throat, feverish sensation in the chest and palms, lumbar debility, scanty coating on the tongue, fine and rapid pulse |
LGDH | Fatigue, bitter mouth, dark urine (jaundice), unsmooth bowel movement, yellow and oily coat on the tongue |
SKD | Poor appetite, urinary frequency or enuresis, loose stool, cold feeling and weakness in the lumbar spine and knee, white slippery coating on the tongue, moderate pulse |
BSBC | Needle-pricking sensation and pain, dark face, dark tongue, fine and sluggish pulse |
BSBC: blood stasis blocking collateral; LGDH: damp-heat in liver-gallbladder; LKD: liver-kidney deficiency; LQD: liver-qi depression; SKD: spleen-kidney deficiency; TCMS: traditional Chinese medicine syndrome.
All serum statistical data are presented as medians with 10%–90% confidence intervals (CIs). Between-group comparisons were carried out by analysis of variance test (ANOVA) with either Mann-Whitney U (in nonparametric test for univariate comparisons) or Kruskal–Wallis (for multivariate comparisons). Categorical data were tested using the
Two hundred and eighty-nine patients with CHB were enrolled in the study and divided into the six groups, representing ITP (
Two hundred and twenty-five patients in the four typical phases did not receive antiviral treatment. For the late or advanced stage groups, 11 cases of HC and 23 cases of PLC were treated with oral antiviral agents. The overall data on patients’ demographics, serum liver biochemical markers, serum qualitative HBeAg status, and HBsAg and HBV DNA quantitation are presented in Table
Patient characteristics based on the six phases in the natural history of CHB.
| | | | | | | |
---|---|---|---|---|---|---|---|
| 36/34 | 42/8 | 54/18 | 29/4 | 17/2 | 38/7 | < 0.001 |
| 27 | 34 | 38 | 41 | 48 | 54 | < 0.001 |
| 38 | 183 | 24 | 175 | 53 | 55 | < 0.001 |
| 27 | 121 | 25 | 97 | 82 | 91 | < 0.001 |
| 7.99 | 6.77 | 2.00 | 5.61 | 4.42 | 3.42 | < 0.001 |
| 4.51 | 3.93 | 3.71 | 3.86 | 3.81 | 3.83 | < 0.001 |
Age is listed as median and min.-max. ALT, AST, HBV DNA, and HBsAg are expressed as the median and 10%-90% confidence interval. ALT: alanine aminotransferase; CHB: chronic hepatitis B virus infection; HBV DNA: hepatitis B virus DNA; HBsAg: hepatitis B surface antigen; HC: hepatic cirrhosis; ICP: immune clearance phase; ITP: immune tolerance phase; LRP: low or nonreplication phase; PLC: primary liver cancer; RAP: reactive phase.
During the natural history of CHB infection, the TCMS patterns varied significantly between patients in different phases of CHB (
In the four typical phases of CHB, ITP and LRP are the inactive states, while ICP and RAP are the active states. Additionally, HBV-associated PLC often presents after HC. Hence, we combined the six groups into three sets, namely, the inactive stage set (IAS), the active stage set (AS), and the late or advanced stage set (LSS). As shown in Figure
The distributive profile of serum HBsAg titers from this study is shown in Figure
Further statistical analysis of the HBsAg titers in Figure
As shown in Figure
As CHB is a highly dynamic pathological process, its trilogy has been used to represent the progressive course across hepatitis, cirrhosis, and PLC since the last century. In recent decades, hepatitis B has been further divided into HBeAg-positive hepatitis and HBeAg-negative hepatitis [
As shown in Figure
Both excess syndrome caused by deficiency and deficiency syndrome caused by excess can be used to explain the change of the nature or mechanism of a disease in TCM. As far as TCMS of CHB is concerned, those results presented in Figure
After reconstruction of the six phases into three bundled sets, the predominant pathogenic factors in the inactive, active and late or advanced stage sets of patients were deficiency (spleen, liver,and kidney), excess (damp-heat) and blood stasis, respectively. Deficiency was the main nature of CHB in ITP and LRP, that is, in the inactive stage sets. A likely reason for this finding is that the patients had a congenital Yang deficiency in spleen and kidney, which also may be the most likely explanation for their being infected by HBV. Another possible explanation is that the HBV infection led patients to the Yang deficiency in spleen and kidney. The factor causing Yin deficiency of liver and kidney in LRP was possibly due to damp-heat consuming Yin. Conversely, excess was the main nature of the disease in ICP and RAP, that is, the active stage sets. Their common reason could be the intensive struggle between the vital and evil qi. Obviously, the reason for explaining blood stasis as the leading pathogenic factor in HC and PLC is that HC and PLC often developed in late-stage CHB.
In summary, the pattern, nature and pathogenic factor varied with the difference of phases or stages. Phases and stages of the natural history of CHB could be considered as indicators of differentiating TCMS. Besides that, the results of this study also verified that TCMS was the dynamic manifestation of the progressive process of disease. This study had significant implications for understanding the natural history of CHB from the TCM perspective, and might provide the joints for the combination of Chinese and Western medicines. Generally, the inactive-stage TCM therapeutic principle should mainly strengthen vital qi, including reinforcement of spleen and kidney Yang in ITP and nourishment of liver and kidney Yin in LRP. And, in active-stage, TCM should mainly eliminate evil qi, clearing damp-heat in liver and gallbladder. Finally, in HC and PLC, TCM should mainly activate blood circulation to dissipate blood stasis.
Studies have also revealed that serological HBsAg titers have a positive association with HBV DNA level, as well as with intrahepatic cccDNA level [
In the future, it may be a meaningful but challenging effort to develop a therapy that will allow for further decrease in HBsAg levels, even down to complete absence, especially in those patients whose HBV DNA has been successfully restrained. TCM, with the Yin-Yang theory, pays attention to reinforcing the vital essence and strengthening the primordial qi. It is no doubt that TCM is very suitable for providing subsequent therapy to those patients who have successfully achieved suppression of HBV replication but who still have high HBsAg titers.
HBsAg titers change with development of the natural history in CHB [
As shown in Figure
HBsAg is not only a pathogenic factor and its titers also are affected by functions of human organs. Thus, the general mechanisms underlying the connection between HBsAg and TCMS may be two-sided. As far as patients are concerned, an abnormal state of physiological functions or substances not only provides opportunities for HBV infection, but also affects the process and outcome of the HBV infection, obviously including the production of HBsAg. Conversely, HBV will disturb and harm the physiological functions and substances of patients since HBV can integrate into the host genome [
The different HBsAg titers also represent different phases or stages of CHB. However, the detailed theories of the interactions among HBV, its markers, and TCMS are not clear. TCMS has its own objective bases and essence [
As a whole, we found positive correlation among the levels of HBsAg, HBV DNA, and HBeAg. Moreover, this correlation existed in SKD and LQD, but not in LGDH and BSBC. Integrating different HBsAg levels in each pattern of TCMS, the present general results suggest that differences in the pattern of TCMS might result from the variation of virological features and viral life cycle during the natural history of CHB. We could not, however, exclude the influence of inherent constitutions in patients. In future research a matched-pair study should be considered, wherein each selected case will be compared with a healthy volunteer without HBV infection.
Our main objective has been achieved in this study. Our findings support the hypothesis that TCMSs vary and associate with HBsAg throughout CHB. There is, however, a particular shortcoming of this study that must be considered when interpreting these findings—the insufficiency of sample size in some phases, specifically the RAP and LC phases, which precluded our ability to analyze the connection of HBsAg and TCMS in each different phase of CHB separately. In order to obtain more powerful evidence to more definitively identify HBsAg, HBeAg, and HBV DNA as indicators of TCMS differentiation, prospective studies are now necessary, as they will allow for assessments of the associations between HBsAg and TCMS in every phase or stage of CHB.
In conclusion, this study found significant differences of TCMS patterns throughout the natural history of CHB. The findings from this study have significant implications for understanding the natural history of CHB from the TCM perspective and might provide the theoretical joints for combining Chinese and Western medicines. Furthermore, HBsAg titers were found to vary among different TCMS patterns of CHB patients. Discovering the association of HBsAg titers and TCMS patterns contributes to our overall understanding the HBV viral life cycle and interaction between pathogenic factors (HBV and HBsAg) and vital essence in the TCM perspective. Knowing baseline HBsAg levels in different TCMS patterns may provide further insight into the therapy of CHB, integrating Chinese and Western medicines.
The data used to support the findings of this study are available from the corresponding author upon request.
All authors declared that there are no conflicts of interest to disclose.
He-Ping Xie and Zhi-Ping Liu contributed equally to this work.
The authors are grateful to Da-Qiao Zhou in Shenzhen Hospital affiliated to Guangzhou University of Chinese Medicine and to the researchers in the Third Affiliated Hospital of Sun Yat-sen University for their help. This work was supported by grants from the National Major Projects for AIDS and Viral Hepatitis of China (no. 2008ZX10005-008), the Guangdong Provincial Administration of Traditional Chinese Medicine (no. 20131142), the National Key Department of Traditional Chinese Medicine (no. ZK1901GB039), and the Guangdong Provincial Traditional Chinese Medicine Heritage Studio Construction Project (no. G00148).
In order to investigate traditional Chinese medicine syndrome (TCMS) patterns and their association with hepatitis B surface antigen (HBsAg) levels during the natural history of chronic hepatitis B virus infection (CHB), patients were categorized according to the disease phase or stage of CHB, as follows: immune tolerance (ITP); immune clearance (ICP); low or nonreplication (LRP); reactivation (RAP); hepatic cirrhosis (HC); and primary liver cancer (PLC). The patients’ TCMS patterns were classified among the following types: spleen-kidney deficiency (SKD); liver-qi depression (LQD); damp-heat in liver-gallbladder (LGDH); liver-kidney deficiency (LKD); and blood stasis blocking collateral (BSBC). HBsAg levels and other serological indicators were quantified for all patients. All clinic data were recorded in the supplementary materials.