Kushenin Combined with Nucleos(t)ide Analogues for Chronic Hepatitis B: A Systematic Review and Meta-Analysis

Objective. To evaluate the efficacy and safety of Kushenin (KS) combined with nucleoside analogues (NAs) for chronic hepatitis B (CHB). Methods. Randomized controlled trials (RCTs) of KS combined with NAs for CHB were identified through 7 databases. Frequencies of loss of serum HBeAg, HBeAg seroconversion, undetectable serum HBV-DNA, ALT normalization, and adverse events at 48 weeks were abstracted by two reviewers. The Cochrane software was performed to assess the risk of bias in the included trials. Data were analyzed with Review Manager 5.3 software. Results. 18 RCTs involving 1684 subjects with CHB were included in the analysis. KS combined with NAs including lamivudine (LAM), entecavir (ETV), adefovir dipivoxil (ADV), and telbivudine (TLV) showed different degree of improvement in CHB indices. KS combined with NAs increased the frequency of loss of serum HBeAg, HBeAg seroconversion, undetectable HBV-DNA levels, and ALT normalization compared with single agents. It also decreased serum ALT and AST level after one-year treatment. However, KS combined with TLV did not show a significant difference in CHB indices. The side-effects of KS combined with NAs were light and of low frequency. Conclusion. KS combined with NAs improves the efficacy of NAs in CHB.


Introduction
Chronic hepatitis B (CHB) is a global health problem with an estimated 400 billion people worldwide being chronically infected with the virus (HBV) [1]. Chronic infection with HBV can significantly impair the quality of life and life expectancy of patients. Disease progression can lead to fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma [2]. Eventually, approximately 25% of the infected patients will die of the liver-related complications if untreated [3]. There is a high frequency of chronic hepatitis B virus infection in China. Approximately 60% of the population have a history of HBV infection. An estimated 7%-10% of people are chronically infected with HBV and are at increased risk of premature death from liver diseases [4].
Currently, available treatments include interferon-alfa, nucleos(t)ide analogue polymerase inhibitors, such as lamivudine (LAM), adefovir (ADV), entecavir (ETV), telbivudine, and tenofovir (TLV), used alone or in combination with 2 or more agents [3]. Interferon is used as a short-term treatment that, if successful, may lead to long-term immune control without the need for further antiviral therapy [5]. Nucleos(t)ide analogues (NAs) directly inhibit HBV reversetranscriptase polymerase. However, with such antiviral therapy HBV replication increases markedly as soon as the treatment is stopped [6]. Furthermore, with long-term use viral 2 Evidence-Based Complementary and Alternative Medicine resistance often emerges and may eventually create serious clinical problems [7]. Therefore, strategies for enhancing the efficacy and safety of these agents are key aspects of CHB treatment.
Traditional Chinese medicine (TCM), one of the complementary and alternative therapies, is often used in cholestasis treatment in China [8]. The Yellow Emperor's Internal Classic, an ancient book containing records of TCM, indicates that TCM was used to treat chronic liver disease in China since 475 BCE at least [9]. The objectives of TCM treatment of CHB are to (i) relieve anxiety and symptoms and hence improve the quality of life of the patients, (ii) alleviate inflammation, (iii) arrest hepatic fibrosis, (iv) improve immune function, and (v) improve lipid metabolism [9].
Kushenin (KS) is a mixture of the alkaloids oxymatrine (the main content > 98%) and oxysophocarpine, extracted from the root of Sophora flavescens Alt. (Kushen in Chinese). Evidence-backed studies reveal its effectiveness in treating hepatocyte injury, chronic hepatitis B, liver fibrosis, and tissue inflammation [10]. It has been reported that a singletherapy treatment may result in the emergence of both viral drug-resistance and dose-dependent side-effects [11]. Under this circumstance, some physicians have attempted to use combinations of TCM with LAM to enhance its curative effect. Recently, we note that the clinical combination of KS with LAM could improve curative effect with regard to HBV infection [12][13][14][15][16][17]. It has been reported that the combination of KS with LAM could significantly increase the negative conversion rate of HBV-DNA and hepatitis B e antigen (HBeAg) in patients compared with LAM treatment. It has also been reported that KS could decrease the development of drug resistance to LAM [10].
Nowadays, the combinations of KS with NAs for CHB treatment have attracted more and more attention. It demonstrates a promising therapy of the combinations of KS with NAs for CHB treatment in clinic. Therefore, this metaanalysis of RCTs was conducted to assess the clinical value of KS combined with NAs for the treatment of CHB, which provides a possible complementary and alternative therapy for global application.

Search Strategy. Comprehensive searches of English and
Chinese databases were performed by two researchers. The databases included PubMed, Embase, Cochrane Library, Chinese Biomedical Database (CBM), Wanfang, VIP medicine information system (VMIS), and China National Knowledge Infrastructure (CNKI), and the dates ranged from the establishment of the different databases through 2015. Search terms included Kushenin, chronic hepatitis B, HBV, CHB, and randomized controlled trial. The first author, year of publication, title, and journal name of the articles were recorded for further screening.

Inclusion
Criteria. The inclusion criteria were as follows. (1) There are randomized controlled trials (RCTs).
(2) The diagnostic criteria for CHB were serum hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) positive for more than 6 months, with elevating levels of serum alanine aminotransferase (ALT). (3) Studies were selected for analysis if there was an objective outcome, including ALT normalization, loss of serum HBeAg, and/or undetectable serum HBV-DNA. (4) Intervention therapies with combinations of KS with NAs or single application were included.

Exclusion Criteria.
The exclusion criteria were as follows.
(1) There are studies of patients who were coinfected with HIV, HCV, or HDV. (2) Patients did not present any severe complications, such as hepatic failure and cirrhosis. (3) Studies did not report any efficacy measures or not conveying sufficient statistical information. (4) There are studies without equal baseline or endpoint of outcome measure.

Data Extraction and Risk of Bias
Assessment. Data extraction and quality assessment were independently performed by two researchers and disagreements were resolved by consensus. Detailed data such as data source, eligibility, methods, participants, interventions, and study results were imported into Cochrane Review Manager 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen, 2014) for further analysis.
The Cochrane risk of bias tool was used to assess the methodological quality of included RCTs. The six domains of this tool include random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome data (attrition bias), incomplete outcome data (attrition bias), and selective reporting (reporting bias). The judgment was marked as "high risk," "unclear risk," or "low risk." Trials that met all the criteria were categorized as low risk of bias, whereas those that met none were categorized as high risk of bias. The others were classified as unclear risk of bias if the information was insufficient to make a judgment.

Data Analysis. Statistical analysis was performed by
Cochrane Review Manager 5.3 (The Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen, 2014). Dichotomous data were presented as odds ratio (OR) and continuous variables as mean difference (MD), with 95% confidence intervals (95% CI). Statistical heterogeneity was assessed by Cochrane's test. Only data with low heterogeneity ( ≥ 0.10 and 2 ≤ 50%) were assessed as a fixed-effects model whereas others were assessed as a random-effects model. A funnel plot was used for assessing the potential publication bias.  were included in this meta-analysis ( Figure 1). There was no significant difference in ages, sex, and course of disease between two groups. Of these 18 articles, 6 were treated with combination of KS and LAM, 4 were treated with combination of KS and ETV, 5 were treated with combination of KS and ADV, and 3 were treated with combination of KS and TLV. Seven of 18 studies were reported with slight adverse events (Table 1).

Undetectable Serum HBV-DNA Rate.
In order to explore the effect on undetectable serum HBV-DNA rate, we studied the combination of KS with four frequently used anti-HBV agents (LAM, ETV, ADV, and TLV). After 48-week treatment, 83.1% of patients in KS + LAM group and 73.5% of patients in LAM group reached undetectable serum HBV-DNA levels, and there was a significant difference between 4 Evidence-Based Complementary and Alternative Medicine   two groups (OR = 1.97, 95% CI = 1.29-3.01; = 0.002). 69.3% of patients in KS + ETV group and 51.5% of patients in ETV group reached undetectable serum HBV-DNA levels, and there was also a significant difference between two groups (OR = 2.38, 95% CI = 1.37-4.12; = 0.002). Moreover, 54.5% of patients in KS + ADV group and 43.5% of patients in ADV group reached the undetectable levels, and KS + ADV group showed a significant effect on undetectable serum HBV-DNA rate at 48 weeks (OR = 1.91, 95% CI = 1.28-2.84; = 0.001), whereas there was no significance in KS + TLV and TLV group (OR = 1.37, 95% CI = 0.70-2.65; = 0.36), which could be caused by small scale of the patients ( Figure 5).

ALT Normalization Rate.
To explore the effect on undetectable serum HBV-DNA rate, we studied the combination of KS with four frequently used anti-HBV agents (LAM, ETV, ADV, and TLV      showed dizziness or fatigue during treatment, but all the symptoms disappeared later. In spite of no difference between trial and control group, further investigation is needed for a systematic safety assessment of combinations of KS with NAs (Table 1).

Discussion
In this study, 18 RCTs involving 891 subjects with CHB were included. The combinations of KS with the main NAs therapy medicine including LAM, ETV, ADV, and TLV were conducted to explore the curative effect. Some CHB indices were selected to represent the curative effect. The loss of serum HBeAg and HBeAg seroconversion rate are considered as indicators of the patients transition to a state of substantially lower HBV replication, which when maintained is likely to be associated with improved long-term clinical outcomes and so are undetectable serum HBV-DNA and ALT normalization rate [31]. The indices of ALT, AST, and TBIL in the research focus more on the degree of liver damage.
In the meta-analysis, KS combined with LAM, ETV, or ADV showed a good curative effect after one-year treatment, which indicated that different improvement of chronic hepatitis B indices included loss of serum HBeAg, HBeAg seroconversion rate, and undetectable serum HBV-DNA. However, KS combined with TLV could enhance the loss of serum HBeAg, HBeAg seroconversion rate, undetectable serum HBV-DNA rate, and ALT normalization rate but did not show a significant difference. Serum ALT and AST level obviously decreased in combination therapy in metaanalysis, which suggested the hepatoprotective effects. This could be caused by small sample size and multicenter and    large-scale randomized trials of KS combined with TLV needed are further carried out. It is still difficult to draw firm conclusions since the studies reviewed here are flawed in some areas. First, the quality of the studies determines the quality of such analyses, and the studies included in this investigation had shortcomings in methodology. Second, the present meta-analysis was based on 18 published RCTs. The relatively small size of the study was a limitation since it restricts statistical power and may explain why some of the changes did not reach statistical significance. Furthermore, only one of the RCTs in this metaanalysis measured follow-up beyond 24 weeks and most of the RCTs did not measure follow-up. Due to this, statistical power of this analysis cannot define the long-term efficacy of combinations of KS with NAs.
Therefore, the methodological quality of clinical trials with KS combined with NAs for chronic hepatitis B needs to be improved. Rigorously designed, large randomized, double  blind, placebo-controlled trials are required to confirm the efficacy of KS combined with NAs in chronic hepatitis B. The outcome measures should include logical changes, liver pathology, and endpoint events. Adverse events should be monitored by a standardized effective reporting system in clinical trials and rare serious adverse events can be observed through epidemiological studies.

Conclusions
The systematic review and meta-analysis indicate that the combinations of KS with NAs not only enhance the indexes of loss of serum HBeAg and HBeAg seroconversion, but also improve the undetectable serum HBV-DNA rate and ALT normalization rate to a certain degree. Meanwhile, there is no obvious adverse effect. In summary, KS combined with NAs could be a beneficial and safe treatment approach for patients to improve the comprehensive efficacy of CHB. Considering being accepted by more and more practitioners, further rigorously designed, multicenter, large-scale trials with higher quality worldwide are required.