Integrative therapy decreases the risk of lupus nephritis in patients with systemic lupus erythematosus: A population-based retrospective cohort study

Author’s Accepted Manuscript Integrative Therapy Decreases the Risk of Lupus Nephritis in Patients with Systemic Lupus Erythematosus: A Population-Based Retrospective Cohort Study Ching-Mao Chang, Po-Chang Wu, Jen-Huai Chiang, Yau-Huei Wei, Fang-Pey Chen, Tzeng-Ji Chen, Tai-Long Pan, Hung-Rong Yen, Hen-Hong Chang

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To appear in: Journal of Ethnopharmacology Received date: 10 July 2016 Revised date: 4 November 2016 Accepted date: 10 December 2016 Cite this article as: Ching-Mao Chang, Po-Chang Wu, Jen-Huai Chiang, YauHuei Wei, Fang-Pey Chen, Tzeng-Ji Chen, Tai-Long Pan, Hung-Rong Yen and Hen-Hong Chang, Integrative Therapy Decreases the Risk of Lupus Nephritis in Patients with Systemic Lupus Erythematosus: A Population-Based Retrospective Cohort Study, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.12.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Integrative Therapy Decreases the Risk of Lupus Nephritis in Patients with Systemic Lupus Erythematosus: A Population-Based Retrospective Cohort Study Ching-Mao Changa,b, Po-Chang Wuc, Jen-Huai Chiangd,e,f, Yau-Huei Weig,h, Fang-Pey Cheni,j, Tzeng-Ji Chenka,l, Tai-Long Panm,n,o, Hung-Rong Yenp,q,r*, Hen-Hong Changp,t,u* a

Center for Traditional Medicine, Taipei Veterans General Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, and Graduate Institute of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan b

c

Division of Rheumatology and Immunology and Department of Education, China Medical University Hospital, Taichung, Taiwan d Management Office for Health Data, China Medical University Hospital, Taichung, Taiwan e

College of Medicine, China Medical University, Taichung, Taiwan Graduate Institute of Integrated Medicine, College of Chinese Medicine, Taichung, Taiwan g Department of Biochemistry and Molecular Biology, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan h Department of Medicine, Mackay Medical College, New Taipei, Taiwan i Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan j Department of Family Medicine, Taipei Veterans General Hospital, Taipei, Taiwan k Institute of Hospital and Health Care Administration, School of Medicine, National Yang-Ming f

University, Taipei, Taiwan l School of Traditional Chinese Medicine, Chang Gung University, Taoyuan, Taiwan m Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan n Liver Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan o Research Center for Chinese Herbal Medicine, and School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan p Research Center for Chinese Medicine & Acupuncture, and School of Post-Baccalaureate Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan q Departments of Chinese Medicine, China Medical University Hospital, Taichung, Taiwan r

Research Center for Traditional Chinese Medicine, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan *

Correspondenceuthor. Prof. Hen-Hong Chang, M.D., Ph.D. School of Post-Baccalaureate Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan No. 91, Hsueh-Shih Road, North District, Taichung 40402, Taiwan, R.O.C Tel.: +886-4-2205-3366 #3001; fax: +886-4-2203-7690; [email protected]

*

Co-correspondence to. Prof. Hung-Rong Yen, M.D., Ph.D. Research Center for Chinese Herbal Medicine, and School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan No. 91, Hsueh-Shih Road, North District, Taichung 40402, Taiwan, R.O.C Tel: +886-4-2205-3366 #3317; fax: +886-4-2236-5141; [email protected] .

Abstract Ethnopharmacological relevance: Evidence on alleviating the risk of lupus nephritis by integrative therapy with conventional medicine (CM) and herbal medicine (HM) had not been addressed. Aim of the study: We investigated the integrative effect associated the risk by a retrospective Systemic Lupus Erythematosus (SLE) cohort from Taiwan National Health Insurance Research Database (NHIRD). Materials and Methods: SLE patients with a catastrophic illness certificate (CIC) were retrospectively enrolled from the SLE cohort of the Taiwan NHIRD between 1997 and 2011. The patients were divided into an integrative medicine (IM: integrated CM plus HM) and a non-IM (CM only) group with 1:1 propensity score matching. Cox proportional regression model and the Kaplan-Meier method were conducted to estimate the hazard ratio (HR) for lupus nephritis in the cohort. Results: Among 16,645 newly diagnosed SLE patients holding a CIC (SLE/CIC), 1,933 had received HM and 1,571 had received no HM treatment. After propensity score matching, there were 273 patients with lupus nephritis-120 in the IM group and 153 in the non-IM group. The adjusted HR (0.68, 95% confidence interval [CI]: 0.54-0.87, p<0.01) for lupus nephritis was lower in the IM group than in the non-IM group. The adjusted HR (0.69, 95% CI: 0.54-0.88, p<0.001) for lupus nephritis was also lower in the group of patients who had received CM plus HM than in the group that received CM only. The core pattern of HM prescriptions, which were integrated with CM for preventing lupus nephritis, was “Sheng-Di-Huang” (raw

Rehmannia glutinosa Libosch.), “Mu-Dan-Pi” (Paeonia suffruticosa Andr.), “Dan-Shan” (Salvia miltiorrhiza Bge.), “Zhi-Bo-Di-Huang-Wan.”, and “Chi-Shao” (Paeoniae lactiflorae Rubra). Conclusion: Integrative therapy decreased the risk of lupus nephritis among SLE patients in Taiwan. Further investigation of the pharmacological mechanism and clinical efficacy are warranted.

Abbreviation: CAM: complementary and alternative medicine; CI: confidence interval; CIC: catastrophic illness certificate; CM: conventional medicine; HM: herbal medicine; HR: hazard ratio; IM: integrative medicine; ICD-9: International Classification of Diseases, Ninth Revision, Clinical Modification; NHI: National Health Insurance; NHIRD: National Health Insurance Research Database; SLE: Systemic lupus erythematosus; TCM: traditional Chinese medicine

Keywords: Complementary and alternative medicine; Herbal medicine; Integrative therapy; lupus nephritis; Systemic Lupus Erythematosus

1. Introduction Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies, which accumulate within tissues and lead to systemic inflammation (Tsokos, 2011). The disease is highly heterogeneous, with individual patients manifesting variable combinations of clinical features. More commonly, these manifestations appear over time as the disease evolves. SLE manifests in the skin, joints, kidneys, and nervous and hematologic systems (Tang et al., 2010); and the pathogenesis of

the condition is thought to involve oxidative stress (Perl, 2013), type I interferon (Crow, 2014), and B/T cell imbalance (Gao et al., 2014; Pernis, 2009; Talaat et al., 2015). Renal involvement is common in SLE and is a significant contribution to morbidity and mortality.(Danila et al., 2009; Tanaka et al., 1993) Deposition of immune complexes in the kidneys is often observed in patients with lupus nephritis, which occurs in 14-55% of patients, with higher rates seen in individuals of Asian, African, and Hispanic lineages than in Caucasians (Huong et al., 1999; Ortega et al., 2010). Medications for the treatment of lupus nephritis include corticosteroids and immunosuppressive drugs (Bertsias et al., 2010), such as cyclophosphamide, azathioprine, and mycophenolate mofetil. However, 10-30% of patients with proliferative lupus nephritis will develop end-stage renal disease despite attempts at optimizing therapy for SLE (Appel et al., 1987). Some SLE patients have a high risk of developing lupus nephritis (Mavragani et al., 2015), and as treatment with conventional medicine (CM), particularly high doses of corticosteroids (Lew et al., 1999) is often accompanied by side-effects, such as fatigue, weight gain, osteoporosis, and cataracts. Therefore, an increasing numbers of SLE patients seek out complementary and alternative medicine (CAM) (Chou, 2010; Greco et al., 2013; Haija and Schulz, 2011) therapies to relieve these symptoms. Some SLE and lupus nephritis patients have received CAM therapy in the form of herbal medicine (HM) (Yap et al., 1999; Zhong et al., 2013), acupuncture (Greco et al., 2008; Mooventhan and Nivethitha, 2014), and moxibustion (Kung et al., 2006). However, few studies have reported the prescription of integrative therapy to SLE patients, and there is little evidence that integrative therapy reduces lupus nephritis and SLE comorbidities. Hence, we

conducted a large population-based retrospective cohort study to evaluate whether integrative therapy combining CM and HM could reduce lupus nephritis among SLE patients in Taiwan.

2. Materials and methods 2.1.Data Sources The mandatory National Health Insurance (NHI) program covers 99% the Taiwanese population and reimburses not only CM but also traditional Chinese medicine (TCM) services-including HM, acupuncture, moxibustion, and manipulative therapy (Lee et al., 2010). The medical claim data including gender, age, dates of visits, disease diagnosis of patients, and prescriptions for all beneficiaries, are recorded in electronic format in the National Health Insurance Research Database (NHIRD) (Cheng and Chiang, 1997), and the details about the structure of claimed visitation files and corresponding prescription files have been described in previous studies (Chang et al., 2015). This study was approved by the Research Ethics Committee of the China Medical University and Hospital (CMUH104-REC2-115). As we conducted this retrospective analysis of the NHIRD and all the individual information were de-identified, we could not obtain informed consent from the included patients. 2.2.Identification of Patients with Systemic Lupus Erythematosus The NHIRD also established a registry for patients with catastrophic illness, including 31 disease categories such as cancer, schizophrenia, rheumatoid arthritis, end-stage renal disease and SLE. SLE patients who received complete clinical and laboratory examinations, followed by careful and routine review under rheumatologists commissioned and the NHI Administration, were granted catastrophic illness

certificates (CIC). The accuracy of the diagnosis of SLE in patients enrolled in the NHIRD is thus highly reliable. We designed a national population-based study to enroll the SLE subjects from the registry of CIC in the Taiwan NHIRD. SLE was identified in individuals with an ICD-9 code of “710.0” who met the American College of Rheumatology revised criteria for the classification and a registry of CIC, and only those with a principal diagnosis of SLE were included in this study. Patients for whom lupus nephritis was diagnosed before SLE were also excluded. Finally, 3,504 subjects with a principal diagnosis of SLE were included in this study from 1997 to 2011. 2.3.Integrative Medicine Group and Non-integrative Medicine Group in Patients with Systemic Lupus Erythematosus The SLE subjects holding a CIC were divided into an IM group (integrated CM plus HM) and a non-IM group (CM only). CM includes prednisolone, methylprednisolone, methotrexate, sulfasalazine, azathioprine, hydroxychloroquine, mycophenolate mofetil or cyclophosphamide. The frequency and patterns of HM usage were analyzed to determine its utilization by subjects with a principal diagnosis of SLE who were treated in TCM outpatient clinics. Using a principal diagnosis of SLE in TCM visits, it was possible to reduce the selection bias that would be imposed by TCM visits for non-SLE treatments. Figure 1 shows the subject recruitment flowchart for SLE patients with a CIC from the NHIRD in Taiwan. 2.4.Outcome Measure of Patients with Systemic Lupus Erythematosus and Lupus Nephritis The outcome measure of patients diagnosed with lupus nephritis (ICD-9: 583.81) should meet the diagnosis of SLE along with clinical or laboratory evidence of active nephritis, such as persistent proteinuria (>500 mg/day), hematuria with red blood cell (RBC) dysmorphism, RBC casts, or consistent

renal biopsy. Lupus nephritis was classified into six stages according to the classification revised by the International Society of Nephrology and the Renal Pathology Society in 2003. 2.5.Propensity Score Matching Propensity score matching was used to select a comparable IM group and non-IM group. We used logistic regression modeling to estimate the propensity score for the predicted probabilities of the IM status. Because nonrandom assignment can lead to selection bias, we performed 1:1 case:control matching based on a propensity score of each subject’s age, gender, urbanization level, comorbidities and CM use to reduce the selection bias. 2.6.Data Analysis Categorical variables were reported as number and percentage and differences in proportions were assessed using a Chi-square test and the t-test was used for continuous variables. Survival was analyzed using a Cox proportional regression model to estimate the hazard ratio (HR) and 95% confidence interval (CI) for lupus nephritis by demographic status, comorbidities, and CM used; these comorbidities were defined on the basis of a previous report (Chiu and Lai, 2010). The Kaplan-Meier method was used to estimate the lupus nephritis-free proportion in the IM group and non-IM group, respectively. Although the method of propensity score matching was conducted in the analysis for SLE subjects, we show the unmatched results in the Supplemental materials. SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyze the frequency and patterns of formulae or single herbs usage and HM utilization per se. In addition, the core patterns of HMs used in treating SLE/CIC patients were identified with an open-sourced freeware NodeXL (http://nodexl.codeplex.com/), as reported previously (Chang et al., 2015). In brief, all the selected

two-drug combinations were applied in this network analysis. Connections between HM and co-prescribed HM were defined as the line width with a width range from 1 to 5 in the network figure; and thicker line width represented stronger prescription patterns. The network analysis revealed the core patterns among the top 100 HM combinations in this survey.

Results There were 24,161 SLE patients with a CIC, including 16,645 newly diagnosed SLE patients; only 1,933 subjects with a principal diagnosis of SLE were treated with HM and 1,571 did not receive any HM treatment (Figure 1). Supplemental materials demonstrates the baseline characteristics, and the crude HR and adjusted HR of lupus nephritis from the enrolled subjects without the propensity score matching were analyzed in the Cox proportional hazard model. After the propensity score analysis, we matched 1,106 SLE patients by age, gender, urbanization level, comorbidities, and CM use in the IM group with those in the non-IM group (Table 1). The study subjects were predominantly women (female:male = 8:1), and there were no significant difference in both groups. Although there were no significant differences between IM and non-IM groups, we still used these factors in the Cox proportional hazard regression, and found that the adjusted HR for lupus nephritis events in the IM group was lower than that for the non-IM group. A total of 273 SLE/CIC subjects were newly diagnosed with lupus nephritis during follow-up, with the events of lupus nephritis were to be 153 in non-IM group and 120 in IM group. Table 2 shows the univariate and multivariate Cox’s proportional hazard model in the cohort undergoing IM treatment vs. the non-IM population, stratified by gender, comorbidities, and CM used during 19972011. The crude HR

and adjusted HR of lupus nephritis were analyzed in the Cox proportional hazard model comparing the IM group and non-IM group. SLE patients receiving IM therapy had a significantly lower risk (adjusted HR: 0.68; 95% CI: 0.540.87, p<0.01) of lupus nephritis compared to non-IM group. Higher adjusted HRs were found in patients with Sjögren’s syndrome (HR: 1.54, 95% CI: 0.455.28), rheumatoid arthritis (HR: 1.28, 95% CI: 0.423.91), and use of methotrexate (HR: 1.52, 95% CI: 0.643.62); however, these factors were all not significantly different between groups. Most of the factors of the comorbidities and CM use had significantly lower adjusted HRs in the IM group. Kaplan-Meier analysis for comparison of the cumulative incidence of lupus nephritis between IM users and non-IM users is shown in Figure 2. The log-rank test showed a higher cumulative incidence of development of lupus nephritis in the non-IM user group than in the IM user group (p<0.001). To compare the individual or integrated effect of HM and CM therapies, we conducted a Cox proportional hazard regression analysis (Table 3). We found a markedly lower HR for lupus nephritis in the combined HM and CM therapy group when compared with those who used only CM (adjusted HR: 0.69, 95% CI: 0.540.88, p<0.001). SLE patients with IM treatment had significant adjusted HR for the risk of lupus nephritis (Table 4). Those who received 30 to 180 days of IM (n=399) had an adjusted HR of 0.49 (95% CI: 0.340.72, p<0.001) for the risk of lupus nephritis, as compared with IM patients who received less than 30 days of IM or non-IM users. The risk of lupus nephritis in IM users who had received more than 6 months’ worth of herbal prescriptions (n=238) was even lower (adjusted HR: 0.47, 95% CI: 0.300.75, p<0.01). As the course of SLE disease progression may affect the development of lupus nephritis, we used a cut-off of a 5-year follow-up period for this purpose. When the follow-up period of SLE disease progression exceeded

5 years, those who had received more than 180 days of IM (n=223) had a lower adjusted HR of 0.46 (95% CI: 0.270.79, p<0.01) than those receiving less than those receiving 30 to 180 days of IM (adjusted HR: 0.58, 95% CI: 0.390.87, p<0.01), or those receiving less than 30 days of IM or non-IM users. The top 10 single herbs and formulae for the treatment of SLE/CIC patients are listed in Table 5 and Table 6. The most commonly used single herb was “Mu-Dan-Pi” (Paeonia suffruticosa, 16.49%), followed by “Dan-Shan” (Salvia miltiorrhiza, 15.04%), “Sheng-Di-Huang” (raw Rehmannia glutinosa,11.55%), “Da-Huang” (Rheum tanguticum, 10.38%), and “Chi-Shao” (Paeoniae lactiflorae, 8.34%). “Jia-Wei-Xiao-Yao-San” (20.99%) was the most commonly used formula, and the following commonly used formulae were “Zhi-Bo-Di-Huang-Wan” (18.89%), “Yu-Nu-Jian” (8.88%), “Liu-Wei-Di-Huang-Wan” (8.57%), and “Gan-Lu-Yin” (8.42%). We also used network analysis to identify the core patterns of formulae and single herbs for treating SLE/CIC patients; Figure 3 demonstrates the composition for the core patterns were “Sheng-Di-Huang” (raw Rehmannia glutinosa Libosch.), “Mu-Dan-Pi” (Paeonia suffruticosa Andr.), “Dan-Shan” (Salvia miltiorrhiza Bge.), “Zhi-Bo-Di-Huang-Wan.”, and “Chi-Shao” (Paeoniae lactiflorae Rubra).

Discussion This is the first nationwide population-based retrospective cohort study to evaluate whether integrative therapy combining CM and HM could reduce the risk of lupus nephritis in patients with SLE in Taiwan. Lin et al. (Lin et al., 2015) reported that HM reduced the risk of end-stage renal disease for patients with chronic kidney disease by 60%, using “dampness-dispelling formulas” or a “harmonizing formula”. Our

study, however, focused on reducing the risk of lupus nephritis in SLE patients, and thus provides evidence in a disease-specific context. Most of our patients were aged around 35 years in both the IM and non-IM groups, consistent with the age of the highest incidence rate in the epidemiologic report for SLE in Taiwan from 2000 to 2007 (Chiu and Lai, 2010). Fluctuating hormone levels and ovarian function are highly associated with SLE disease activity (Shabanova et al., 2008). Hence, females of childbearing age may need more health care with IM, while older SLE patients may have a comparatively more stable condition (Pego-Reigosa et al., 2015). Table 2 indicates that SLE/CIC patients with comorbidities of Sjögren’s syndrome or rheumatoid arthritis who were undergoing IM treatment did not show any significant effect of IM. These two diseases are more complex, and their etiologies are not fully understood (Glocker et al., 2006; Maslinska et al., 2015), which could influence the effects of IM treatment. The effect of IM in SLE/CIC patients with comorbidities of essential hypertension and Raynaud’s syndrome was significant lower; individuals with comorbid Raynaud’s syndrome were less likely to have nephritis and nephrotic syndrome (Dimant et al., 1979; Lavras Costallat and Valente Coimbra, 1995), Some HMs can inhibit the production of endothelin-1 and have a prolonged anti-vasoconstrictive effect (Cho et al., 2014), which can reduce inflammation. SLE/CIC patients with comorbid essential hypertension had a high risk for developing lupus nephritis (Costenbader et al., 2011; Fanouriakis et al., 2012), but hydroxychloroquine may reduce hypertension along with ameliorating endothelial dysfunction (Gomez-Guzman et al., 2014). HM not combined with any CM for the treatment of SLE could reduce the risk of lupus nephritis; patients receiving most of these CMs were at a slightly higher risk, but the risk was still lower when HM and CM therapies were integrated. Therefore, HM combined with prednisolone, methylprednisolone, or

hydroxychloroquine could potentially reduce the risk of lupus nephritis. This demonstrated that these CMs may not exert protective effects in our patients, but actually serve as high-risk surrogates for lupus nephritis (Chan, 2015). There was a lower HR for lupus nephritis in patients under combined HM and CM than in patients using neither HM nor CM. This may mean that SLE patients who willingly receive multiple therapies may have a reduced lupus nephritis risk; which may also mean that integrative therapies combined HM and CM may play an important role in preventing lupus nephritis in SLE patients (Chou, 2010; Greco et al., 2013; Haija and Schulz, 2011; Yap et al., 1999; Zhong et al., 2013). Most of the top 10 formulae and single herbs that were found to be commonly prescribed in this study had antioxidant, anti-inflammatory, and immune-modulating effects. But the core pattern, as determined by network analysis, was “Sheng-Di-Huang”, “Mu-Dan-Pi”, “Dan-Shan”, “Zhi-Bo-Di-Huang-Wan”, and “Chi-Shao”, which may not only have antioxidant, anti-inflammatory, and immune-modulatory functions, but may also protect against epithelial injury and result in nephritis improvement. “Sheng-Di-Huang” (raw Rehmannia glutinosa) can reduce the interaction between eosinophil cationic protein and epithelial cells for anti- inflammation,(Chang et al., 2010) enhance endothelial progenitor cells, proliferation and migration for protecting against epithelial injury (Wang, Y.B. et al., 2013), and result in proteinuria improvement (Qiu et al., 2014). “Mu-Dan-Pi” (Paeonia suffruticosa) can inhibit the iNOS signaling pathways (Chen, C. et al., 2015) for antioxidant capacity and anti-inflammatory effects, and regulate the inflammation and immune response-related genes for immune-modulation (Yun et al., 2013). “Chi-Shao” (Paeoniae lactiflorae) has the effect on reducing NO and MDA for antioxidant capacity (Zhao et al., 2013), and Cyr61 expression down-regulation for alleviating inflammation (Li et al., 2015). “Dan-Shan” (Salvia miltiorrhiza) can enhance the activities about CAT, SOD and GPX for antioxidant

capacity (Chung et al., 2006), anti-inflammatory capacity (Ji et al., 2010), and promote macrophage synthesis of C4 for immune-modulation (Ling et al., 1993), while it also protects against epithelial injury (Ji et al., 2010) and improve nephritis (Zhang et al., 1997). There is no clear evidence of the effects of “Zhi-Bo-Di-Huang-Wan”, which is derived from “Liu-Wei-Di-Huang-Wan”. “Liu-Wei-Di-Huang-Wan” also has antioxidant capacity by increasing SOD and GSH (Tseng et al., 2014), down-regulates the mRNA expression of caspase-3, capase-9 and RANKL for anti-inflammation (Li, M. et al., 2010), and enriches neuroendocrine-immune pathways for immune-modulatory effects (Li, S. et al., 2010), thus resulting in nephritis improvement (Hsu et al., 2014). Moreover, “Zhi-Bo-Di-Huang-Wan” is composed of “Liu-Wei-Di-Huang-Wan”, “Zhi-Mu” (Anemarrhena asphodeloides), and “Huang-Bo” (Phellodendron chinense). “Zhi-Mu” has the effects on scavenging oxidative stress (Chae et al., 2011), regulating AMPK activation for anti-inflammation (Zhao et al., 2014), and immune-modulation (Kiyohara et al., 2013). “Huang-Bo” can inhibit the mRNA levels of TNF-α, IL-1β, IL-6 and COX-2 for anti-inflammation (Xian et al., 2011). The herb pair of “Zhi-Mu” plus “Huang-Bo” can regulate the Akt/mTOR/FoxO3 signaling pathways (Zhang et al., 2014), which may have anti-inflammatory functions. In our previous study (Chang et al., 2015), we analyzed the core pattern of HM prescriptions for Sjögren’s syndrome with network analysis, and it was found to be consistent with the most commonly used prescriptions. While in the present study, even though “Jia-Wei-Xiao-Yao-San” was the most commonly used formula, it was not highlighted in the core pattern prescriptions for SLE. Furthermore, “Jia-Wei-Xiao-Yao-San” was also the top most commonly used formula in the total statistics of the NHIRD (Hsieh et al., 2008), given its broad spectrum therapeutic indications such as sleep disorder, psychosomatic disorder (Chen, Y.L. et al., 2015), and menopausal syndrome (Yang et al., 2009), which has

a high comorbidity with SLE. In the last 10 years, studies on the Taiwan NHIRD tended to investigate the frequency of prescription usage for various diseases; yet these results may not reflect the core pattern and, more importantly, therapeutic prescriptions. Hence, the results may mislead subsequent basic research and clinical trials if the researchers simply used these data. Data mining using network analysis in this study could explore the infrastructure of the clinical logic and group recognition among TCM doctors, and this type of evidence-based approach is required for future HM studies. Total 1,933 SLE patients in IM group and 1,571 in non-IM group were revealed before propensity score matching, although many characteristics had significant differences in the baseline, these unmatched populations still conducted a significant lower HR for lupus nephritis events in the IM group when compared with non-IM group. The method of propensity score matching could decrease the selection bias in the baseline, but it may also reduce the sample size in the control and case group (each 1106 subjects), which resulted in reducing the number of SLE patients from 3,504 to 2,212 and the number of lupus events from 475 to 273. This kind of method may influence the outcomes when the study subjects were totally enrolled. Some new methods may need to develop for resolving this problem about initial characteristic difference among the entire study subjects in the future. However, there are some limitations to this study. (1) SLE patients with CIC are one of the criteria in this study. The patients without CIC were included and indicated the minor activity to develop lupus nephritis. Therefore, the integrative effects in lupus nephritis prevention may be underestimated if they were not included. (2) Although we conducted the propensity score matching analysis and adjusted for the variables in the Cox proportional hazard regression, some potential confounding factors that were not recorded in the NHIRD (such as life style, health-promoting behaviors, or environmental factors etc.) may

have influenced our findings. (3) Only the utilization of HM was investigated in this study, but the utilization of acupuncture, tuina, or other CAMs were rarely applied for SLE treatment in Taiwan. This should not have any impact on our risk estimation for lupus nephritis. (4) Because of our results were generated from the NHIRD, which was constructed by the following systems including: A. Relatively consistent medicine from a TCM pharmaceutical factory with a good manufacturing practice certificate. B. Clinical guideline, continuing medical education, and peer review system of TCM doctors’ association and TCM teaching hospitals/departments. C. A reporting system of HM adverse drug reactions. The aforementioned findings may not be easily inferred to other regions where TCM or CAM is not administrated under these guidelines.

Conclusions The first nationwide population-based retrospective cohort study in Taiwan shows the risk of lupus nephritis in SLE could be reduced by the integrative therapy with CM and the core HM pattern prescriptions “Sheng-Di-Huang” (raw Rehmannia glutinosa), “Mu-Dan-Pi” (Paeonia suffruticosa), “Dan-Shan” (Salvia miltiorrhiza), “Zhi-Bo-Di-Huang-Wan”, and “Chi-Shao” (Paeoniae lactiflorae). The adjusted HR in the IM group was lower than that of the non-IM group, which indicated the protection of lupus nephritis from SLE patients. Hence, HM integrated with prednisolone, methylprednisolone, or hydroxychloroquine may have potential protections for lupus nephritis. However, the therapeutic effects and safety of these integrative HMs for treating SLE should be clarified and the clinical trials should also be conducted in the future.

Ethics approval This study was approved by the Research Ethics Committee of the China Medical University and Hospital (CMUH104-REC2-115)

Author’s contributions CMC, HRY, and HHC were responsible for study concept and design, modification of study design, and review and interpretation of data. CMC, HRY, and HHC were also responsible for drafting the manuscript. PCW, JHC, and YHW provided modifications of the study design and revised the manuscript. FPC, TJC, and TLP contributed to collection, analysis, and interpretation of data and revised the manuscript. All authors read and approved the final manuscript.

Conflict of interests The authors declare that they have no competing interests.

Ethics approval This study was approved by the Research Ethics Committee of the China Medical University and Hospital (CMUH104-REC2-115)

Acknowledgments This study utilized data that were extracted from the National Health Insurance Research Database provided by the Bureau of National Health Insurance, Ministry of Health and Welfare, Taiwan. This study

was supported by the National Research Program for Biopharmaceuticals (NRPB), Ministry of Science and Technology (MOST-104-2325-B-039-009, MOST-105-2325-B-039-006), and China Medical University under the Aim for the Top University Plan of the Ministry of Education, Taiwan. This study is supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212-133019), China Medical University Hospital, Academia Sinica Taiwan Biobank Stroke Biosignature Project (BM10501010037), NRPB Stroke Clinical Trial Consortium (MOST 105-2325-B-039-003), Tseng-Lien Lin Foundation, Taichung, Taiwan, Taiwan Brain DiseaseFoundation, Taipei, Taiwan, and Katsuzo and Kiyo AoshimaMemorial Funds, Japan.

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Fig 1. Flowchart of the recruitment of patients for systemic lupus erythematosus with a catastrophic illness certificate in Taiwan from year 1997 to 2011. Fig 2. The estimated cumulative incidence of lupus nephritis between the IM user and non-IM user from matched systemic lupus erythematosus cohort by Kaplan-Meier analysis. Fig 3. The core pattern in top 100 combinations of formulae and single herbs for the treatment of matched systemic lupus erythematosus.

Table 1. Characteristics of systemic lupus erythematosus patients according to use of integrative medicine with propensity score matching. Patients with SLE (N=2212)

Gender Women Men

Integrative medicine (IM) No (N=1106) Yes (N=1106) p-value N % N % 0.892 983 88.88 985 89.06 123 11.12 121 10.94

Age Mean ± SD a (years)

35.44 ± 16.87 35.01 ± 14.39 0.5128

Urbanization level 1 (highest) 2 3 4 (lowest) Comorbidities Sjögren's syndrome Essential hypertension Raynaud’s syndrome Rheumatoid arthritis Conventional medicine use

0.2 354 331

32.01 29.93

350 373

31.65 33.73

193 228

17.45 20.61

168 215

15.19 19.44

105 87 51 115

9.49 7.87 4.61 10.4

96 78 50 115

8.68 7.05 4.52 10.4

0.5056 0.4664 0.9189 0.99

Prednisolone Methylprednisolone Methotrexate

1012 91.5 358 32.37 141 12.75

1017 91.95 354 32.01 159 14.38

0.6996 0.8556 0.2637

Sulfasalazine Azathioprine Hydroxychloroquine Mycophenolate mofetil Cyclophosphamide

71 498 997 31 215

79 495 990 33 232

0.4987 0.898 0.6225 0.7997 0.368

6.42 45.03 90.14 2.8 19.44

7.14 44.76 89.51 2.98 20.98

Chi-Square Test, a t-test †

: The urbanization level was categorized by the population density of the residential area into 4 levels,

with level 1 as the most urbanized and level 4 as the least urbanized.

Table 2. Incidence rates and hazard ratio of lupus nephritis for matched systemic lupus erythematosus patients with/without integrative medicine stratified by demographic, comorbidities and conventional medicine usage. Risk of Lupus nephritis Adjusted HR (95%CI) Variables

IM Not IM Favor Favor

Total

Non-IM (N=1106)

IM (N=1106 )

Even

Eve

t 153

IR 20. 38

nt 120

IR

Crude HR

Adjusted HR

(95%CI)

(95%CI)

13.6 0.66(0.52-0. 0.68(0.54-0. 8 84)*** 87)**

Gender Female

137

Male

16

20. 4 20. 2

13.1 2 18.4 17 5

0.64(0.5-0.8 3)*** 0.76(0.38-1. 53)

0.68(0.52-0. 88)** 0.76(0.36-1. 62)

13.9 5 10.4 7 5

0.63(0.5-0.8 1)*** 1.72(0.53-5. 53)

0.67(0.52-0. 86)** 1.54(0.45-5. 28)

103

Comorbidities Sjögren's syndrome No

147

Yes

6

Essential hypertension No

21. 6 8.5 3

144 20.

113

119 14.4 0.69(0.54-0. 0.71(0.56-0.

Yes

9

49 18. 7

1

88)** 91)** 0.13(0.02-1. 0.07(0.01-0. 1 1.96 03) 87)*

Raynaud’s syndrome No

147

Yes

6

20. 6 16. 08

14.0 0.67(0.52-0. 0.70(0.55-0. 2 85)** 89)** 0.39(0.08-1. 0.20(0.02-1. 2 5.64 94) 98)

118

Rheumatoid arthritis No

147

21. 98

109

13.7 0.62(0.48-0. 0.67(0.52-0. 3 79)*** 86)**

Yes

6

7.3 2

11

13.2 1.91(0.71-5. 1.28(0.42-3. 5 19) 91)

Conventional medicine use Prednisolone 23. 85 20. 142 15

13.4 0.41(0.13-1. 0.5(0.2-1.24) 3 3) 0.68(0.53-0. 0.70(0.54-0. 112 13.7 87)** 9)**

No

115

24

94

Yes

38

13. 99

No Yes

11

8

Methylpredniso lone 16.5 0.68(0.52-0. 0.71(0.54-0. 2 89)** 94)* 0.6(0.36-0.9 0.58(0.35-0. 26 8.44 8)* 97)*

Methotrexate No

145

Yes

8

22. 88 6.8 4

13.6 6 13.8 20 1

100

0.59(0.46-0. 76)*** 1.85(0.81-4. 24)

0.63(0.48-0. 81)*** 1.52(0.64-3. 62)

Sulfasalazine No

149

Yes

4

21. 64 6.4 3

14.3 0.65(0.51-0. 0.67(0.52-0. 3 83)*** 86)** 1.04(0.28-3. 0.80(0.19-3. 5 6.72 87) 31)

115

Azathioprine No

71

17. 96

49

10.4 0.60(0.42-0. 0.69(0.47-1. 8 87)** 01)

Yes

82

23. 07

71

17.3 0.72(0.53-1) 0.75(0.54-1. 4 * 03)

Hydroxychloro quine 37. 01 18. 128 73

No

25

Yes

17.7 7 13.2 105 5 15

0.63(0.33-1. 22) 0.67(0.52-0. 87)**

0.58(0.28-1. 19) 0.70(0.54-0. 91)**

Mycophenolate mofetil No

146

Yes

7

20. 03 31. 99

114 6

13.3 0.66(0.51-0. 0.68(0.53-0. 7 84)*** 87)** 24.6 1.06(0.32-3. 0.52(0.11-2. 1

5)

58)

0.65(0.49-0. 87)** 0.71(0.47-1. 08)

0.66(0.49-0. 89)** 0.73(0.47-1. 13)

Cyclophospha mide No

103

Yes

50

17. 66 29. 85

11.7 9 20.1 40 3 80

IM Not IM Favor Favor

Abbreviations: IR: incidence rates, per 1,000 person-years; HR: hazard ratio; CI: confidence interval Crude HR: represented relative hazard ratio; Adjusted HR: represented adjusted hazard ratio: mutually adjusted for IM use, age, gender, urbanization level, Sjögren's syndrome, essential hypertension, Raynaud’s syndrome, rheumatoid arthritis and conventional medicine use in Cox proportional hazard regression. The crude HR and adjusted HR were analyzed under IM treatment in the Cox proportional hazard model. *:<0.05; **:<0.01; *** p<0.001 Table 3. Cox proportional hazard regression analysis for the lupus nephritis risk – associated integrative therapy of conventional medicine and herbal medicine among matched systemic lupus erythematosus patients Treatment

N

HM CM No No 17 Yes No 15 No Yes 1089 Yes Yes 1091

Lupus nephritis

Adjusted HR¶

Adjusted HR¶

(N=273)

(95% CI)

(95% CI)

5 2 148 118

1(reference) 0.11(0.02-0.59)** 0.09(0.03-0.21)*** 1(reference) 0.06(0.02-0.15)*** 0.69 (0.54-0.88)**

Abbreviations: HR: hazard ratio; CI: confidence interval

Risk of Lupus nephritis Adjusted HR (95%CI) IM Favor

Not IM Favor

¶Adjusted HR represented adjusted hazard ratio: mutually adjusted for age, gender, urbanization level, Sjögren's syndrome, essential hypertension, Raynaud’s syndrome and rheumatoid arthritis in Cox proportional hazard regression. Conventional medicine included prednisolone, methylprednisolone, methotrexate, sulfasalazine, azathioprine, hydroxychloroquine, mycophenolate mofetil and cyclophosphamide. *:<0.05; **:<0.01; *** p<0.001 Table 4. Hazard ratios and 95% confidence intervals of lupus nephritis risk associated with cumulative use day of integrative therapy combining conventional medicine and herbal medicine among matched systemic lupus erythematosus patients Lupus SLE nephritis (N=2212) (N=273) Non-IM users or IM users<30days

Hazard Ratio(95% CI) Crude

Adjusted†

1575

221

1(reference)

1(reference)

399 238

32 20

0.51(0.35-0.74)*** 0.49(0.34-0.72)*** 0.46(0.29-0.73)*** 0.47(0.30-0.75)**

1514

61

1(reference)

1(reference)

395 233

4 5

0.24(0.09-0.67)** 0.48(0.19-1.20)

0.24(0.09-0.65)** 0.52(0.21-1.29)

1415

160

1(reference)

1(reference)

371 223

28 15

0.61(0.41-0.91)* 0.46(0.27-0.78)**

0.58(0.39-0.87)** 0.46(0.27-0.79)**

IM users(≥30 days) 30-180 days >180 days <5year Non-IM users or IM users<30days 30-180 days >180 days ≥5 year Non-IM users or IM users<30days

Crude HR＊

30-180 days >180 days represented relative hazard ratio.

Adjusted HR† represented adjusted hazard ratio: mutually adjusted for age, gender, urbanization level, Sjögren's syndrome, essential hypertension, Raynaud’s syndrome and CM use in Cox proportional hazard regression. *p < 0.05, **p < 0.01, ***p < 0.001

Table 5. The top 10 single herbs of matched systemic lupus erythematosus patients for preventing the lupus events in Taiwan

Single herb

Ingredients

Therapeutic actions and indications

Average Frequency daily dose N (%) (g) (Mean ±

SD) Mu-Dan-Pi

Paeonia suffruticosa Clear heat and cool the blood Andr. A (Chen, C. et al., 2015; Nuong et al., 2014), B

(Chen, C. et al., 2015; Nuong et al., 2014) and C (Yun et al., 2013) Dan-Shan Salvia miltiorrhiza Clear heat, quicken the blood and transform stasis Bge. A (Chung et al., 2006; Ji et al., 2010), B (Ji et al., 2010), C (Gao et al., 2012; Ling et al., 1993), D (Ji et al., 2010) and E (Zhang et al., 1997) Sheng-Di-Huang raw Rehmannia Enrich yin and clear heat glutinosa Libosch. B (Chang et al., 2010), D (Wang, Y.B. et al., 2013) and E (Qiu et al., 2013; Qiu et al., 2014) Da-Huang Rheum tanguticum Clear heat and drain fire Maxim. ex Balf.

Chi-Shao Huang-Qin

Huang-Qi

A (Liu, L.N. et al., 2005), B (Chang et al., 2010; Liu et al., 2008), C (Liu, L. et al., 2005; Liu et al., 2003; Liu et al., 2012), D (Liu, L.N. et al., 2005) and E

(Hu et al., 2013) Paeoniae lactiflorae Clear heat and cool the blood Rubra A (Zhao et al., 2013) and B (Li et al., 2015) Scutellaria Clear heat and drain fire baicalensis Georgi A (Wang, S.C. et al., 2013), B (Dong et al., 2015; Kim et al., 2014; Yao et al., 2014), C (Dong et al., 2015; Yao et al., 2014) Astragalus Supplement qi and secure the exterior

2305 (16.49)

1.31 ± 0.48

2103 (15.04)

1.40 ± 0.61

1615 (11.55)

1.55 ± 0.70

1451

0.65 ± 0.51

(10.38)

1166 (8.34) 1.28 ± 0.51 1149 (8.22) 1.17 ± 0.65

1122 (8.02) 1.37 ± 0.65

membranaceus (Fisch.) Bge.

Zhi-Gan-Cao

Ye-Jiao-Teng

Xuan-Shen

A (Chen et al., 2011; Guan et al., 2015), B (Li et al., 2007), C (Chen et al., 2014; Yang et al., 2013), D (Qin et al., 2015) and E (Ahmed et al., 2007; Zhang et al., 2009) Glycyrrhiza glabra Fortify the spleen and boost qi 1076 (7.70) 1.14 ± 0.66 L. A (Fukai et al., 2003; Hong et al., 2009), B (Li et al., 2014), C (Bordbar et al., 2012; Li et al., 2014) and E (Fukai et al., 2003) Polygonum Nourish the heart and quiet the spirit 1050 (7.51) 1.26 ± 0.58 multiflorum Thunb. A (Ahn et al., 2015; Lee et al., 2012; Um et al., 2006; Zhang et al., 2012), B (Lee et al., 2012; Zhang et al., 2007) Scrophularia Enrich yin and clear heat 945 (6.76) 1.15 ± 0.43 ningpoensis Hemsl. B (Shen et al., 2012)

A: Antioxidant capacity, B: Anti-inflammation, C: Immune modulation, D: Protect epithelial injury, E: Nephritis improvement

Table 6. The top 10 formulae of matched systemic lupus erythematosus patients for preventing the lupus events in Taiwan Average Formula

Jia-Wei-Xiao-Yao-San

Ingredients

Zhi-Gan-Cao (Glycyrrhiza glabra L.), Dang-Gui (Angelica sinensis (Oliv.) Diels), Fu-Ling (Poria cocos (Schw.) Wolff), Bai-Shao-Yao (Paeonia lactiflora Pall.) , Bai-Zhu (Atractylodes

Therapeutic actions and Frequency indications N (%)

daily dose (g) (Mean ± SD)

Course the liver and 2537 (20.99) 4.63 ± 1.65 resolve depression A (Chien et al., 2014), B (Yasui et al., 2011) and C (Ushiroyama et al., 2004)

macrocephala Koidz.), Chai-Hu (Bupleurum chinense DC.), Sheng-Jiang (Zingiber officinale Rosc.), Bo-He (Mentha haplocalyx Briq.), Mu-Dan-Pi (Paeonia suffruticosa Andr.), Zhi-Zi (Gardenia jasminoides Ellis)

Zhi-Bo-Di-Huang-Wan

Zhi-Mu (Anemarrhena asphodeloides

Enrich yin and clear heat 2283 (18.89) 4.57 ± 1.91

Bunge), Huang-Bo (Phellodendron chinense Schneid.), Shou-Di-Huang (Rehmannia glutinosa Libosch.)

Yu-Nu-Jian

Shi-Gao (Gypsum Fibrosum), Shou-Di-Huang (Rehmannia glutinosa

Clear stomach heat and enrich kidney yin

1073 (8.88) 4.66 ± 1.58

Libosch.), Mai-Men-Dong (Ophiopogon japonicus (L. f.) Ker-Gawl.), Zhi-Mu (Anemarrhena asphodeloides Bunge), Niu-Xi (Achyranthes bidentata Blume)

Liu-Wei-Di-Huang-Wan

Enrich the yin of liver and 1036 (8.57) 4.78 ± 2.36 Libosch.), Shan-Zhu-Yu (Cornus kidney officinalis Sieb. et Zucc.), Shan-Yao A (Sangha et al., 2012; (Dioscorea opposita Thunb.), Ze-Xie Szeto et al., 2009; Tseng (Alisma orientalis (Sam.) Juzep.), Fu-Ling et al., 2014), B (Li, M. et (Poria cocos (Schw.) Wolff), Mu-Dan-Pi al., 2010; Perry et al., Shou-Di-Huang (Rehmannia glutinosa

(Paeonia suffruticosa Andr.)

Gan-Lu-Yin

Sheng-Di-Huang (raw Rehmannia glutinosa Libosch.), Shou-Di-Huang (Rehmannia glutinosa Libosch.),

2014), C (Dong et al., 2010; Fang et al., 2001; Li, S. et al., 2010; Shen et al., 2003) and E (Hsu et al., 2014) Enrich yin and clear heat 1018 (8.42) 4.57 ± 1.88 A (Lin, 2000) and C (Yang et al., 2015)

Tian-Men-Dong (Asparagus cochinchinensis (Lour.) Merr.), Mai-Men-Dong (Ophiopogon japonicus (L. f.) Ker-Gawl.), Shi-Hu (Dendrobium chrysanthum Wall.), Yin-Chen-Hao (Artemisia capillaris Thunb.), Huang-Qin (Scutellaria baicalensis Georgi), Zhi-Ke (Citrus aurantium L.), Pi-Pa-Ye (Eriobotrya japonica (Thunb.) Lindl.), Zhi-Gan-Cao (Glycyrrhiza glabra L.)

Long-Dan-Xie-Gan-Tang

Long-Dan-Cao (Gentiana scabra Bge.), Zhi-Zi (Gardenia jasminoides Ellis), Huang-Qin (Scutellaria baicalensis Georgi), Chai-Hu (Bupleurum chinense DC.), Sheng-Di-Huang (raw Rehmannia glutinosa Libosch.), Ze-Xie (Alisma

Clear damp-heat and 949 (7.85) 4.38 ± 1.71 drain fire A (Lee and Chang, 2010), B (Lee and Chang, 2010) and C (Lee and Chang, 2010)

orientalis (Sam.) Juzep.), Dang-Gui (Angelica sinensis (Oliv.) Diels), Che-Qian-Zi (Plantago asiatica L.), Mu-Tong (Clematis montana Buch.-Ham.), Zhi-Gan-Cao (Glycyrrhiza glabra L.)

Qi-Ju-Di-Huang-Wan

Ju-Hua (Chrysanthemum morifolium (Ramat.) Tzvel.), Gou-Qi (Lycium

Nourish the liver and brighten the eyes

904 (7.48) 4.32 ± 1.75

barbarum L.), Shou-Di-Huang (Rehmannia glutinosa Libosch.), Shan-Zhu-Yu (Cornus officinalis Sieb. et Zucc.), Shan-Yao (Dioscorea opposita Thunb.), Ze-Xie (Alisma orientalis (Sam.) Juzep.), Fu-Ling (Poria cocos (Schw.) Wolff), Mu-Dan-Pi (Paeonia suffruticosa Andr.)

Ma-Zi-Ren-Wan

Ma-Zi-Ren (Cannabis sativa L.), Bai-Shao-Yao (Paeonia lactiflora Pall.),

Moisten the intestines and 858 (7.10) 2.43 ± 1.70 drain fire

Zhi-Shi (Citrus aurantium L.), Da-Huang (Rheum tanguticum Maxim. ex Balf.), Hou-Po (Magnolia officinalis Rehd. et Wils.), Xing-Ren (Prunus armeniaca L.)

Xue-Fu-Zhu-Yu-Decoction Dang-Gui (Angelica sinensis (Oliv.)

Quicken the blood and Diels), Sheng-Di-Huang (raw Rehmannia dispel stasis glutinosa Libosch.), Tao-Ren (Prunus C (Ji et al., 1999) persica (L.) Batsch), Hong-Hua (Carthamus tinctorius L.), Zhi-Ke (Citrus aurantium L.), Chi-Shao (Paeoniae

751 (6.21) 4.07 ± 1.84

lactiflorae Rubra), Chai-Hu (Bupleurum chinense DC.), Zhi-Gan-Cao (Glycyrrhiza glabra L.), Jie-Geng (Platycodon grandiflorum (Jacq.) A. DC.), Chuan-Qiong (Ligusticum chuanxiong Hortorum), Niu-Xi (Achyranthes bidentata Blume)

Dang-Gui-Nian-Tong-Tang Dang-Gui (Angelica sinensis (Oliv.) Diels), Ze-Xie (Alisma orientalis (Sam.) Juzep.), Zhi-Mu (Anemarrhena asphodeloides Bunge), Huang-Qin (Scutellaria baicalensis Georgi), Bai-Zhu (Atractylodes macrocephala Koidz.),

Clear heat and disinhibit dampness B (Chun-juan and Rong, 2010) and C (Chou and Kuo, 1995; Chun-juan and Rong, 2010)

675 (5.59) 4.53 ± 2.10

Zhi-Gan-Cao (Glycyrrhiza glabra L.), Yin-Chen-Hao (Artemisia capillaris Thunb.), Cang-Zhu (Atractylodes lancea (Thunb.) DC.), Qiang-Huo (Notopterygium incisum Ting ex H. T. Chang), Fang-Feng (Saposhnikovia divaricata (Thurcz.) Schischk.), Sheng-Ma (Cimicifuga foetida L.), Ge-Gen (Pueraria lobata (Willd.) Ohwi), Ku-Shan (Sophora flavescens Ait.), Zhu-Ling (Polyporus umbellatus (Pers.) Fries) A: Antioxidant capacity, B: Anti-inflammation, C: Immune modulation, D: Protect epithelial injury, E: Nephritis improvemen

Grpahical Abstract