A Randomized, Placebo-controlled, Double-blind Study on the Effects of SZL on Patients With Mild to Moderate Depressive Disorder With Comparison to Fluoxetine

Objective: Kaixinsan (KXS) decoction, as an ancient’s herbal formula, has been demonstrated to be active in various animal models resembling human depression with multi-target effects. This very rst study evaluated the ecacy and tolerability of Shen Zhi Ling (SZL) tables (KXS preparation), compared with uoxetine (FLX, positive comparator), in patients with mild to moderate depressive disorder. Methods: In this randomized double-blind parallel-group study, 156 patients with mild to moderate depression without taken any antidepressants in the past 6 months or 4 straight weeks were randomized to receive either 3.2g/d SZL plus 20mg/d FLX placebo (SZL group) or 20mg/d FLX plus 3.2g/d SZL placebo (FLX group), for 8 weeks. Their clinical presentations and some metabolic indexes were assessed during the 8 weeks visiting period. Results: Patients in SZL group showed a statistically signicant improvement after 8 weeks of treatment in HAMD-17 score (18.79±2.09 to 4.43±4.71, p<0.001) and self-rating depression scale (SDS) score (58.49±8.89 to 39.84±12.09, p<0.001), but not in N-back total respond time (1145.55±608.26 to 1128.47±387.49, p>0.05). In addition, no signicant difference at 8 weeks of treatment was found between SZL and FLX groups in SDS score (39.84±12.09 vs. 36.63±12.44) and N-back respond time (1128.47±387.49 vs. 1089.43±352.08) as well as reduction of HAMD-17 score (14.79±4.88 vs. 15.24±4.29) (p>0.05 for all). However, the serum APOB, APOC3 and ALB levels and HDL-C/LDL-C ratio decreased signicantly in patients after SZL treatment, while only APOB/APOA1 ratio decreased signicantly in FLX group. Other metabolic indexes did not alter signicantly after treated with SZL or FLX. Conclusion: The ecacy and safety prole of SZL are comparable to that of uoxetine in patients with mild to moderate depression. The benecial effect of SZL is probably associated with improvement of lipid metabolic balance.

hypotheses in this regard. First, cortisol and glucocorticoid receptor (GR) as the main elements of the HPA-axis can increase the risk of hyperlipidemia and play important roles in depression [6,7] . Similar hypothesis that elevated basal cortisol concentrations and lower circadian cortisol variability induce dyslipidemia in patients with depressive or anxiety disorders has also been proposed [8] . Secondly, changes in the lipid composition of cell membrane can modulate the microenvironment and consequently the function of its proteins, e.g. neurotransmission in brain, an organ with the highest lipid concentration in the body. The effect of ω-3 polyunsaturated fatty acids (ω-3-PUFA) in affective disorders attributes to their action on neurotransmission [9] . At last, adipose tissue produced cytokines like IL-6 and TNFα, which activate nuclear factor-kappa B (NF-kB) pathways involving in in ammation, can modulate lipid metabolism, hyperglycemia and depression [5,9] .
Currently, the pharmacotherapy of depression includes antidepressants such as selective serotonin reuptake inhibitors (SSRIs). However, the above agents are associated with multiple adverse effects, such as insomnia, sexual dysfunction, weight gain, diarrhea, constipation, headache, and nausea, and thereby have poor tolerability in depressed patients [10,11] . In addition, as one of SSRIs, uoxetine (FLX) exerts a side effect on lipid index and balance [12,13] . Traditional Chinese medicine is one of the commonly used complementary and alternative medicine therapies for depression [14] . It is especially popular in treatment for some comorbidity of depression and metabolic syndrome.
Shen Zhi Ling (SZL) tablet, a proprietary Chinese medicine formulated from a Kaixinsan (KXS) decoction, is composed of Renshen (Panax Ginseng), Fuling (Poria Cocos), Yuanzhi (Polygala Tenuifolia), and Shichangpu (Acorus Tatarinowii) at a ratio of 3:3:2:2 [15,16,17] . Its treatment has been demonstrated to be active in various animal experimental models resembling some features of human depression [18,19] and a small size clinical study [20] . Researchers have found that KXS has multi-effect targets and could promote synaptogenesis via inducing synaptic protein expressions and increasing nerve growth factor binding in rat brain and improve the feedback control of HPA axis response by restoring corticosterone level in basal and stress conditions [18,21] . In addition, proteomic and metabolomics analysis has indicated that SZL regulates several signal pathways and targets involved in lipid metabolism disorders, reduces the levels of serum TC, TG, free fatty acid (FFA) and apolipoprotein C3 (APOC3), and elevates the level of HDL-C in depression animal models [22,23] .
Based on the above, we, for the rst time, conducted a randomized, double-blind, parallel-group case study to 1) evaluate the e cacy and tolerability of SZL in patients with mild to moderate depressive disorder, 2) compare its effects with positive comparator uoxetine (FLX) and 3) investigate its additional advantages on lipid metabolic index in patients.

Study design
The present trial is a randomized, controlled, intervention, double-blind study with 2 parallel groups including a total of 156 individuals. The study protocol was approved by Ethics Committee of Xijing Hospital of The Fourth Military Medical University of the Chinese people's Liberation Army (Xi'an China, Ethical approval No. YS20150507-4), and written informed consent was obtained from all patients. The eligible patients suffering from mild to moderate depression were equally randomized into the following two groups, SZL Group (SZL plus FLX placebo) vs. FLX group (FLX plus SZL placebo). In observation period of 8 weeks, the therapeutic e cacy was evaluated by HAMD [24] , SDS [25] , and N-back task score. Besides, the serum lipid indexes were also evaluated at baseline and 8 weeks.

Participants recruitment
From December 2015 to April 2018, 180 patients were treated in the Department of Psychosomatic Xijing Hospital of the Fourth Military Medical University and the 261 Hospital of the Chinese people's Liberation Army (PLA Mental Health Center). Patients were required to provide their medical history, receive a physical examination and laboratory safety tests. The inclusion and exclusion criteria are shown in Table 1. All studyrelated information was stored securely with limited access. Laboratory specimens were identi ed by a coded identi cation to maintain participant con dentiality [26] . Table 1 Inclusion and exclusion criteria 3 In accordance with the diagnostic criteria of TCM syndrome of de ciency of heart and spleen; 4 Conform to clinical laboratory tests within the normal range (e.g., blood pressure, heart rate, blood and urine tests, hepatic and renal function tests, and electrocardiograms).
Exclusion criteria 1 History of suicide or any other serious mental illness; 2 Take any antidepressants or lipid-lowering drugs for 4 consecutive weeks or in the past 6 months; 3 Pregnant or lactating women or those planning pregnancy; 4 Active psychotherapy is not allowed before and during the trial; Serious cardiovascular, cerebrovascular, hepatic, renal, or thyroid diseases; Hypersensitivity to uoxetine or herbal compounds; 5 History of addiction or substance abuse; 6 Those who have participated in clinical trials of other drugs within 30 days.

Randomization
Randomization was computer-generated with a 1:1 allocation ratio and a block size range of 4. The drug numbers were obtained in the order from small to large according to the order of joining the group in treatment. In the process of the above experiments, researchers, data managers, statisticians and monitors were all in a blind state. There is no difference in shape, size, texture, color and smell between uoxetine and placebo tablets, SZL and its placebo as well.

Interventions and group assessment
Participants were randomized into SZL and FLX groups on a 1:1 ratio. Based on previous dose response results from preclinical studies and TCM clinical trials [27,28] [29] , the dosage of uoxetine and SZL tablets had been proved as a relatively safe and e cient standard. Patients in SZL groups received 3.2 g/d SZL (0.8 g, BID, No. 141001, provided by Beijing YueKangKaiYue pharmaceutical co. LTD) and 20 mg/d FLX placebo. Patients in FLX groups were given 3.2 g/d SZL placebo and 20 mg/d FLX (10 mg, BID, No. 201410102, provided by ChangZhouSiYao pharmaceutical co. LTD). Both the groups' safety was evaluated by changes in vital signs and adverse events reported by the patient or observed by the psychiatrist during 8 weeks periods.

Clinical Evaluation
All the researchers were trained in the use of diagnostic procedures and rating scales in a standardized manner by the same expert before the study. Most researchers have previously experience in diagnosis of mental illness and have participated in clinical trials of depression disorders.

Primary outcomes
The severity of depressive symptoms was measured by trained psychiatrists using the 17-item HAMD [19] at baseline (week 0), week 4, and week 8. Besides, the curative effect of each treatment stage was analyzed and evaluated in accordance with HAMD. The changes of the HAMD-17 score between baseline and the nal examination at week 8 were speci ed as primary outcome measures of treatment e cacy.

Secondary outcomes
Further standardized scales assessed as secondary e cacy outcome measures included the self-rating depression scale (SDS) as a subjective feelings re ection of the subjects which was mainly applicable to adults with depressive symptoms, and the N-back evaluation, as a cognitive function assessment for the performances of working memory by paradigm a visual letter task with up to six load factors [30] , at the right time line.

Statistical Analysis
Statistical analysis was performed using SAS software version 9.4 (SAS Institute Inc). Generally, differences of continuous variables between groups were assessed by Student's t-test, and differences of continuous variables within a group were compared by paired t-test. Differences of discrete variables were analyzed by Wilcoxon test or chi-squared test. Primary e cacy analysis was carried out with the full analysis set (FAS) comprising all patients randomized with at least one post-baseline e cacy rating (intention-to-treat principle) and per-protocol analysis (PP) was also conducted. The missing data was estimated by the last observed carried forward (LOCF) with at least one post-treatment evaluation [31] . Data are reported as mean ± standard deviation. p < 0.05 was considered statistically signi cant for all analysis.

Baseline data: SZL vs FLX
A total of 156 patients were included and assessed for eligibility. Among them, 144 (SZL 73 vs. FLX 71) were randomized and treated, 129 patients were enrolled in the complete analysis set during treatment and 121 patients in the per-protocol analysis. Figure 1 shows the participants' assessment owchart and Table 2 summarizes the basic demographic and clinical features of patients in the two groups. The results showed no signi cant difference at baseline between SZL and FLX groups in age (47.14 ± 14.29 vs. 46.03 ± 11.92 years old, p > 0.05), HAMD score (18.79 ± 2.09 vs.18.53 ± 1.95, p > 0.05) and other indexes.

Changes of HAM-D scores and SDS scores after SZL or FLX treatment
In both SZL and FLX groups, a general improvement was seen continuously in the clinical features throughout the entire duration of treatment, as re ected as obvious reduction in the HAMD scores at both 4 and 8 weeks in comparison with baseline obtained with either paired t-test (4 weeks) or Wilcoxon-test (8 weeks) ( Fig. 2A).
Working memory is a memory system that temporarily processes and stores information with limited energy. Nback task can manipulate the working memory load by controlling the interval value of current value "N", so as to investigate the processing mechanism of working memory under different memory loads. Western blot analysis revealed that serum APOC3 level decreased signi cantly after 8 weeks of treatment in SZL group (p < 0.05), but not in FLX group (p > 0.05). In addition, there was no statistical difference in APOC3 between the two groups at the end of the treatment (Fig. 3).

Safety analysis
Among the 156 enrolled subjects, 144 subjects including 73 in SZL group and 71 in FLX group who had complete documented data including at least one follow-up were eligible for safety evaluation. Out of the 73 patients in SZL group, adverse events occurred in 6 patients and adverse drug reactions occurred in 3 patients.
Out of the 71 patients in FLX group, adverse events occurred in 12 patients and adverse drug reactions occurred in 10 patients. There were no severe adverse events taking place in both groups (Table 5). Safety set(SS), a data set for safety analysis, including all randomly enrolled, drugged, and documented at least one safety follow-up. SS contained 144 cases, 73 cases in trial group, 71 cases in control group.

Discussion
The results obtained in this randomized, placebo-controlled, double-blind study provide insights into the use of SZL to treat patients with depression. First, the results indicated that SZL is an effective and safe drug for patients with mild to moderate depression. Second, SZL exhibits an antidepressant effect comparable to FLX at 8 weeks of treatment and is well tolerated by patients with mild to moderate depressive disorder. In addition, SZL treatment signi cantly reduces the levels of serum ALB, APOB and APOC3 as well as HDL-C/LDL-C ratio, suggesting that it might help balance the disorder of lipid metabolism caused by depression.
Previous studies have shown that natural plants or traditional Chinese medicine (TCM) alone or in combination with routine treatments are very effective in treatment of depression as they can signi cantly reduce HAMD scores. Xiaoyao pills combined with uoxetine signi cantly decreased HAMD scores from 8.43 ± 2.01 to 3.50 ± 1.53 in 6 weeks [32] . Shuganjieyu capsule was more effective than Citalopram and reduced HAMD scores from 22.7 ± 2.8 to 6.5 ± 4.3 in 6 weeks [33] . Bushenshuganhuajifang also signi cant decreased HAMD scores from 26.97 ± 8.46 to 16.94 ± 7.05 in 8 weeks [34] . However, most TCM studies were small size studies without placebo control. KXS is a famous ancient herb and has been widely used to treat mental illness since Tang Dynasty in China. Many pharmacological and mechanism studies in recent two decades have approved its anti-depressive effect in animals and patients and demonstrated its multi-target regulation on traditional neurotransmitters, HPA axis and CREB-BDNF pathway [16,35,36] . As an easy taking preparation of KXS, SZL is prepared using new crude prescription according to the characteristics of modern clinical medicines and improved traditional preparation technology. To our best knowledge, this study is the very rst randomized, With our understanding on anti-depressive mechanisms deepening, it is well accepted that depression or antidepressive treatment is related to metabolic abnormalities. FLX has side effects to increase serum TG, TC and LDL-C levels in depression mice model [12] , accumulate lipids in primary mouse hepatocytes via enhancing acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) expression [13] and alter human metabolism [13,37] . Clomipramine intervenes glycolysis and glycolysis in mice model [38] . Paroxetine is a kind of SSRIs that mediate SERT de ciency can lead to adiposity and abnormal glucose metabolism [39,40] . Different from the above study on FLX, our results showed that FLX treatment signi cantly decreased APOB/APOA1 ratio while did not alter lipid indexes TG, TC, HDL-C, LDL-C, FPG, ALB, TBIL, UA, APOA1, APOB and APOE.
TCM with the advantages of multi-components, multi-targets and fewer adverse reactions might have desirable therapeutic effects on complicated depression with metabolic diseases. TCM can not only improve depression, but also regulate serum lipids and glucose levels, anti-lipid peroxidation and anti-atherosclerosis, in addition to enhancing cognition [41][42][43][44][45] . Our previous studies using iTRAQ and RNA sequencing methods found that KXS (SZL) regulates not only the brain neural pathway and target, but also many genes and proteins related to lipid metabolism and cardiovascular system, such as thymosin Beta-4 (a biomarker of heart protection and heart failure)), ADIPOQ (a target of blood lipid, diabetes and vascular endothelial damage), and apolipoproteins (APOA1, APOC1, APOC3, etc) [22,23] . Partly consistent with previous reports, our current study showed that SZL reduced the level of APOB and APOC3, as well as the ratios of HDL-C/LDL-C and APOB/APOA1. High APOB level is corresponding to high CES-D score in type 1 diabetes patients with mild to moderate or severe depressive symptoms [46] . In addition, high serum ApoB level is also negatively correlated with delayed memory decline owing to its relation to the dysfunctions of lipid peroxidation, malondialdehyde (MDA) and immune system [47] . The ratios of HDL-C/LDL-C and APOB/APOA1 are signi cantly correlated with depression and cardiovascular diseases [48][49][50] . Abundant serum albumin which is related to cardiovascular disease [51] is signi cantly decreased in depressive patients [52] . APOC3 is correlated with risk of adverse events (AD) [53] and associated with depression [54] . These together indicate that SZL could improve the abnormalities of lipid metabolism in patients with depression to some extent and suggest that KXS exerts its antidepressant effects by regulating the signaling pathways involved in lipid metabolism disorders.

Limitations
Although our study provided an available assessment of SZL in the treatment of depression, it still had some defects. In spite of the advantage of placebo control used in the experiment, the number of cases included in the trail was limited. In addition, lipid-related evaluation was insu cient and the correlation between lipid level and response rate was absent. Due to ethical reasons, subjects with different degrees of mild to moderate depression were included, which could lead to a certain degree of deviation. Therefore, it is unclear whether patients with severe or other types of depression can also bene t from SZL treatment. Moreover, the period to observe metabolic indexes was short. Therefore, whether it may have a prognostic value in predicting SZL treatment responses needs to be investigated in a larger patient population.

Conclusions
To sum up, we found that SZL has e cacy and safety pro le comparable to FLX in treatment of patients with mild to moderate depression. But in contrast to FLX, which did not signi cantly improve the levels of TC, TG,   HDL-C, LDL-C, APOA1, APOB, APOE, TBIL,  Availability of data and material The datasets used and analysed during the current study are available from the corresponding author on reasonable request.