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Clinical Efficacy and Transcriptomic Analysis of Congrong Shujing Granules (苁蓉舒痉颗粒) in Patients with Parkinson's Disease of Shen (Kidney) Essence Deficiency Syndrome

CHEN Shi-ya1, XIAO Shao-jian1, LIN You-ning1, LI Xi-yu1, XU Qian2,

YANG Sha-sha2, HUANG Lian-hong3,4, and CAI Jing1,2

ABSTRACT  Objective: To evaluate the clinical efficacy and safety of Congrong Shujing Granules (苁蓉舒痉颗粒, CSGs) in treating patients with Parkinson's disease (PD) and Chinese medicine (CM) syndrome of Shen (Kidney) essence deficiency, and to investigate the potential mechanism involving efficacy through a transcriptome sequencing approach. Methods: Eligible PD patients with the syndrome of Shen essence deficiency were randomly assigned to a treatment group or a control group by a random number table, and were treated with CSGs combined with Western medicine (WM), or placebo combined with WM, respectively. Both courses of treatment lasted for 12 weeks. The Unified Parkinson's Disease Rating Scale (UPDRS) score, the PD Question-39 (PDQ-39) score, CM Syndrome Scale score, and drug usage of all patients were evaluated before and after treatment. Safety was evaluated by clinical laboratory tests and electrocardiographs. Blood samples from 6 patients in each group were collected before and after the trial and used for transcriptomic analysis by gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Differentially expressed genes were validated using reverse transcription-polymerase chain reaction. Results: A total of 86 PD patients were selected from the Third Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine between January 2017 and December 2017. Finally, 72 patients completed the trial, including 35 in the treatment group and 37 in the control group. When compared with the control group after treatment, patients in the treatment group showed significant decreases in UPDRS sub-Ⅱ score, PDQ-39 score, CM syndrome score, and Levodopa equivalent dose (P<0.05). During the treatment course, no significant changes were observed in safety indicators between the two groups (P>0.05). A possible mechanism of clinical efficacy was proposed that involved regulating cell metabolism-related processes and ribosome-related pathways. Treatment with CSGs had shown to affect relevant gene loci for PD, including AIDA, ANKRD36BP2, BCL2A1, BCL2L11, FTH1P2, GCH1, HPRT1, NFE2L2, RMRP, RPS7, TGFBR1, WIPF2, and COX7B. Conclusions: CSGs combined with WM can be used to treat PD patients with CM syndrome of Shen essence deficiency with good safety. The possible mechanism of action and relevant gene loci were proposed. (Registration No. ChiCTR-IOR-16008394)

KEYWORDS Congrong Shujing Granules, Parkinson's disease, randomized controlled trial, RNA-sequencing, transcriptomics, Chinese medicine

Parkinson's disease (PD) is a neurodegenerative disease that occurs in both the middle-aged and elderly populations. Its prevalence, morbidity, and associated economic burden have steadily increased. In the general population, the estimated prevalence of PDn in industrialized countries is 0.3%. In the year 2030, the number of patients with PD is expected to reach 4,940,000 in China and 8,670,000 worldwide. The relative risk of death in patients with PD is about twice as high compared to the total population. However, current treatment regimens cannot impede the progression of the condition. With disease progression, the clinical effectiveness of the medication regime often decreases, and daily functioning and social participation are significantly compromised.) Thus, long-term care of PD patients brings a significant economic burden to individuals, families, and society.

Levodopa, the "gold standard" for PD treatment, is generally effective for 3–5 years. After that, patients commonly experience serious motor complications, including end of dose phenomenon, morning stiffness, freezing, and dyskinesia. Treatment of PD with Chinese medicines (CMs) can improve patients' clinical symptoms, and corresponding side effects, thereby improving PD patients' quality of life. 

Congrongjing Granules (苁蓉精颗粒, CRJ) is mainly composed of Herba Cistanches and Polygonatum sibiricum, which stems from the famous classical CM prescription Dihuang Decoction (地黄饮子) that used for the treatment of PD. In our previous experimental studies, the beneficial effects of CRJ were confirmed. The data showed that Polygonatum sibiricum and Herba Cistanches, the main component of CRJ, improved the expression of dopamine, reduced the content of related apoptosis factors (caspase-3, FasL), increased the expressions of neurotrophic factors, and decreased apoptosis of dopaminergic nerve cells. The study also showed that CRJ protected dopaminergic neurons by regulating apoptosis and glial cell-derived neurotrophic factor. All of these mentioned above suggested that CRJ was a safe and valuable therapeutic medicine for the treatment of PD.

Based on our clinical experience and experimental research, CRJ was modified by adding Radix Paeoniae Rubra, Cortex Moutan and Salvia miltiorrhiza Bge., thus Congrong Shujing Granules (苁蓉舒痉颗粒, CSGs) were composed. This study observed the clinical efficacy of CSGs on treating PD patients with CM syndrome of Shen (Kidney) essence deficiency and the genetic changes in peripheral blood mRNA of patients. To investigate the underlying mechanism of action of CSGs in treating PD, an RNA-sequencing (RNA-seq) approach was used to comprehensively analyze the patients' gene transcripts before and after the trial, and to identify and validate differentially expressed genes (DEGs).

METHODS

Diagnostic Criteria

The Western medicine (WM) diagnostic criteria referred to the latest diagnostic standards of PD published by the International Parkinson and Movement Disorder Society in 2015. The CM diagnostic criteria of Shen essence deficiency syndrome referred to the related standards published by the China Press of Traditional Chinese Medicine.

Inclusion and Exclusion Criteria

Patients who met the above WM diagnostic criteria as well as the CM syndrome differentiation criteria for Shen essence deficiency, 50–80 years old, had a Hoehn-Yahr (H-Y) staging scale of 4, and signed written informed consents were included in the study. Patients who were diagnosed with major diseases, including severe organ dysfunction, mental or neurological diseases, severe infections, tumors, encephalitis, or allergic constitution, received CM treatment for PD within 3 months, or participated in other clinical trials in the last 3 months were excluded.

Drop-Out and Termination Criteria

Patients who voluntarily withdrew from the trial or failed to be contacted were considered drop-outs. Patients who experienced severe adverse effects or sudden severe diseases and complications, and were unable to continue the trial, had poor subject compliance, received less than 80% or more than 120% of the designated treatment doses, violated trial protocols, provided incomplete information that affected the evaluation of efficacy were excluded from the study.

Participants

Eligible PD outpatients and inpatients from the Third Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine were enrolled. The experimental plan, informed consent, and case report forms received written approval from the Medical Ethics Committee of the Third Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine (No. 2017KS-6-1), and was registered at the Chinese Clinical Trial Registry (registration No. ChiCTR-IOR-16008394).

Estimation of Sample Size

The sample size was calculated based on the data presented in the previous study, and the sample• 414 • Chin J Integr Med 2020 Jun;26(6):412-419 size estimation method was established by the China Clinical Trials Statistical Working Group. Using the sample content design formula of a fully randomized design, and considering a drop-out rate of 20%, it was determined that the treatment group and control group should include 43 subjects, respectively. 

Randomization, Allocation Concealment, and Blinding

In this study, a fully randomized double-blinded method was employed. SPSS software was used to randomly assign subjects to the two groups, and to generate a random numbered table. Pharmacists dispensed drugs according to the table. Subjects, doctors, and reviewers were all blinded to the assignment of subjects and drugs.

Drugs

Each dose of CSGs was composed of the extractive of Herba Cistanches 6 g, Polygonatum sibiricum 12 g, Salvia miltiorrhiza Bge. 15 g, Radix Paeoniae rubra 12 g, and Cortex Moutan 10 g. The placebo is comprised of 5% Radix Bupleuri with starch, dextrin, etc. Its appearance, smell and taste are similar to those of CSGs. Both CSGs (batch No. 16004691) and placebo (batch No. 16004728) were provided by Beijing Kangrentang Pharmaceutical Co., Ltd. (Beijing, China), and distributed by the Pharmacy Department of the Third Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine.

Treatment

Subjects in the treatment group were treated with WM combined with CSGs, whereas subjects in the control group were treated with WM combined with placebos. The WMs were selected according to the guideline. Among them, the most commonly used drugs were Madopar (batch No. H10930198, Shanghai Roche Pharmaceutical Co. Ltd., China) and Sifrol (Boehringer Ingelheim Pharma GmbH, Germany). According to the guideline provided, during treatment, primary physicians would adjust the medication dosage based on the patient's condition. Each dose of CSGs and placebo was divided into two equal packages, taken 0.5 h after breakfast and dinner, respectively, for 12 consecutive weeks.

Outcome Measures

For outcome measures, including drug usage rating scales, and related indices, subjects from both groups were evaluated by trained physicians both before the study and at 3 months after administration of the initial dose.

Primary Outcome Measures

The Unified Parkinson's Disease Rating Scale (UPDRS) was composed by scores of Ⅰ (mental behavior, active behavior, and emotion), Ⅱ (daily life activities), Ⅲ (motor examination), and Ⅳ (motor complications). The higher the score, the more severe the condition.

Secondary Outcome Measures

The secondary outcome measures included Parkinson's Disease Question-39 (PDQ-39) score,(21) CM Syndrome Scale score, and drug usage of WM. PDQ-39 evaluates the quality of daily life of PD patients by scoring their actions, emotions, daily life, and social support. CM Syndrome Scale scores the related CM syndromes of PD patients, so as to evaluate the severity of the disease (Appendix 1). The dosage of WM was calculated as Levodopa equivalent dose (LED). For drugs related to the treatment of PD and its complications that were not included in the LED formula (i.e. benzhexol hydrochloride), corresponding remarks were made.

Safety Evaluation

The clinical laboratory tests including 3 major routines (blood/urine/stool), liver function (aspartate transaminase (AST) and alanine aminotransferase (ALT)) and kidney function (blood urea nitrogen (BUN) and serum creatinine (Cr)) as well as electrocardiograph (ECG) of patients were evaluated before and after the trial. Any adverse events were recorded with details of timing, degree of severity, symptoms, duration, measures taken, and outcomes.

Statistical Analysis

SPSS version 24.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Normally distributed measurement data were expressed as the mean ± standard deviation (x–± s), whereas non-normally distributed data were expressed as the median and interquartile range. Counts were expressed as absolute numbers and rates. When comparing measurement data between groups, the two-sample t-test was applied if the data/transformed data followed a normal distribution. In other cases, the two-sample rank-sum test was applied. When comparing measurement data before and after the trial, a paired-sample t-test was applied if the data/transformed data followed a normal distribution, whereas the paired-sample rank-sum test was applied. For the comparison of counting data, the Chi-square test was employed. P<0.05 was considered statistically significant. 

Transcriptome Sequencing Experiments

RNA-seq Blood samples from 6 patients in each group were randomly selected before and after the trial, and RNA was extracted, purified, and evaluated. To complete the sequencing sample library construction, total RNA underwent rRNA removal, fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, terminal repairing, the addition of A at the 3' terminus, joint connecting, and enrichment. The constructed library was validated by Qubit® 2.0 Fluorometer and Agilent 2100. Sequencing was performed after validation of the library.

Bioinformatics Analysis

For bioinformatics analysis, the raw reads derived from sequencing analysis were first purified to obtain high-quality and clean reads. Subsequently, a spliced mapping algorithm of His at 2 (version 2.04) was used for genome mapping of the clean reads. The number of reads was converted into fragments per kilo-base of exon model per million (FPKM) mapped reads using Stringtie (version 1.3.0), and the trimmed mean of M values methods to standardize gene expression. Next, the foldchange in differential gene expression was calculated from the FPKM. Genes with a q value 0.05 and foldchange 2 were considered DEGs. To DEGs from the corresponding group with clinical efficacy, gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway

enrichment analysis was applied. After adjusting the variation criteria, the DEGs of each group before and after the trial were selected, which were the effect gene loci of the Cistanche deserticola extract. Selected genes were further validated by reverse transcriptionpolymerase chain reaction (RT-PCR).

RESULTS

Trial Completion Conditions

A total of 86 PD patients were selected from the Third Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine between January 2017 and December 2017, 43 patients in each group. Finally, 72 patients completed the trial following the protocol, including 35 in the treatment group and 37 in the control group. The study flow diagram is presented in Figure 1. Between groups, no statistically significant difference was observed in age, disease stage, and H-Y stage (P>0.05, Table 1). 

Comparison of UPDRS Scores between the Two Groups

There was no significant difference between the two groups before treatment in UPDRS total score and subsection score (P>0.05). Compared with the scores before treatment, the UPDRS total score and sub-Ⅱ score were significantly decreased in the treatment group after treatment (P<0.05). Moreover, when compared with the control group after treatment, the UPDRS sub-Ⅱ score was significantly decreased in the treatment group (P<0.05). However, there was no significant difference in UPDRS total score between the two groups after treatment (P>0.05, Table 2). 

Comparison of PDQ-39 Scores, CM Syndrome Scores and Drug Usage between the Two Groups

There was no significant difference between the two groups before treatment in PDQ-39 scores, CM syndrome scores, and drug dosage (P>0.05). Compared with before treatment, the PDQ-39 scores, CM syndrome score, and LED were significantly decreased in the treatment group after treatment (P<0.05). Moreover, when compared with the control group after treatment, the PDQ-39 score, CM symptom score and LED were significantly decreased in the treatment group (P<0.05, Table 3).

Safety Evaluation

During the trial, 2 cases in the treatment group with adverse events were observed. Both adverse reactions were light in grade and occurred on day 4 after the start of the trial, with symptoms of internal heat, thirsty, and dryness heat. One of them was not willing to continue the trial and dropped out. One week after terminating the CM granules, the symptoms reduced and then disappeared. Regarding the other subject, after Paeoniae Rubra and Salvia Miltiorrhiza were added to the CSGs, symptoms reduced after 4 days and disappeared. No changes were observed in the clinical laboratory tests and ECG that were directly related to the trial. In addition, no statistically significant differences were observed between the two groups in any of the indices before and after the trial (P>0.05).

Genome Comparison

Of the six samples in each group, the range of the number of reads in the reference gene was 74452867–117774869. Mapping ratios of the sample transcriptome sequences were 87.30%–96.14%.

Functional Analysis of DEGs GO Analysis of DEGs
DEGs of the treatment group before and after the trial were grouped into 47 functional clusters (Figure 2). In addition, DEGs between the treatment group and the control group after the trial were grouped into 35 functional clusters (Figure 3). The DEGs were mainly related to cell regulation and metabolism processes.

KEGG Enrichment Analysis of DEGs
Pathway enrichment analysis using the KEGG public database of DEGs of the corresponding group with clinical statistical significance of P<0.05 of the efficacy evaluation indices above suggested that PD-related DEGs were likely to be enriched in the following 4 signaling pathways: (1) ribosome-related signaling pathways, (2) endocytosis-related signaling pathways, (3) FoxO signaling pathways, and (4) were identified. Moreover, 1 up-regulated gene and 12 down-regulated genes were identified in the control group. According to the quantitative analysis of DEGs, 13 highly DEGs related to CSGs had an effect in the PD patients screened, including AIDA, ANKRD36BP2, BCL2A1, BCL2L11, FTH1P2, GCH1, HPRT1, NFE2L2, RMRP, RPS7, TGFBR1, WIPF2, and COX7B.

RT-PCR Analysis
The genes mentioned above were validated using RT-PCR analysis. Among them, AIDA, ANKRD36BP2, BCL2A1, GCH1, HPRT1, NFE2L2, RMRP, RPS7, and COX7B were up-regulated genes, whereas TGFBR1 was down-regulated (Appendix 3). Moreover, there was no significant difference in the change of the other three genes.

DISCUSSION

A total of 95% of PD patients are between 40 and 70 years old. The age at onset is mostly around 50 years and the incidence increases with age. In previous studies, it was suggested that active ingredients in CSGs reduced oxidation-related damage of PD and decreased apoptosis of dopamine neurons. The results of this trial indicated that CSGs reduced the medicine dosage and improved the quality of life of PD patients. In this study, a total of 72 out of 86 subjects completed the trial. The completion rate was only 83.7%, which was relatively low when compared to other similar clinical trials The reasons may involve the two sets of comprehensive tests of the subjects before and after the trial, complicated questionnaires, and regular 3-month follow-up visits for dose adjustment. Further efforts are required to improve the compliance of subjects and the completion rate of trials. 

There are however more questions to be answered. Can CSGs substitute medication, such as dopamine, in the early stage of PD treatment and delay medication intake? To which stage of PD does CSGs have a significant effect on reducing the dose of medication? Can CSGs extend the effective period of medication? To address these questions, further efforts, including additional case studies, longer treatment periods, extended follow-up after treatment, increased detailed severity and age stratification, and more comprehensive statistical analysis are warranted. 

Based on the observation of the effectiveness of CSGs in the treatment of PD patients, in this study, we investigated the possible mechanism of the clinical efficacy of CSGs and related genes through RNA-seq technology. The transcript, GO function analysis results related to the clinical efficacy, KEGG pathway analysis results, and the acting gene loci in the CSGs treatment on PD were obtained. However, further efforts will be needed to identify the primary pathway that is involved in CSGs activity, the active ingredients and monomers in CSGs for treatment of PD, and the corresponding symptoms of gene loci that CSGs act on. Future
studies should focus on integrating CM theories with bioinformatics and modern molecular biology to investigate the effects and mechanism of action of Shen reinforcing therapy on the treatment of PD.

In summary, the findings indicated that CSGs were effective in improving the quality of life, alleviating CM symptoms, and reducing drug dosage in PD patients with Shen essence deficiency syndrome. No serious side effect was observed during the trial. Our study indicated that CSGs might be a potential new drug for the treatment of PD. However, a larger sample size, multicenter studies are needed to confirm these findings, which is within our future research plan.

Excerpted from: Clinical Efficacy and Transcriptomic Analysis of Congrong Shujing Granules (苁蓉舒痉颗粒) in Patients with Parkinson's Disease of Shen (Kidney) Essence Deficiency Syndrome  By CHEN Shi-ya1 etc.

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