Advances in Omics Studies, Biological and Clinical Potentialities of Cistanches Herba (Rou Cong Rong)

  


    
 

    
 

    
C. deserticola 

    
C. tubulosa 

    
C. salsa 

    
C. sinensis 

    
References

  

  


    
Total size (bp)

    
Plastomes

    
102,657

    
94,123

    
101,776

    
87,707

    
[6]

  

  


    
Cp genome

    
109,495

    
75,375

    
111,710

    
111,500

    
[8,9]

  

  


    
Mit genome

    
1,860,774

    
3,978,341

    
1,708,661

    
-

    
[11]

  

  


    
LSC


      (bp)

    
Plastomes

    
48,350

    
31,017

    
46,579

    
26,435

    
[6]

  

  


    
Cp genome

    
48,352

    
32,470

    
50,497

    
52,005

    
[8,9]

  

  


    
Mit genome

    
-

    
-

    
-

    
-

    
[11]

  

  


    
IRs


      (bp)

    
Plastomes

    
45,507

    
54,559

    
46,729

    
49,407

    
[6]

  

  


    
Cp genome

    
30,352

    
6,593

    
30,389

    
27,458

    
[8,9]

  

  


    
Mit genome

    
-

    
-

    
-

    
-

    
[11]

  

  


    
SSC


      (bp)

    
Plastomes

    
8,800

    
8,547

    
8,468

    
11,865

    
[6]

  

  


    
Cp genome

    
398

    
29,719

    
435

    
4,579

    
[8,9]

  

  


    
Mit genome

    
-

    
-

    
-

    
-

    
[11]

  

  


    
GC content (%)

    
Plastomes

    
37.95

    
36.53

    
37.26

    
36.95

    
[6]

  

  


    
Cp genome

    
36.27

    
34.95

    
36.11

    
36.75

    
[8,9]

  

  


    
Mit genome

    
44.59

    
44.57

    
44.52

    
-

    
[11]

  

  


    
Total number of genes

    
Plastomes

    
89

    
72

    
89

    
68

    
[6]

  

  


    
Cp genome

    
60

    
53

    
61

    
60

    
[8,9]

  

  


    
Mit genome

    
82

    
126

    
75

    
-

    
[11]

  

  


    
Total number of protein coding genes

    
Plastomes

    
27

    
22

    
20

    
23

    
[6]

  

  


    
Cp genome

    
21

    
20

    
21

    
21

    
[8,9]

  

  


    
Mit genome

    
37

    
65

    
41

    
-

    
[11]

  

  


    
tRNA

    
Plastomes

    
30

    
26

    
30

    
22

    
[6]

  

  


    
Cp genome

    
29

    
25

    
28

    
28

    
[8,9]

  

  


    
Mit genome

    
39

    
58

    
31

    
-

    
[11]

  

  


    
rRNA

    
Plastomes

    
4

    
4

    
4

    
4

    
[6]

  

  


    
Cp genome

    
4

    
4

    
4

    
4

    
[8,9]

  

  


    
Mit genome

    
5

    
4

    
3

    
-

    
[11]

  

  


    
Cp:    Chloroplast; Mit: Mitochondrial; LSC: Large Single-Copy region; SSC:    Single-Copy region; IRs: Inverted Repeats; C. deserticola: Cistanches    Deserticola; C. salsa: Cistanches salsa; C. sinensis: Cistanches sinensis; C.    tubulosa: Cistanches tubulosa; rRNA: Ribosomal RNA; tRNA: Transfer RNA 

  

Table 1: Characteristics of the four Cistanche herba organelle genomes.

Chloroplasts (Cp) are the places where plants perform photosynthesis and have attracted more attention than other plastids because of their important role in plant physiological development [7]. The chloroplasts genome of C. deserticola is 109,495 bp in length, consisting of one LSC (48,352 bp), one SSC (398 bp), and two IRs (30,352 bp) [8]. It contained 27 protein-coding genes, four rRNA genes, and 29 tRNA genes. Additionally, genes required for photosynthesis suffer from gene loss and pseudogenization, except for psbM [8]. As for the other Cistanche herba, the chloroplasts genome size was 111,710 bp in C. salsa, 111,500 bp in C. sinensis, and 75,375 bp in C. tubulosa [9]. Almost all genes related to photosynthesis were pseudogenized or lost, with the most severe loss occurring in C. tubulosa [9]. This suggests that Cistanche herba cannot photosynthesize independently, and its photosynthesis-related genes or protein products may come from the host Haloxylon ammodendron. Mitochondria (Mit) are essential for various metabolic processes, such as cellular respiration and ATP synthesis [10]. The mitochondria genome of C. tubulosa was significantly larger than other species (3,978,341 bp). The mitogenomic lengths of the C. deserticola and C. salsa were 1,860,774 bp and 1,708,661 bp, respectively [11]. It is worth mentioning that C. tubulosa has a higher number of genes than C. deserticola and C. salsa [11].

Gene Expression Profiles

For non-model organisms without reference gene sequences, transcriptome data can be used to discover gene expression profiles and biological characteristics of species on a larger scale [12]. At present, the nuclear genome data of Cistanche herba have not been published, but their gene expression profiles are crucial to elucidate its parasitic mechanism, compound synthesis pathway, and environmental adaptation.

Li Y et al. performed deep transcriptome sequencing in fleshy stem of C. deserticola using Illumina HiSeq2000 platform [13]. Using trinity assembler, they obtained 95,787 transcript sequences ranging from 200 bp to 15,698 bp, having an average length of 950 bases and the N50 length of 1,519 bases. 30098 identified actively expressed transcripts were annotated to a wide range of GO categories and KEGG pathways, such as terpenoid backbone biosynthesis, phenylpropanoid biosynthesis, and carotenoid biosynthesis, indicating that active metabolic processes were underway in the C. deserticola stem tissue [13]. A recent study by Hou et al. performed the full-length transcriptome sequencing and gene expression profiling of C. tubulosa using PacBio combined with BGISEQ-500 RNA-seq technology [14]. A total of 237,772 unique transcripts were obtained, ranging from 199 bp to 31,857 bp. Among the unique transcripts, 188,135 (79.12%) transcripts were annotated [14]. It is worth mentioning that 1080 transcripts were annotated for 22 enzymes related to PhGs biosynthesis [14].

Synthesis of Important Compounds

Traditional natural extraction or artificial chemical synthesis methods are difficult to meet the needs of scientific research and new drug development. Genomic and transcriptomic studies could be used to identify key enzyme coding genes of specific metabolic pathways and optimize these pathways by improving the expression of key enzymes encoding genes to obtain the required components [15]. Based on de novo transcriptome of C. deserticola, PhGs has two different biosynthesis pathways and 17 enzyme genes [13,14]. The possible post-caffeic acid/ferulic acid reaction process was published for the first time, in which the caffeic/ferulic acid would be first oxidized into phenylpyruvate derivate, then the carboxyl group was removed by decarboxylases, and finally, aldehyde group was converted back into alcohol group by dehydrogenase [13,14]. Another is based on phenylalanine metabolism pathway, in which the phenylalanine to phenylethanol was achieved by a known ‘Enrlich pathway’, then phenylethanol is converted to phenylethanol aglycone by monooxygenase or methyltransferase, and further phenylethyl alcohol glycoside is synthesized [13]. The complete picture of lignin biosynthesis pathways in C. deserticola was also presented by Li YL et al. [13], in which the lignin monomers are biosynthesized from phenylalanine through a series of enzymatic reactions, including hydroxylation, methylation, reduction, and oxidative polymerization process [16,17]. Phenylalanine Ammonialyas (PAL) is the first key enzyme in this pathway to convert phenylalanine to cinnamic acid by non-oxidative deamination [18,19]. 4-Coumarate-CoA Ligase (4CL) and trans-Cinnamate 4-Monooxygenase (CYP73A) subsequently convert cinnamic acid to coumaroyl-CoA. Finally, key enzymes Cinnamoyl-CoA Reductase (CCR), shikimate o-Hydroxycinnamoyl Transferase (HCT), and Ferulate-5-Hydroxylase (F5H) regulate the synthesis of different types of lignin by coumaroyl-CoA [20,21].

Biological Activities

Neuroprotection effects: AD is an irreversible and progressive neurodegenerative disorder whose pathogenesis is complex and not completely clear, including oxidative stress, mitochondrial abnormalities and neuroinflammation [22]. In vitro experiments, PhGs of Cistanche herba could reduce oxidative stress, scour free radicals, inhibit neural cell apoptosis, and promote neural cell growth and repair [23-25]. C. Tubulosa aqueous Extract (CTE) could improve memory loss in AD-like rats [24]. Specifically, Echinacoside (ECH), and Acteoside (ACT), the major components of CTE [26], could significantly improve the learning and memory ability of AD animal models, and the mechanism is related to inhibiting neural cell apoptosis and reducing oxidative stress [23,27-30]. Additionally, PhGs of Cistanche herba could regulate brain energy metabolism by regulating insulin signaling [31], and ACT is preferable to total phenylethanoid glycosides in anti-AD [32].

The typical neuropathological features of PD are degeneration of dopaminergic neurons in the substantia nigra [33], accompanied by chronic neuroinflammation [34]. Neurotoxic substances produced by activated microglia are key molecules that mediate the occurrence. ECH of Cistanche herba could significantly inhibit the activation of microglia cells induced by inhibiting the inflammatory signaling pathway NLRP3/CASP-1/IL-1β [35]. Additionally, ECH showed neuroprotective effects on PD pathological mice via regulating the IL-6/JAK2/STAT3 pathway to inhibit the activation of microglia [36]. ACT effectively alleviated rotational behavior in PD rats, and its anti-PD mechanism is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and the activation of the Nrf2/ARE signaling pathway [37-39].

PhGs from C. tubulosa could regulate neuroinflammation via microglial M1-M2 polarization and alleviate ischemic stroke caused by Middle Cerebral Artery Occlusion (MCAO) [40]. PhGs from C. deserticola could improve the stroke through enhancing endogenous neural stem cells proliferation via activating Wnt/β-catenin signaling pathway [41]. Therefore, Cistanche tubulosa and C. deserticola could be a candidate therapeutic agent or supplements for treating neuroinflammation related to ischemic stroke. Specifically, ACT could alleviate ischemic stroke by inhibiting microglial HMGB1/TLR4/NLRP3 signaling pathway in vivo [42]. Vascular Dementia (VD) is a severe cognitive disorder mainly caused by ischemic stroke. PhGs of Cistanche herba exerts its learning and memory-promoting effects in rat models of VD by weakening the accumulation of toxic proteins (Aβ and p-tau) and promoting neuronal cytoskeleton regeneration [43]. ECH could exert anti-VD activity by upregulating the expression of brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) in the hippocampus of VD rats and alleviating the ischemic injury of neurons [44].

Gastrointestinal Tract Protection Effects

Cistanche herba extract could significantly improve the constipation of yang deficiency constipation rats and its mechanism may be related to the regulation of gastrointestinal hormone levels [45]. It could effectively improve the water holding capacity of feces, which is conducive to increasing the excretion of mice [46]. Furthermore, C. deserticola extract could improve defecation via SCF/C-kit signaling pathway [47]. In addition, CTE could regulate the composition of intestinal microbiota in rats [48] and restore their disordered intestinal microbiota [49]. Moreover, CTE could restore the growth of lactic acid bacteria and then regulate the structure of intestinal microbiota in mice with intestinal diseases [50]. Polysaccharide, as the main active ingredient of CTE [51], also played intestinal regulatory function mainly by maintaining the balance of intestinal microbiota composition, improving its metabolic function, or synergistic interaction with intestinal microbiota mediated drugs [52]. Additionally, alditol extract from C. Aeserticola (CDAE) attenuate functional constipation by regulating bile acid metabolism. It could increase the expression of CYP8B1, FGF15, TGR5, and FXR, thereby modulating bile acid synthesis and enterohepatic circulation [53].

Anti-Osteoporosis Effects

Osteoporosis is a chronic disease characterized by deterioration of the bone structure and low bone mineral density [54]. Many studies have demonstrated that C. deserticola extract have significant anti-osteoporosis activity [55-57]. Ethanol extract of C. Deserticola (CDHE) improves bone loss in Postmenopausal Osteoporosis (PMOP) mainly through regulating lipid metabolism pathways in vivo [58]. ECH and ACT from C. tubulosa have shown significant anti-osteoporosis activities in the rat model of osteoporosis combined with AD [59]. ECH and ACT could significantly improve the bone quality and total bone mineral density of the femur in rats and promote bone formation and inhibit bone resorption [60]. ACT from Cistanche may exert osteoprotective effects by activating the PI3K/AKT/mTOR signaling pathway to alleviate Dex-induced osteoporosis [61]. In addition, Cistanoside A is able to promote bone formation and prevent bone resorption by interfering with NF-?B and PI3K/Akt pathways [62]. Moreover, total glycosides and polysaccharides from C. deserticola could promote osteogenesis formation and improve bone microstructure damage in mice by activating the Wnt/β-catenin signaling pathway [63].

Anti-Depressant Effects

The decoction of C. deserticola and C. tubulosa could improve spatial learning and memory in mice models of depression, and the mechanism may be related to the ‘gut-brain’ axis [64]. C. Deserticola Polysaccharides (CDPs) showed significant effects on improving abnormal behaviors of depressed rats by maintaining Th17/Treg balance and modulating gut immunity [65]. CTE could significantly improve depression-like behaviors in rats by regulating the structure of intestinal microbiota and restoring the levels of Hydroxytryptamine (5-HT) and BDNF through the ‘gut-brain’ axis [48]. Caffeic acid is the main degradation product of CTE in depression model rats [66], which could Produce antidepressant effects [67]. These metabolites have good intestinal absorption and bioavailability and are more likely to be absorbed into the blood [68,69]. In addition, ECH could exert antidepressant effects by activating the AMPAR-Akt/ERK-mTOR signaling pathway, in which mTOR is a key signaling target for rapid antidepressant effects [70].

Anti-Tumor Effects

Previous studies have demonstrated that PhGs of Cistanche herba exhibit antitumor effects on a variety of tumor cells through different signaling pathways. PhGs of C. tubulosa induced apoptosis in Hepatocellular Carcinoma (HCC) cells through both the mitochondria-dependent and MAPK signaling pathways in vitro [71]. In addition, PhGs of C. tubulosa could inhibit the growth of B16-F10 cells and esophageal cancer ECA-109 cells both in vitro and in vivo via mitochondria-dependent pathway [72,73]. PhGs of C. deserticola could significantly inhibit the mitochondrial respiration and glycolysis functions of HepG2 cells, increase the level of ROS, and inhibit cell proliferation [74]. ECH exerted antiproliferative and proapoptotic functions on HepG2 cells via decreasing TREM2 expression and inactivating the AKT pathway as well as miR-503-3p/TGF-β1/Smad axis [75,76]. Moreover, ECH could not only inhibit the growth of pancreatic cancer SW1990 cells by promoting ROS generation and MAPK signaling pathway [77], but also promote pyroptosis of non-small cell lung cancer cells through Raf/MEK/ERK signaling pathway [78].

Anti-Oxidant Effects

ECH could significantly attenuate ethanol-induced oxidative stress by enhancing the levels of antioxidants and reducing the level of ROS via SREBP-1c/FASN pathway [79]. ECH could also ameliorate oxidative stress and alcohol-induced liver injury by increasing the activity of erythroid 2-related factor 2 (Nrf2), which is promising for the treatment of Alcoholic Liver Disease (ALD) [80]. Additionally, Cistanoside could markedly attenuate the harmful effects of hypoxia-induced oxidative stress by affecting antioxidant enzyme activities in testes and GC-1 cells [81]. Moreover, Cistanche polysaccharide is also an effective antioxidant, and its phenolic hydroxyl structure could directly bind to free radicals and the activate antioxidant defense system [82]. The polysaccharide of C. deserticola could induce melanogenesis in melanocytes and reduce oxidative stress via activating NRF2/HO-1 pathway, which could be used as a new drug for the treatment of decolorization disease [83].

Immune Regulation Effects

PhGs of C. Tubulosa (CTPG) had an immune enhancement function, which could not only significantly increase the numbers of CD4+ and CD8+ T cells but also enhance the activation state of CD4+ T cells [71]. Moreover, CTPG could inhibit the apoptosis of splenocytes induced by cisplatin, which is characterized by the increased numbers of T cells and the decreased numbers of MDSCs and Tregs [71]. Polysaccharides of C. deserticola (WPCD) could modulate immune responses in vitro and in vivo. WPCD significantly promoted the maturation and function of murine Marrow-Derived Cendritic Cells (BM-DCs) through up-regulating the expression levels of MHC-II and allogenic T cell proliferation, as indicated by in vitro experiments [84]. Moreover, WPCD could enhance immunogenicity through a balanced Th1-/Th2-type response and an effective T-cell response, which could be used as a polysaccharide adjuvant on seasonal influenza vaccines [85].

Clinical Application

Digestive System

The clinical treatment of Cistanche herba on the digestive system is limited to constipation. Congrong tongbian decoction (Chinese herba preparation in hospital) has good therapeutic effects on yang-deficiency constipation, which refers to the patients with dry stool but difficult defecation, accompanied by cold and waist and knee soreness. After seven days of treatment, the defecation time of patients with constipation significantly shortened, and the defecation effect was strengthened with the increase in dose [86]. Cistanche herba decoction (Chinese native medicine preparation made by the hospital) could not only improve constipation caused by hemodialysis but also have long-lasting effects and no adverse reactions [87]. Moreover, Cistanche decoction (Chinese native medicine preparation made by the hospital) could effectively improve the constipation symptoms of Parkinson's patients, and the efficacy is better than that of the western medicine group [88].

Nervous System

Cistanche herba is clinically used for the treatment of mild AD. After agents treatment of C. deserticola (3222002216000), the cognitive status of mild AD patients has been significantly improved, and the mechanism is mainly related to the reduction of t-tau, TNF-a, and IL-1β in cerebrospinal fluid and delayed hippocampal atrophy [89]. Cistanche total glycoside capsules (Z20080047) also had good therapeutic effects on AD. After 12 weeks of treatment, the cognitive function of AD patients was significantly improved and the adverse reactions were less than those of the western medicine group [90]. Cistanche herba is usually combined with western medicine in the treatment of PD. Congrongjing Granules (CRJG) (Chinese native medicine preparation made by the hospital) could reduce the side effects of western medicine and improve the quality of life in patients with PD, and its efficacy and safety are better [91]. Moreover, Cistanche herba (Chinese native medicine preparation made by the hospital) combined with acupuncture and moxibustion could not only improve the motor ability of early PD patients [92], but also improve the non-motor symptoms of PD patients [93]. Additionally, the other two medicines of Cistanche could effectively improve life quality of PD patients, Cistanche Shujing granule (Chinese native medicine preparation made by the hospital) and Cistanche Yizhi (Z20194044) capsule could significantly relieve the depressive symptoms of PD patients [94,95].

Other Diseases

C. deserticola has two-way regulation of bone formation and bone resorption, and could effectively resist osteoporosis. After six months of C. deserticola treatment (Chinese native medicine preparation made by the hospital) in patients with primary osteoporosis, the achieved effects are significant, such as improved serum alkaline phosphatase, calcium, and phosphorus metabolism, increased bone density [96]. Additionally, CTE (Chinese native medicine preparation made by the hospital) is helpful to prevent motor organ dysfunction. After 12 weeks of CTE treatment, the stride length and gait speed of patients with locomotive syndrome increased, and no obvious adverse reactions were observed [97]. Moreover, CTE could effectively relieve tinnitus symptoms caused by chronic nephritis. After three months of treatment, the tinnitus symptoms of adult patients with chronic glomerulonephritis were significantly relieved [97].

Conclusions

This article summarized the progress in omics research including the organelle genome and transcriptome of Cistanche herba for the first time. We found significant differences in the genomic structure of important organelles of different Cistanche herba (Table 1), suggesting that genomic data could solve the long-controversial problem of Cistanche herba identification in the future. In addition, the synthesis pathway, biological function and clinical application of PhGs, an important compound of Cistanche herba, were reviewed. Future studies should focus on overcoming the technical problems of assembling genome with high heterozygosity, high repeat sequences and high GC content to construct high-quality nuclear genome profiles of Cistanche herba, so as to solve the difficult situation of qualitative characterization of transcriptome. Moreover, researchers should integrate the multi-omics data of Cistanche herba to elucidate the total synthesis pathways of its important compounds (taking PhGs as an example) and further explore new active compounds. In conclusion, this article provides a new insight for further pharmacological research and clinical application of Cistanche herba, and provides a high-level theoretical basis for its industrial transformation.

Author Statements

Funding

This study was supported by grants from Science and Technology Innovation Action Plan Startup Project (Sail Special) of Shanghai (grant number: 22YF1420500), the Fundamental Research Funds for the Central Universities (grant numbers: KLSB2022QN-01), Medical-Industrial Crossover Research Fund of Shanghai Jiao Tong University (grant numbers: YG2022QN070 and 19X190020005), and the National Natural Science Foundation of China (grant number: 81872274).

Authors Contributions

Huang J were responsible for study concept and design. Zou R and Huang J were responsible for drafting the manuscript. Huang WQ and Xu J participated in the review & editing. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Consent to Publish

The Author confirms that the work described has not been published before.

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