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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 8  |  Issue : 1  |  Page : 115-122

Quercetin-3-O-β-D-glucuronide inhibits mitochondria pathway-mediated platelet apoptosis via the phosphatidylinositol-3-kinase/AKT pathway in immunological bone marrow failure


1 Department of Hematology, Jing' an District Centre Hospital of Shanghai (Jing' an Branch, Huashan Hospital, Fudan University); Department of Hematology, Shanghai Bao' shan Hospital of Integrated Traditional Chinese and Western Medicine (Bao' shan Branch of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine), Shanghai, China
2 Department of Hematology, Shanghai Bao' shan Hospital of Integrated Traditional Chinese and Western Medicine (Bao' shan Branch of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine), Shanghai, China
3 Department of Hematology, Jing' an District Centre Hospital of Shanghai (Jing' an Branch, Huashan Hospital, Fudan University), Shanghai, China
4 Department of Hematology Laboratory, Singapore General Hospital, Singapore

Date of Submission20-Dec-2020
Date of Acceptance07-Feb-2021
Date of Web Publication27-Sep-2021

Correspondence Address:
Prof. He-Ping Yu
Department of Hematology, Jing' an District Centre Hospital of Shanghai (Jing'an Branch, Huashan Hospital, Fudan University), Shanghai 200040
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_44_21

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  Abstract 


Objective: Quercetin-3-O-β-D-glucuronide (QG) can alleviate immunological bone marrow failure (BMF) by increasing platelet counts. However, the principal mechanism is less known. This study aimed at deciphering the possible underlying mechanism of QG that is indicated in thrombocytopenic purpura. Methods: In vitro and in vivo experiments were carried out for investigating the mechanism behind QG-facilitated inhibition of mitochondrial pathway-mediated excessive apoptosis of platelets through the phosphatidylinositol-3-kinase (PI3K)/AKT pathway. Results: Our results revealed that QG, the main effective ingredient of Herba Sarcandrae, increases the number of platelets and decreases the expression of Bax, Bad, Bid, and caspase-9 in immunological BMF, indicating the inhibition of mitochondrial pathway-mediated apoptosis. Moreover, we found that the protein and mRNA expressions, as well as the phosphorylated levels of PI3K and AKT, were increased significantly by QG, suggesting the activation of the PI3K/AKT pathway. Furthermore, the inhibition of the PI3K/AKT pathway by LY294002 antagonizes the effects of QG on platelet counts and mitochondrial pathway-mediated apoptosis. Conclusion: We demonstrate that QG inhibits the mitochondria pathway-mediated platelet apoptosis via the PI3K/AKT pathway in immunological BMF. This study thus sheds light on exploring the possible regulatory mechanism of traditional Chinese medicine in the treatment of thrombocytopenia induced by BMF.

Keywords: Apoptosis; mitochondrial pathway; phosphatidylinositol-3-kinase/AKT; platelets; Quercetin-3-O-β-D-glucuronide


How to cite this article:
Xia LM, Zhang AP, Zheng Q, Ding J, Jin Z, Yu H, Wong WH, Yu HP. Quercetin-3-O-β-D-glucuronide inhibits mitochondria pathway-mediated platelet apoptosis via the phosphatidylinositol-3-kinase/AKT pathway in immunological bone marrow failure. World J Tradit Chin Med 2022;8:115-22

How to cite this URL:
Xia LM, Zhang AP, Zheng Q, Ding J, Jin Z, Yu H, Wong WH, Yu HP. Quercetin-3-O-β-D-glucuronide inhibits mitochondria pathway-mediated platelet apoptosis via the phosphatidylinositol-3-kinase/AKT pathway in immunological bone marrow failure. World J Tradit Chin Med [serial online] 2022 [cited 2022 May 18];8:115-22. Available from: https://www.wjtcm.net/text.asp?2022/8/1/115/336832




  Introduction Top


Quercetin-3-O-β-D-glucuronide (QG), a flavonoid found in Herba Sarcandrae, can promote the proliferation of bone marrow megakaryocytes and raise the number of platelets.[1] Studies have shown that QG significantly increases the number of leukocytes and platelets in bone marrow-suppressed mice induced by cytarabine.[2] However, the specific mechanism of QG in elevating the platelet number remains unclear.

The main physiological function of platelets is hemostasis. Abnormalities in the number of platelets are the main cause behind most platelet-related diseases. Despite lacking nuclei, platelets have been shown to undergo apoptosis via the extrinsic and intrinsic apoptotic pathways recently.[3] A study has also indicated that among the two apoptotic pathways, the intrinsic pathway (i.e., the mitochondrial pathway) plays an important role in regulating the apoptosis in platelets.[4] To the best of our knowledge, depolarization of mitochondrial transmembrane potential (ΔΨm) is an early manifestation and characteristic change during apoptosis via the mitochondrial pathway. Further release of cytochrome c is induced by the opening of mitochondrial permeability transition pore (MPTP) while a variety of mitochondrial proteins rapidly transfer to cytoplasm and regulate the platelets apoptosis, including the expression, activation and translocation of pro-apoptotic protein (Bax, Bad and Bid) to mitochondria. Subsquently, the downstream gene expression changes, such as Caspase-9 activation and phosphatidylserine (PS) exposure.[5],[6],[7]

It has been found that the mouse model used for studying immunologic bone marrow failure (BMF) exhibits thrombocytopenia accompanied by decreased expression of caspase-8 and caspase-3 and alterations in the expression of other apoptotic markers.[8] The pathogenesis of thrombocytopenia in the immunologic BMF mouse model is closely related to platelet apoptosis. Our previous studies have found that QG demonstrates mechanisms similar to cyclosporine A (CSA).[9] One of the mechanisms of CSA in treating thrombocytopenia is increasing platelet survival time by inhibiting the mitochondrial pathway-mediated platelet apoptosis.[10] However, CSA is not appropriate for the treatment of acute bleeding due to its long onset time.

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is a classical signaling pathway involved in cell metabolism, proliferation, apoptosis, and energy metabolism that has attracted increasing attention.[11] Activation of PI3K/AKT signaling has a peculiar mechanism with platelet-related signal transduction, as both of them are located at the junction of mitochondria-mediated platelet apoptosis pathway. The mitochondrial pathway can regulate platelet apoptosis directly or indirectly through phosphorylation or interaction with related apoptosis markers. However, it remains to be uncovered whether the mechanism of QG behind the treatment of thrombocytopenia involves the PI3K/AKT pathway-mediated mitochondrial pathway.

In this study, in vitro and in vivo experiments were carried out for investigating whether QG inhibits the excessive apoptosis of platelets mediated by mitochondrial apoptosis through the PI3K/AKT pathway and explores the possible regulatory mechanism of the traditional Chinese medicine, Herba Sarcandrae, in the treatment of thrombocytopenia induced by BMF.


  Methods Top


Experimental animals

The research was approved by the Ethics Committee of Laboratory Animals of Jing'an District Center Hospital of Shanghai, Shanghai. Clean C57BL/6 mice (half male and female) aged from 8 to 12 weeks, weighing 20 ± 2 g, were supplied by Shanghai SLAC Laboratory Animal Co., Ltd. and housed in specific pathogen-free environment at room temperature 22 ± 2°C, humidity 55%, and 12 h/12 h light/dark cycle with free access to food and water. The animal experimentation project approval number was SCXK (Shanghai) 2012-0002. Appropriate housing, feeding, and care along with all interventions relating to the animal welfare were carried out in conformity with the stipulations of Regulations for the Administration of Affairs Concerning Experimental Animals (People's Republic of China).

Mouse model for bone marrow failure

DBA/2 mice (SLAC Laboratory Animal Co., Shanghai, People's Republic of China) were euthanized and then soaked in 75% ethanol for 5 min. After routine disinfection, the thymus was removed and saline was used to wash away the blood. The thymus was tenderly ground and carefully filtered with a nylon filter and then passed through a size 4-gauge needle to form a single-cell suspension. Trypan blue staining was used to evaluate cell viability (up to 95% viable cells). After cell counting, the single-cell suspension was adjusted to an appropriate concentration. The thymus and lymph node cell mix from DBA/2 mice (1 × 106/mouse in 0.2 ml) was injected into the clean C57BL/6 mice (SLAC Laboratory Animal Co., Shanghai, People's Republic of China), which were irradiated with different doses of 60Co-γ rays, by the tail vein within 4 h. After 3 days, peripheral blood was taken from the tail vein of mice and routine blood examination was performed by the automatic blood cell analyzer. A decrease of the total amount of blood cells suggested the successful generation of the BMF mouse model.[12],[13]

Drug preparation and animals

QG standard preparation (purity >98%) was obtained from Shanghai Yansheng Industrial Co. Ltd. (Shanghai, People's Republic of China). The CSA capsule (S0408, 25mg × 50 granules) was purchased from Novartis German Pharmaceutical Co., Ltd. CSA was reconstituted to 4 mg/mL solution using sterile saline. Second, the lavage solution was diluted to 0.1 Ml/10 g of mouse body weight.

Forty mice were assigned into four groups with 10 mice in each group at random: the control group, BMF group, BMF + CSA group (CSA group), and BMF + QG group. The control mice were not subjected to any treatment. Based on our previous research, mice in the QG group were intragastrically administered with 0.2 g/kg QG daily after BMF for 3 consecutive days. Mice in the CSA group were intragastrically administered with 0.027 g/kg cyclosporine daily after BMF for 3 consecutive days, as the positive control. Post 3 days of lavage, the mice were injected with 0.005 mL/g of 10% hydrochloride for anesthesia. Blood samples were then drawn from the tail venous plexus for the complete blood count and preparation of washed platelets.

Preparation of washed platelets and cell culture

The washed platelets were obtained from the BMF mouse model. Briefly, the platelet-rich plasma (PRP) was prepared by combining 1 mL venous blood with 7 mL anticoagulant citrate dextrose solution (2.5% sodium citrate, 2% glucose, 1.5% citric acid) and centrifuged at 1300 rpm for 20 min. The supernatant was then discarded and the PRP was further centrifuged at 2500 rpm for 20 min. Subsequently, the platelets were washed with a CGS buffer (0.123 M sodium chloride, 0.033 M glucose, and 0.013 M sodium citrate, pH 6.5) and resuspended with Tyrode's buffer (2.5 mM HEPES, 150 mM sodium chloride, 2.5 mM potassium chloride, 1 mM calcium chloride, 1 mM magnesium chloride, 12 mM sodium bicarbonate, and 5.5 mM glucose, pH 7.4). Next, the cell-count boards were used to count the platelet number. Subsequently, the washed platelet suspension was adjusted to a concentration of 3 × 108/mL and rested at room temperature for 60 min. In order to conduct in vitro tests, it was later randomly divided into six groups (10 samples per group), including the control group, BMF group, QG group (washed platelets diluted and incubated with 250 μg/mL QG), and QG + LY294002 (EKEAR Biotechnology Co., Shanghai, People's Republic of China) group at doses of 10, 25, and 50 μmol/L, respectively.

Flow cytometry

As previously described,[14] washed platelets were, respectively, incubated with apoptosis markers, Bax, Bad, Bid, and caspase-9 antibodies (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), and fluorescein isothiocyanate-conjugated Annexin V (Bender Medsystem Co., Vienna, Austria) at 37°C for 20 min or for the indicated time interval. Subsequently, the cells were assessed for apoptosis by flow cytometry (CYTOMICS FC500, Beckman Coulter Co., Ltd.).

Western blotting

With the presence of 1 mM phenylmethanesulfonyl fluoride and 0.1–2.0 mM sodium orthovanadate (Beyotime Institute of Biotechnology), total protein extracts were prepared from the washed platelets with RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, People's Republic of China). Total protein extracts were blotted onto nitrocellulose membranes (Bio-Rad, Inc., Hercules, CA, USA) post resolving in a 10% sodium dodecyl sulfate -polyacrylamide gel using electrophoresis. The membranes were immunoblotted with antibodies specific to the following proteins: Bax, Bad, Bid, caspase-9, PI3K, phosphorylated-PI3K (p-PI3K), AKT, and p-AKT (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a protein loading control, while anti-mouse immunoglobulin G (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as the secondary antibody. BandScan software was then used for analyzing the signal intensity of each protein band detected via chemiluminescence and for calculating the level of expression. The experiments were repeated three times and the mean expression levels were calculated.

Detection of the expression of phosphatidylinositol-3-kinase and AKT by quantitative polymerase chain reaction

According to the manufacturer's instructions, total RNA from the washed platelets was extracted by TRIzol reagent (Thermo Fisher Scientific, Inc., USA). Next, the Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., USA) was used to synthesize cDNA from total RNA. The synthesized cDNA was then used to perform quantitative polymerase chain reaction (PCR) on a CFX96 Touch real-time PCR Detection System (Bio-Rad, USA) using Power Up SYBR® Green Master Mix (Thermo Fisher Scientific, Inc., USA). The indicated primers [Table 1] were used to determine the expression of indicated genes. The polymerase chain reaction program used for amplification was as follows: 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 35 s. 2ΔΔCt method was used to calculate the relative amount of specific mRNA. The ΔCt was calculated by subtracting the Ct of the internal control from the CT of the target gene.
Table 1: Primes

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Statistical analysis

All the data were analyzed by SPSS19.0 software (SPSS, Chicago, Illinois, USA). The data were presented as mean ± standard deviation. The differences were compared with an analysis of variance. The statistical difference between groups was further performed by the Student–Newman–Keuls method. P < 0.05 was considered as statistically significant.


  Results Top


Quercetin-3-O-β-D-glucuronide increases the number of platelets in immunological bone marrow failure

QG was isolated from Herba Sarcandrae, having a chemical structure, as shown in [Figure 1]. To explore the effect of QG on platelet number in immunological BMF, mice suffering from immunological BMF were treated with QG at the dose of 0.2 g/kg body weight daily. CSA treatment was set as a positive control. After 3 days, the platelet number was measured by the auto-analyzer (Sysmex XE-2100, Sysmex Co., Ltd., Kobe, Japan). The results showed that the platelet number after BMF was significantly lower than that in the control group (P < 0.05, n = 10), indicating the successful establishment of immunological BMF mouse model. Furthermore, our results showed that QG evidently increased the platelet number significantly, similar to CSA (P < 0.05, n = 10) [Figure 2].
Figure 1: Chemical structure of quercetin-3-O-β-D-glucuronide

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Figure 2: The number of platelets in immunological BMF mice under different treatments in vivo. CTL: CTL group; BMF: Bone marrow failure group, QG: Quercetin-3-O-α-D-glucuronide group, CSA: Cyclosporine A group. aP < 0.05 versus the CTL group; bP < 0.05 versus the BMF group

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Quercetin-3-O-β-D-glucuronide inhibits the mitochondrial pathway-mediated platelet apoptosis in immunological bone marrow failure

To explore the mechanism of QG in augmenting the platelet number, we further tested whether the QG treatment affects platelet apoptosis. Flow cytometry was used to detect the expression levels of Bax, Bad, Bid, and caspase-9 in washed platelets derived from the four groups described previously. Our results revealed that the expression levels of Bax, Bad, Bid, and caspase-9 in the washed platelets derived from the BMF group were increased significantly (P < 0.05, n = 10) as compared with those in the control group indicating that the mitochondria pathway plays a significant role in inducing platelet apoptosis in BMF. Moreover, these protein levels decreased significantly in QG and CSA groups compared with those in the BMF group (P < 0.05, n = 10). These findings signify that QG inhibits excessive platelet apoptosis via the mitochondria pathway in BMF [Figure 3].
Figure 3: The expression levels of Bax, Bad, Bid, and caspase-9 in different groups in vivo. (a-d) The expression levels of Bax, Bad, Bid, and caspase-9, respectively, under different treatments measured by flow cytometer (Left). The quantitative analysis of Bax, Bad, Bid, and caspase-9 respectively (Right). CTL: CTL group, BMF: Bone marrow failure group, QG: Quercetin-3-O-β-D-glucuronide group, CSA: Cyclosporine A group. aP < 0.05 versus the CTL group; bP < 0.05 versus the BMF group; cP < 0.05 versus the CSA group

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Quercetin-3-O-β-D-glucuronide activates the phosphatidylinositol-3-kinase/AKT pathway

The PI3K/AKT pathway is an important pathway in inhibiting the mitochondrial pathway of platelet apoptosis. To identify whether QG inhibits mitochondria pathway-mediated platelet apoptosis through the PI3K/AKT pathway, the total and phosphorylated protein levels of PI3K and AKT of washed platelets in the four groups were detected by Western blotting and PCR. Our results showed that the levels of total PI3K, phosphorylated-PI3K (p-PI3K), total AKT, and p-AKT were reduced significantly (P < 0.05, n = 10) in the BMF group as compared to those in the control group [Figure 4]a. The protein and mRNA levels of PI3K and AKT in the QG and CSA groups were also increased significantly as compared to those in the BMF group (P < 0.05, n = 10) [Figure 4], suggesting that QG activates the PI3K/AKT pathway.
Figure 4: The protein levels of PI3K, p-PI3K, AKT, and p-AKT under different treatments in vivo. (a) The protein levels of PI3K, p-PI3K, AKT, and p-AKT were detected by Western blot; (b) The mRNA expression levels of PI3K and AKT were detected by PCR. CTL: CTL group, BMF: Bone marrow failure group; QG: Quercetin-3-O-β-D-glucuronide group, CSA: Cyclosporine A group, PI3K: Phosphatidylinositol-3-kinase. aP < 0.05 versus the CTL group; bP < 0.05 versus the BMF group; cP < 0.05 versus the CSA group

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LY294002 inhibits the activation of the phosphatidylinositol-3-kinase/AKT pathway under quercetin-3-O-β-D-glucuronide treatment

To identify if QG inhibits the excessive platelet apoptosis through the PI3K/AKT pathway, the washed platelets were prepared from the BMF mice and then treated with different drug interventions as indicated. The results revealed that the activation of the PI3K/AKT pathway by QG is significantly weakened upon incubation with different concentrations of LY294002, a PI3K/AKT inhibitor, especially LY25 (P < 0.05) [Figure 5].
Figure 5: The protein levels of PI3K, p-PI3K, AKT, and p-AKT in the presentence of LY294002 under quercetin-3-O-β-D-glucuronide treatment in vitro. The protein levels of PI3K, p-PI3K, AKT, and p-AKT were detected under quercetin-3-O-β-D-glucuronide treatment by Western blot (a) and quantified by statistical analysis (b). CTL: CTL group; BMF: Bone marrow failure group, LY10: 10 μmol/L LY294002, LY25: 25 μmol/L LY294002, LY50: 50 μmol/L LY294002, PI3K: Phosphatidylinositol-3-kinase. aP < 0.05 versus the CTL group; bP < 0.05 versus the BMF group; cP < 0.05 versus the quercetin-3-O-β-D-glucuronide group

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The inhibition of the phosphatidylinositol-3-kinase/AKT pathway decreases the number of platelets enhanced by quercetin-3-O-β-D-glucuronide in bone marrow failure

To identify whether QG increases the number of platelets through PI3K/AKT, the washed platelets were taken from the BMF mice and treated with various drug interventions. Flow cytometry assay results showed that LY294002 prevents the augmentation of QG-induced platelet number [Figure 6].
Figure 6: The platelet counts were detected by flow cytometry under quercetin-3-O-β-D-glucuronide treatment in vitro. CTL: CTL group, BMF: Bone marrow failure group, LY10: 10 μmol/L LY294002; LY25: 25 μmol/L LY294002; LY50: 50 μmol/L LY294002. aP < 0.05 versus the CTL group; bP < 0.05 versus the BMF group; cP < 0.05 versus the quercetin-3-O-β-D-glucuronide group

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The inhibition of the phosphatidylinositol-3-kinase/AKT pathway promotes mitochondrial pathway-mediated platelet apoptosis inhibited by quercetin-3-O-β-D-glucuronide in bone marrow failure

To address whether QG inhibits mitochondrial pathway-mediated platelet apoptosis in immunological BMF through the PI3K/AKT pathway, the washed platelets were drawn from the BMF mice and then treated with different drug interventions. The results showed that the decreased expression of Bax, Bad, Bid, and caspase-9 in QG group was reversed upon treatment with LY294002 (P < 0.05) [Figure 7]. These data indicate that QG inhibits mitochondrial pathway-mediated platelet apoptosis through the PI3K/AKT pathway.
Figure 7: The expression of Bax, Bad, Bid, and caspase-9 in the presentence of LY294002 under quercetin-3-O-β-D-glucuronide treatment in vitro. The expression levels of Bax, Bad, Bid, and caspase-9 were detected under different treatments by Western blot (a) and quantified by statistical analysis (b). CTL: CTL group, BMF: Bone marrow failure group, LY10: 10 μmol/L LY294002, LY25: 25 μmol/L LY294002, LY50: 50 μmol/L LY294002. aP < 0.05 versus the CTL group; abP < 0.05 versus the BMF group; acP < 0.05 versus the quercetin-3-O-β-D-glucuronide group

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  Discussion Top


In clinical practice, it is a quite challenging to treat a hemorrhage caused by the thrombocytopenia of BMF. The bleeding generally occurs in the skin, mucosa, and viscera with repeated incidences. Particularly, it may even lead to life-threatening intracranial hemorrhages. For now, owing to the high cost, strong side effects in a dose-dependent manner, drugs for the treatment of BMF such as immunosuppressive agents and anti-thymic globulin, and the treatment of thrombocytopenia, such as glucocorticoid and transfusion of platelets, are difficult in popularization and application worldwide.[15] Hence, it is particularly important to explore new and effective hemostasis methods. Studies have shown that the effect of traditional Chinese medicine, with blood cooling for hemostasis, on thrombocytopenia is significant.[16] Our previous studies have surprisingly found that the ancient traditional herbal medicine Herba Sarcandrae, which has the effect of cooling blood and hemostasis, could improve the bleeding of acute BMF mice models effectively.[1] In addition, QG, a glycoside derivative of quercetin, is a major active component in Herba Sarcandrae.[17],[18]

The PI3K/AKT pathway regulates the platelet life cycle through several pro-apoptotic proteins. Bax is made up of 192 amino acids with a relative molecular mass of 21 kDa. 21% of amino acids in Bax are homologous to Bcl-2 and are centered on the C-terminal conserved domain (BH1 and BH2). Studies have shown that Bax and Bcl-2 can form homodimers or interact as heterodimers. The levels of Bax and Bcl-2 are related to the regulation of apoptosis directly, as the elevated Bax levels promote platelet apoptosis while elevated Bcl-2 levels inhibit it.[19] Bad (including BH1 and BH2 domain) consists of a 204-amino acid domain with a relative molecular mass of 22 kDa. The functional regulation of Bad is mainly achieved through phosphorylation of two fixed sites, Ser112 and Ser136.[20] Phosphorylation of Bad promotes the recruitment of Bad into the cytoplasm and binding to the 14-3-3 protein, which prevents its binding to Bcl-2 and Bcl-xL. Free Bcl-2 and Bcl-xL can form heterodimers with Bax which prevents Bax oligomers from self-formation and inhibits its pro-apoptotic function. Bid proteins belong to the BH3-only subclass of the Bcl-2 superfamily, which contains only the BH3 domain in the conserved BH domain. Bid exists in a form with barely any apoptosis-inducing activity under normal physiological conditions. Few studies focusing on Fas-mediated apoptosis of rheumatoid synovial fibroblast cells have revealed that the PI3K inhibitors such as Wortmannin and LY294002 block the phosphorylation of AKT resulting in the increased lysis of Bid significantly. The cleaved 15 kDa c-segment of Bid transfers from the cytoplasm to the mitochondria, promoting the conformational changes of Bax and Bak to form oligomers. Oligomerization of Bax and Bak forms mitochondrial membrane channels that induce the release of cytochrome c from mitochondria to activate the mitochondrial pathway of cellular apoptosis.[21]

Caspase proteins are a class of proteases with sequence and structural homology to CED-3, which are all synthesized in the form of caspase proenzyme in platelets and consist of three domains: the N-terminal pro-domain, a small subunit (approximately 10 kDa), and a large subunit (approximately 20 kDa) corresponding to the mature protein. As the initiator of the mitochondrial pathway in platelet apoptosis, caspase-9 combines with cytochrome c and Apaf-1 to form complexes, which is an activated form transformed from a precursor enzyme. Caspase-9 is further cleaved by caspase-3 and caspase-7, as the process of apoptosis begins. It has been found that activated Akt can phosphorylate the Ser196 residue of caspase-9, inactivating it and inhibiting its pro-apoptotic activity. Studies on gastric cancer and colon cancer tissues using immunohistochemistry have shown that activated AKT could further decrease phosphorylated caspase-9 level and the phosphorylation of caspase-9 was negatively correlated with p-Akt, indicating that the activity of caspase-9 is regulated by the PI3K/AKT pathway. Hence, the PI3K/AKT pathway is involved in the regulation of platelet apoptosis at different stages by regulating mitochondria directly or indirectly.[22]

In the present study, we have found that QG could increase the number of platelets and decrease the expression of Bax, Bad, Bid, and caspase-9 in immunological BMF, indicating the inhibition of mitochondrial pathway-mediated apoptosis. Moreover, we have shown that protein and mRNA expressions, as well as the phosphorylated levels of PI3K and AKT, are increased significantly upon QG treatment, suggesting the activation of the PI3K/AKT pathway. The inhibition of the PI3K/AKT pathway by LY294002 antagonizes the effects of QG on platelet counts and mitochondrial pathway-mediated apoptosis.


  Conclusion Top


In this study, for the first time, we have illustrated that QG increases the number of platelets and inhibits the expression of apoptotic proteins Bax, Bad, Bid, and caspase-9 by the PI3K/AKT pathway both in vitro and in vivo. It was elucidated that QG can inhibit platelet apoptosis by regulating the mitochondrial pathway, mediated by PI3K/AKT, to increase the platelet count in BMF. We aim to carry out human studies to further clarify the mechanism of QG in the treatment of thrombocytopenia arising from BMF and provide traditional Chinese medicine a new scientific connotation.

Financial support and sponsorship

This study was supported by the research project of health sciences of Shanghai Jing'an District (2019MS02) and Shanghai Bao'shan science and technology commission (18-E-10), the research project of Shanghai municipal commission of health and family planning (201640144, 20184Y0094), and Shanghai science and technology commission (18401903800).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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