1.Analysis of Chaihu Jia Longgu Mulitang Based on Trinity Life View of ''Physique, Qi, and Spirit''
Jiaxin ZHOU ; Jianping HU ; Zhengqiang LIU ; Xiyang LIU ; Dingding ZHANG ; Yaya DANG ; Wenchun ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(16):225-234
		                        		
		                        			
		                        			Based on the trinity life view of ''physique, Qi, and spirit'', Chaihu Jia Longgu Mulitang treats the patient's physical symptoms, disorders of Qi movement, and disorders of consciousness, covering the overall treatment and comprehensive nursing of physique, Qi, and spirit. It is widely applied and recognized for its efficacy in modern clinical practice. This paper explored the treatment effect of Chaihu Jia Longgu Mulitang from the trinity life view of ''physique, Qi, and spirit''. This formula mainly targeted patients with Qi deficiency caused by cold, leading to a syndrome of Qi stagnation and water retention in the Triple Energizer Meridian of Hand Lesser Yang (TE), as well as fire-heat syndrome in the Large Intestine Meridian of Hand Yang Brightness (LI) and Stomach Meridian of Foot Yang Brightness (ST), accompanied by disorder of nutrient-blood and subsequent spirit and soul unrest. Accurately judging the imbalance of the patient's physique, Qi, and spirit and using an appropriate combination of medicinals can achieve balance among the three to achieve the best effect. The treatment strategy of Chaihu Jia Longgu Mulitang is as follows: For disorders of Qi movement, such as Qi deficiency, Qi stagnation, and gastrointestinal fire-heat, Ginseng Radix et Rhizoma and Bupleuri Radix-Scutellariae Radix, and Rhei Radix et Rhizoma are used in combination. For physical symptoms such as water retention and disorder of nutrient-blood, Poria-Pinelliae Rhizoma-Zingiberis Rhizoma Recens, as well as Cinnamomi Ramulus-Jujubae Fructus are used in combination. Finally, Os Draconis-Ostreae Concha-Plumbum Rubrum is used to calm the spirit and soothe the soul. According to existing research, Chaihu Jia Longgu Mulitang has shown good efficacy in treating a variety of complex clinical diseases. This article provides a comprehensive interpretation of Chaihu Jia Longgu Mulitang from the perspective of the trinity life view of ''physique, Qi, and spirit'', offering new insights for clinical syndrome differentiation, treatment, and prescription. 
		                        		
		                        		
		                        		
		                        	
2.Advances in the role of ketone body metabolism in the pathogenesis of diabetic retinopathy
Jiaxin LI ; Yuanyuan ZHANG ; Yan SHAO
International Eye Science 2025;25(10):1623-1627
		                        		
		                        			
		                        			 Ketone body metabolism plays a significant role in the development and progression of diabetic retinopathy(DR), which closely related to the system and local metabolic disorders as a major microvascular complication of diabetes mellitus. Previous research has established a close relationship between dyslipidemia and DR progression. Ketone bodies, comprising β-hydroxybutyrate, acetoacetate, and acetone, are metabolic products generated from fat breakdown when glucose metabolism is impaired. Studies have revealed that ketone body metabolism is intricately linked to multiple pathophysiological processes in DR, including oxidative stress, inflammatory responses, and neurodegeneration within retinal cells. This article provides a review exploring the impact of ketone body metabolism on the pathogenesis of DR, and systematically reviews the latest research progress on the impact of ketone bodies on the core pathological links such as retinal vascular barrier destruction, glial cell activation and angiogenesis through metabolic reprogramming, epigenetic modification and cell signal transduction, so as to provide a theoretical basis for in-depth understanding of the metabolic driving mechanism of DR. 
		                        		
		                        		
		                        		
		                        	
3.Association between mental health and muscle strength among Chinese adolescents aged 13-18
Chinese Journal of School Health 2025;46(9):1232-1236
		                        		
		                        			Objective:
		                        			To explore the association between mental health and muscle strength among Chinese adolescents aged  13- 18, providing a theoretical foundation and intervention strategies for mental health promotion.
		                        		
		                        			Methods:
		                        			Data were obtained from the 2019 Chinese National Survey on Students  Constitution and Health, including 98 631 Chinese adolescents aged  13- 18. Psychological distress was assessed by using the Kessler Psychological Distress Scale (K10), and mental well being was measured with the Warwick-Edinburgh Mental Well being Scale (WEMWBS). Based on the gender  and age specific  Z scores of various test items [grip strength, standing long jump, pull ups (for males), and sit ups (for females)], muscle strength index (MSI) was constructed to evaluate the comprehensive level of muscle strength in adolescents. According to the Dual factor Model (DFM) of mental health, participants were categorized into four groups:troubled, symptomatic but content, vulnerable, and complete mental health. Gender differences were analyzed by using  Chi-square tests, trends were tested with Cochran-Armitage tests, and multinomial Logistic regression models were applied to assess associations between muscle strength and mental health among adolescents.
		                        		
		                        			Results:
		                        			In 2019, 37.4% of Chinese adolescents aged 13-18 were reported of high mental distress, and 59.9% were reported of low mental well being. Boys had significantly lower rates of high mental distress (35.3%) and low mental well being (55.6%) compared to girls (39.4%, 64.3%), and the differences were of statistical significance ( χ 2=176.13, 780.42, both  P <0.05). In 2019, the rate of complete mental health among adolescents showed a downward trend with increasing age ( χ 2 trend = 258.47) and a gradual upward trend with increasing muscle strength levels ( χ 2 trend =123.14),and both boys and girls exhibited similar trends ( χ 2 trend =103.83, 168.46;  57.00 , 67.34) (all  P <0.05). The results of the unordered multiclass Logistic regression model showed that after controlling for confounding factors such as age and gender, when the completely pathological group as a reference, for every 1 unit increase in MSI in adolescents, the likelihood of being in a completely mental health state increased by 29% ( OR = 1.29); for every unit increase in the  Z-score for pull ups, the likelihood of being in a completely mental health state increased by 6% ( OR =1.06) among boys; for every 1 unit increase in sit up  Z score, the likelihood of being in a completely mental health state increased by 19% ( OR =1.19) among girls (all  P <0.05).
		                        		
		                        			Conclusions
		                        			The mental health status of Chinese adolescents is not good enough. Muscle strength is positively associated with mental health.
		                        		
		                        		
		                        		
		                        	
4.Evolution and development of mental health policies for children and adolescents in China
Chinese Journal of School Health 2025;46(9):1246-1251
		                        		
		                        			Objective:
		                        			To systematically review the development and changes in mental health policies within the National Outline for Children s Development in China from 1992 to 2030, providing a reference basis for future formulation of mental health policies among children and adolescent in China.
		                        		
		                        			Methods:
		                        			Based on the four editions of the National Outline for Children s Development in China across different periods from 1992 to 2030, word frequency analysis was used to reveal shifts in policy priorities, and an internationally recognized framework for adolescent health policy analysis was applied to conduct a textual review.
		                        		
		                        			Results:
		                        			Word frequency analysis revealed that the term "psychological" appeared 6 times in the National Outline for Children s Development in China (2001-2010) but increased to 20 times in the National Outline for Children s Development in China (2021-2030) (abbreviated as the National Outline of 2021), while the term "health" rose from 4 times in the National Outline for Children s  Development Plan in China in the 1990s to 68 times in the National Outline of 2021. The scope of mental health policy interventions expanded to encompass five key areas:health, safety, education, welfare and legal protection. Textual analysis highlighted that the policies of the National Outline for Children s Development in China were demand driven, prioritized vulnerable groups and continuously broadened their coverage, emphasizing sustainability and appropriateness, and monitoring/evaluation mechanisms. By 2023, 42.3% of primary schools and 64.8% of secondary schools employed full time mental health education teachers. However, the National Outline for Children s Development in China lacked direct evidence of children and adolescents participation in policy formulation, and publicly available mental health data disaggregated by age and gender remained limited.
		                        		
		                        			Conclusion
		                        			Mental health policies of children and adolescents in China have evolved from nonexistence to gradual refinement, yet institutionalized channels for youth involvement in policy development and evaluation remain insufficient, and transparency in age  and gender specific mental health data needs improvement.
		                        		
		                        		
		                        		
		                        	
5.Optimization of osmotic pressure swelling method in the process of hemoglobin extraction from red blood cells
Honghui ZHANG ; Wentao ZHOU ; Shasha HAO ; Hong WANG ; Jiaxin LIU ; Chengmin YANG ; Shen LI ; Fengjuan LI
Chinese Journal of Blood Transfusion 2025;38(1):91-96
		                        		
		                        			
		                        			[Objective] To extract hemoglobin (Hb) from red blood cells using osmotic pressure swelling method, expected to achieve a hemoglobin dissolution rate of ≥80% and a cell membrane integrity rate of ≥70%. [Methods] Human umbilical cord blood red blood cells were used as raw materials and phosphate buffer solution was used as the swelling solution for red blood cells. A three factor three-level orthogonal experiment (n=3) was conducted to determine the optimal matching conditions for selecting the osmolality molar concentration of phosphate buffer solution, pH value of hypotonic phosphate buffer solution and volume ratio of hypotonic phosphate buffer solution to washed red blood cells. Red blood cell swelling solution samples (n=6) were prepared by the optimal matching conditions and the original process conditions. The hemoglobin dissolution rate and cell membrane integrity rate were checked. In the expanded comparative experiment, red blood cell swelling solution samples (n=6) were prepared by the optimal matching conditions and the original process conditions, which was filtered by ultrafiltration membranes. The filtration time and hemoglobin yield were checked. [Results] The optimal matching conditions for preparing red blood cell swelling solution were obtained through orthogonal experiment as follows: osmotic pressure molar concentration was 30 mOsmol/Kg, pH was 7.8, and phosphate buffer to red blood cell volume ratio was 6∶1. On the basis of the above conditions, the red blood cell swelling solution sample was compared with the original process sample: the hemoglobin dissolution rate was (82.4±1.8)% vs (78.6±3.0)% (P<0.05), and the cell membrane integrity rate was (65.8±4.0)% vs (28.7±2.3)% (P<0.05). In the expanded comparative experiment, the optimal matching conditions were compared with the original process conditions: filtration time(s) (327±9) vs (434±13) (P<0.05), and hemoglobin yield was (72.3±1.2)% vs (66.0±1.4)% (P<0.05). [Conclusion] Compared with the original preparation process, the hemoglobin extraction process which optimized through orthogonal experiments greatly reduces the cell membrane fragmentation rate and minimizes the entry of cell membrane matrix into the target solution, ensuring a slightly higher hemoglobin dissolution rate, and reducing the preparation difficulty for the subsequent cell membrane separation and further purification.
		                        		
		                        		
		                        		
		                        	
6.Application of single-cell RNA sequencing technology in Parkinson's disease
Ziyu LIU ; Dandan GENG ; Runjiao ZHANG ; Qing LIU ; Yibo LI ; Hongfang WANG ; Wenmeng XIE ; Wenyu WANG ; Jiaxin HAO ; Lei WANG
Chinese Journal of Tissue Engineering Research 2025;29(1):193-201
		                        		
		                        			
		                        			BACKGROUND:Parkinson's disease has the main pathological changes in the midbrain,especially in the dense substantia nigra,leading to impaired motor and non-motor function in patients.At present,research is limited by cellular heterogeneity,and its pathogenesis still needs to be further elucidated.In recent years,single-cell RNA sequencing(scRNA-seq)has gradually been applied in neurodegenerative diseases,which is of great significance for understanding intercellular heterogeneity,disease development mechanisms,and treatment strategies. OBJECTIVE:To review the research progress of scRNA-seq technology applied to Parkinson's disease in recent years,providing a theoretical basis for the application of scRNA-seq in the treatment and diagnosis of Parkinson's disease. METHODS:The first author used a computer system to search for relevant literature in the CNKI,WanFang,PubMed,and Web of Science databases,with the Chinese search terms"single-cell RNA sequencing,Parkinson's disease,cell heterogeneity,cell subtypes,dopaminergic neurons,glial cells"and English search terms"single-cell RNA seq,Parkinson disease,heterogenicity,subtypes,dopaminergic neurons,glial cells."71 articles were ultimately included for review and analysis. RESULTS AND CONCLUSION:(1)scRNA-seq is a high-throughput experimental technique that utilizes RNA sequencing at the single-cell level to quantify gene expression profiles in specific cell populations,revealing cellular mysteries at the molecular level.Compared with traditional sequencing techniques,scRNA-seq technology is used to reveal the diversity of cell types and changes in specific gene expression in complex tissues under various physiological and pathological conditions through automatic clustering analysis of cell transcriptome.(2)By using scRNA-seq,the development process of dopaminergic neurons and the unique functional characteristics of various cell subtypes are elucidated,in order to better understand potential therapeutic molecular targets.(3)The use of scRNA-seq analysis has improved our understanding of the response of Parkinson's disease glial cells,enabling us to comprehensively map and characterize different cell type populations,identify specific glial cell subpopulations related to neurodegeneration,and draw valuable single cell maps as reference data for future research.(4)The application of scRNA-seq to detect embryonic mice and stem cells will help improve the in vitro differentiation protocol and quality control of cell therapy,as well as evaluate the overall cell quality and developmental stage of dopaminergic neurons derived from stem cells.
		                        		
		                        		
		                        		
		                        	
7.The renoprotective effect of Salvia miltiorrhiza injection on HBOC-CHP01 resuscitated haemorrhagic shock rats
Shasha HAO ; Xintong XIE ; Shen LI ; Honghui ZHANG ; Hong WANG ; Chengmin YANG ; Wentao ZHOU ; Jiaxin LIU
Chinese Journal of Blood Transfusion 2025;38(2):172-180
		                        		
		                        			
		                        			[Objective] To investigate the protective effect of Salvia miltiorrhiza injection (SMI) on the kidneys of HBOC-CHP01 resuscitated haemorrhagic shock rats. [Methods] A 50% haemorrhagic shock rat model was established, with 12 rats divided into two groups: SMI + HBOC-CHP01 group and HBOC-CHP01 group, with 6 rats in each group. The rats in the SMI+ HBOC-CHP01 group were given an equal volume of HBOC-CHP01 for resuscitation after haemorrhagic shock, and an 8 mL/kg dose of SMI. Rats in the HBOC-CHP01 group were resuscitated by administering an equilibrium blood loss volume of HBOC-CHP01 and given an 8 mL/kg dose of 0.9% NaCl solution. Blood was taken from rats at five points: before bloodletting (baseline), during haemorrhagic shock (HS), immediately after resuscitation (RS0h), 1 h after resuscitation (RS1h), and 24 h after resuscitation (RS24h). A blood gas analyser was used to detect the lactate level (Lac), glucose content (Glu), residual base (BEecf), pH, bicarbonate (HCO3-), high iron haemoglobin (MetHb). White blood cells (WBC), platelets (PLT), haemoglobin content (Hb), carboxyhaemoglobin (COHb) were detected using a quintuple classification. Blood creatinine (SCr), uric acid (UA), kidney-related indexes were detected using biochemistry instrument. Kidney tissues of the rats were taken after 24 h of resuscitation and after execution, and the inflammation of kidneys of the rats of the two groups was analyzed using HE staining. Fluorescence staining was used to detect the level of ROS in the kidneys of rats in both groups. [Results] At RS 0h, the Beecf, Glu and Lac levels of rats in the SMI+HBOC-CHP01 group were significantly lower than those of rats in the HBOC-CHP01 group, and the pH level of rats in the SMI+HBOC-CHP01 group was significantly higher than that of rats in the HBOC-CHP01 group, and the Glu levels of rats in the SMI+HBOC-CHP01 group were significantly lower than those of rats in the HBOC-CHP01 group at RS 1h. At RS 0h, the WBC, PLT and COHb contents of rats in the SMI+HBOC-CHP01 group were all significantly higher than those of rats in the HBOC-CHP01 group, and at RS 1h, the WBC content of rats in the SMI+HBOC-CHP01 group was significantly higher than that of rats in the HBOC-CHP01 group; at RS 1h, the UA content of rats in the SMI+HBOC-CHP01 group was significantly lower than that of rats in the HBOC-CHP01 group; at RS 24h, the SCr content of rats in the SMI+HBOC-CHP01 group was significantly lower than that of rats in the HBOC-CHP01 group; at RS 24h, the inflammation level of kidney tissues of rats in the SMI+HBOC-CHP01 group was significantly lower than that of rats in the HBOC -CHP01 group rats, and the ROS and MPO levels in the kidney tissues of rats in the SMI+HBOC-CHP01 group were significantly lower than those of rats in the HBOC-CHP01 group. [Conclusion] The combination of Salvia miltiorrhiza injection during the resuscitation of rats with severe haemorrhagic shock by HBOC-CHP01 can alleviate renal injury by reducing inflammatory response and oxidative stress.
		                        		
		                        		
		                        		
		                        	
8.Study on soluble microneedles loaded with aPexos and EGCG for healing of burn wounds in rats
Along ZHANG ; Wanjing LI ; Hong WANG ; Jiaxin LIU ; Ye CAO ; Rui ZHONG
Chinese Journal of Blood Transfusion 2025;38(3):333-342
		                        		
		                        			
		                        			 [Objective] To use Chitosan Methacryloyl (CSMA) loaded with artificial platelet-derived exosomes (aPexos) as the needle body material, and gelatin and carboxymethyl chitosan (CMCS) loaded with Epigallocatechin gallate(EGCG) as the backing material to prepare microneedles, aims to investigate the therapeutic effect and mechanism of aPexos-EGCG microneedles in the treatment of burn wound healing. [Methods] First, aPexos were extracted using ultrasound and gradient ultracentrifugation. The concentration, morphology, and growth factor content (TGF-β1, PDGF-BB, VEGF) of the exosomes were assessed using NTA, transmission electron microscopy, and ELISA kits. The aPexos and EGCG were then dissolved in the needle body and backing materials, respectively. The aPexos and EGCG were loaded into the needle body and backing layer, respectively. The morphology, mechanical properties, and puncture performance of the microneedles were examined, and the preparation conditions for the microneedles loaded with aPexos-EGCG were optimized. Finally, animal experiments and tissue staining were conducted to assess the efficacy of the aPexos-EGCG microneedles in promoting burn wound healing in rats. [Results] The size distribution of aPexos was mainly in the range of 50-150 nm, with an average diameter of 132.7±3.8 nm and an average concentration of approximately 2.88×1013±3.62×1012 particles/mL. The concentrations of TGF-β1, PDGF-BB, and VEGF were approximately 1 363±135.9 ng/mL, 63.5±14.8 ng/mL, and 1 606.0±77.5 pg/mL, respectively. Mechanical property testing of the microneedles indicated that, compared with the blank microneedles, the drug-loaded microneedles had sufficient piercing ability to penetrate the stratum corneum of the skin. The results of the rat burn wound healing experiment showed that the wound healing rates of the d 3, d 7, aPexos-EGCG microneedle group compared to the control group showed significant differences, with respective values of (47.64±12.5)% vs (18.11±6.40)%, (87.45±5.57)% vs (79.85±5.03)%(P<0.05). Hematoxylin-eosin (HE) staining revealed more intact epidermal layers in the aPexos-EGCG microneedle group. Masson staining showed that the collagen deposition percentage in the aPexos-EGCG microneedle group was higher than in the control group. Immunofluorescence staining results indicated that the aPexos-EGCG microneedle group had a reduced content of M1-type macrophages and an increased content of M2-type macrophages. Additionally, the vascular markers CD31 and α-SMA showed elevated expression in this group, with significant differences compared to the other groups (P<0.05). [Conclusion] The aPexos-EGCG microneedle developed in this study exhibits good mechanical properties, capable of successfully penetrating the skin's stratum corneum and delivering aPexos and EGCG. aPexos promotes wound repair, while EGCG regulates the ratio of M1 and M2 macrophages at the wound site and suppresses the inflammatory response. This microneedle can effectively promote wound healing in rats with burn injuries, offering a novel approach for tissue repair.
		                        		
		                        		
		                        		
		                        	
9.Exploring Molecular Mechanism of Gypenoside L against Ovarian Cancer Based on Ferroptosis Pathway Mediated by Mature-tRNA-Asp-GTC/ATF3-LPCAT3
Jingxuan ZHU ; Jiao ZHAO ; Qun WANG ; Xiaofei SUN ; Jiaxin WANG ; Hongda ZHANG ; Nan SONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):107-117
		                        		
		                        			
		                        			ObjectiveTo investigate the role of mature-tRNA-Asp-GTC and pre-tRNA-Arg-TCT in the ferroptosis phenotype of ovarian cancer (OC) cells and the regulatory mechanism of gypenoside L (Gyp-L) on mature-tRNA-Asp-GTC and pre-tRNA-Arg-TCT in OC cells. MethodsThe proliferation of human ovarian adenocarcinoma OVCAR3 cells was detected by cell counting kit-8 (CCK-8) assay, and the half-maximal inhibitory concentration (IC50) values of cisplatin (DDP), Gyp-L, and DDP in the presence of Gyp-L were calculated to determine the intervention concentration for subsequent experiments. Cell cloning assay and scratch assay reflected the proliferation and migration ability of OVCAR3 cells. PANDORA-seq small RNA sequencing was used to detect the differentially expressed transfer RNA-derived small RNAs (tsRNAs) in the cells after Gyp-L intervention, and the corresponding target genes of the tsRNAs were found by the RNAhybrid software. Malondialdehyde (MDA), glutathione (GSH), and lipid peroxide (LPO) levels were measured by colorimetry or enzyme linked immunosorbent assay (ELISA) method, Fe2+ content by FerroOrange fluorescent probe, and reactive oxygen species (ROS) content by DCFH-DA fluorescent probe to reflect the occurrence of ferroptosis in OVCAR3 cells. OVCAR3 cells were divided into a control group, a 50 µmol·L-1 Gyp-L group, and a 100 µmol·L-1 Gyp-L group. Quantitative real-time polymerase chain reaction (PCR) was performed to detect the expression of mature-tRNA-Asp-GTC, mature-tRNA-Leu-CAA, mature-mt_tRNA-Tyr-GTA_5_end, mature-tRNA-Val-CAC, mature-mt_tRNA-Glu-TTC, pre-tRNA-Arg-TCT, mature-tRNA-Asn-GTT, hydroxymethylbilane synthase (HMBS), Wnt, β-catenin, glutathione peroxidase 4 (GPX4), Kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid 2-related factor 2 (Nrf2), activating transcription factor 3 (ATF3), cystine/glutamate antiporter xCT, lysophosphatidylcholine acyltransferase 3 (LPCAT3), and arachidonate 15-lipoxygenase (ALOX15). Western blot was performed to detect the expression of HMBS, Wnt, β-catenin, GPX4, KEAP1, Nrf2, ATF3, xCT, LPCAT3, and ALOX15 proteins. ResultsThe 50 µmol·L-1 Gyp-L, 100 µmol·L-1 Gyp-L, DDP, 50 µmol·L-1 Gyp-L+DDP, and 100 µmol·L-1 Gyp-L+DDP groups showed significantly inhibited proliferation and migration of OVCAR3 cells (P<0.05) and exacerbated cell ferroptosis as reflected by the increase in the content of ROS, MDA, LPO, and Fe2+, as well as a decrease in the content of GSH (P<0.05). Compared with the control group, Gyp-L effectively interfered with the expression of 25 tsRNAs in OVCAR3 cells (P<0.05, |log2Fc|>1). Pre-tRNA-Arg-TCT/HMBS/Wnt/β-catenin/GPX4, pre-tRNA-Arg-TCT/KEAP1/NRF2/xCT, mature-tRNA-Asp-GTC/ATF3/KEAP1/NRF2/xCT, and mature-tRNA-Asp-GTC/LPCAT3/ALOX15 axial expression was significantly aberrant after Gyp-L intervention (P<0.05). ConclusionThe pre-tRNA-Arg-TCT/HMBS/Wnt/β-catenin/GPX4, pre-tRNA-Arg-TCT/KEAP1/Nrf2/xCT, mature-tRNA-Asp-GTC/ATF3/KEAP1/Nrf2/xCT, and mature-tRNA-Asp-GTC/LPCAT3/ALOX15 signaling pathways are involved in OC development. Gyp-L inhibits OC development by activating OVCAR3 cell ferroptosis onset mainly through the mature-tRNA-Asp-GTC/ATF3/KEAP1/Nrf2/xCT and mature-tRNA-Asp-GTC/LPCAT3/ALOX15 signaling axes. 
		                        		
		                        		
		                        		
		                        	
10.Exploring Molecular Mechanism of Gypenoside L against Ovarian Cancer Based on Ferroptosis Pathway Mediated by Mature-tRNA-Asp-GTC/ATF3-LPCAT3
Jingxuan ZHU ; Jiao ZHAO ; Qun WANG ; Xiaofei SUN ; Jiaxin WANG ; Hongda ZHANG ; Nan SONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(11):107-117
		                        		
		                        			
		                        			ObjectiveTo investigate the role of mature-tRNA-Asp-GTC and pre-tRNA-Arg-TCT in the ferroptosis phenotype of ovarian cancer (OC) cells and the regulatory mechanism of gypenoside L (Gyp-L) on mature-tRNA-Asp-GTC and pre-tRNA-Arg-TCT in OC cells. MethodsThe proliferation of human ovarian adenocarcinoma OVCAR3 cells was detected by cell counting kit-8 (CCK-8) assay, and the half-maximal inhibitory concentration (IC50) values of cisplatin (DDP), Gyp-L, and DDP in the presence of Gyp-L were calculated to determine the intervention concentration for subsequent experiments. Cell cloning assay and scratch assay reflected the proliferation and migration ability of OVCAR3 cells. PANDORA-seq small RNA sequencing was used to detect the differentially expressed transfer RNA-derived small RNAs (tsRNAs) in the cells after Gyp-L intervention, and the corresponding target genes of the tsRNAs were found by the RNAhybrid software. Malondialdehyde (MDA), glutathione (GSH), and lipid peroxide (LPO) levels were measured by colorimetry or enzyme linked immunosorbent assay (ELISA) method, Fe2+ content by FerroOrange fluorescent probe, and reactive oxygen species (ROS) content by DCFH-DA fluorescent probe to reflect the occurrence of ferroptosis in OVCAR3 cells. OVCAR3 cells were divided into a control group, a 50 µmol·L-1 Gyp-L group, and a 100 µmol·L-1 Gyp-L group. Quantitative real-time polymerase chain reaction (PCR) was performed to detect the expression of mature-tRNA-Asp-GTC, mature-tRNA-Leu-CAA, mature-mt_tRNA-Tyr-GTA_5_end, mature-tRNA-Val-CAC, mature-mt_tRNA-Glu-TTC, pre-tRNA-Arg-TCT, mature-tRNA-Asn-GTT, hydroxymethylbilane synthase (HMBS), Wnt, β-catenin, glutathione peroxidase 4 (GPX4), Kelch-like ECH-associated protein 1 (KEAP1), nuclear factor erythroid 2-related factor 2 (Nrf2), activating transcription factor 3 (ATF3), cystine/glutamate antiporter xCT, lysophosphatidylcholine acyltransferase 3 (LPCAT3), and arachidonate 15-lipoxygenase (ALOX15). Western blot was performed to detect the expression of HMBS, Wnt, β-catenin, GPX4, KEAP1, Nrf2, ATF3, xCT, LPCAT3, and ALOX15 proteins. ResultsThe 50 µmol·L-1 Gyp-L, 100 µmol·L-1 Gyp-L, DDP, 50 µmol·L-1 Gyp-L+DDP, and 100 µmol·L-1 Gyp-L+DDP groups showed significantly inhibited proliferation and migration of OVCAR3 cells (P<0.05) and exacerbated cell ferroptosis as reflected by the increase in the content of ROS, MDA, LPO, and Fe2+, as well as a decrease in the content of GSH (P<0.05). Compared with the control group, Gyp-L effectively interfered with the expression of 25 tsRNAs in OVCAR3 cells (P<0.05, |log2Fc|>1). Pre-tRNA-Arg-TCT/HMBS/Wnt/β-catenin/GPX4, pre-tRNA-Arg-TCT/KEAP1/NRF2/xCT, mature-tRNA-Asp-GTC/ATF3/KEAP1/NRF2/xCT, and mature-tRNA-Asp-GTC/LPCAT3/ALOX15 axial expression was significantly aberrant after Gyp-L intervention (P<0.05). ConclusionThe pre-tRNA-Arg-TCT/HMBS/Wnt/β-catenin/GPX4, pre-tRNA-Arg-TCT/KEAP1/Nrf2/xCT, mature-tRNA-Asp-GTC/ATF3/KEAP1/Nrf2/xCT, and mature-tRNA-Asp-GTC/LPCAT3/ALOX15 signaling pathways are involved in OC development. Gyp-L inhibits OC development by activating OVCAR3 cell ferroptosis onset mainly through the mature-tRNA-Asp-GTC/ATF3/KEAP1/Nrf2/xCT and mature-tRNA-Asp-GTC/LPCAT3/ALOX15 signaling axes. 
		                        		
		                        		
		                        		
		                        	
            

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