1.Erjingwan Alleviate Inflammatory Response and Apoptosis in Skeletal Muscle Cells of Sarcopenia via SIRT1/Nrf2/HO-1 Signaling Pathway
Long SHI ; Yang LI ; Hongyu YAN ; Tianle ZHOU ; Zhiwen ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(3):57-66
ObjectiveTo investigate the effects of the classical Chinese medicine compound prescription Erjingwan on the inflammatory response and apoptosis of skeletal muscle cells in a mouse model of sarcopenia and decipher the mechanism based on the silent information regulator 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. MethodsForty C57/BL6 male mice were randomized into a control group, a model group, and groups with different doses of Erjingwan (8,16,32 g·kg-1). The mouse model of sarcopenia was established by D-gal-induced skeletal muscle senescence. The body weight and grip strength of mice treated with different doses of Erjingwan were examined to evaluate their physiological functions. Hematoxylin-eosin (HE) staining and Masson staining were used to observe the pathological changes and fibrosis in the skeletal muscle of mice. Enzyme-linked immunosorbent assay (ELISA) was adopted to determine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum samples of mice, and biochemical tests were conducted to quantify the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) in the serum. The protein and mRNA levels of SIRT1, Nrf2, B-cell lymphoma (Bcl-2), and Bcl-2-associated X protein (Bax) were determined by Western blot and Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), respectively. ResultsAfter 4 weeks of drug intervention, the model group exhibited significant reductions in body weight and grip strength (P0.01) compared with the control group. Compared with the model group, all doses of Erjingwan increased the body weight in mice at week 8 (P0.01) and grip strength from week 6 (P0.01). HE staining revealed clear muscle fiber structure in the control group, muscle fiber rupture and atrophy in the model group, and dose-dependent repair of muscle fiber structure in the Erjingwan groups. Masson staining showed minimal collagen fibers and mild fibrosis in the control group, collagen fiber proliferation and severe fibrosis in the model group, and collagen proliferation with dose-dependent inhibition of fibrosis in the Erjingwan groups. ELISA results showed that serum levels of TNF-α and IL-6 were elevated in the model group compared with those in the control group (P0.01). After intervention, the low-dose Erjingwan group exhibited a decreased TNF-α level (P0.05), while the medium and high-dose groups showed decreases in both TNF-α and IL-6 levels (P0.01). Biochemical assays revealed that the model group had decreased SOD and GSH levels (P0.01) and an increased MDA level (P0.01) compared with the control group. The medium and high-dose Erjingwan groups exhibited increases in SOD and GSH levels (P0.01) and decreases in MDA level (P0.01), compared with the model group. WB and Real-time PCR results showed that compared with the control group, the model group presented down-regulated protein and mRNA levels of SIRT1, Nrf2, HO-1, and Bcl-2 in the muscle tissue (P0.01) and up-regulated protein and mRNA levels of Bax (P0.01). Compared with the model group, Erjingwan at different doses up-regulated the protein levels of SIRT1, Nrf2, HO-1, and Bcl-2 (P0.01) and down-regulated the protein and mRNA levels of Bax (P0.01) in the muscle tissue. Low-dose Erjingwan elevated the mRNA levels of Nrf2 and HO-1 (P0.05, P0.01), and medium and high-dose Erjingwan up-regulated the mRNA levels of SIRT1, Nrf2, HO-1, and Bcl-2 (P0.01). ConclusionErjingwan reduced the content of inflammatory factors in skeletal muscle cells, improved the antioxidant capacity, and attenuated pathological changes and fibrosis in the muscle of the mouse model of sarcopenia by regulating the SIRT1/Nrf2/HO-1 pathway, inflammatory response, and apoptosis network.
2.Erjingwan Alleviate Inflammatory Response and Apoptosis in Skeletal Muscle Cells of Sarcopenia via SIRT1/Nrf2/HO-1 Signaling Pathway
Long SHI ; Yang LI ; Hongyu YAN ; Tianle ZHOU ; Zhiwen ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(3):57-66
ObjectiveTo investigate the effects of the classical Chinese medicine compound prescription Erjingwan on the inflammatory response and apoptosis of skeletal muscle cells in a mouse model of sarcopenia and decipher the mechanism based on the silent information regulator 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. MethodsForty C57/BL6 male mice were randomized into a control group, a model group, and groups with different doses of Erjingwan (8,16,32 g·kg-1). The mouse model of sarcopenia was established by D-gal-induced skeletal muscle senescence. The body weight and grip strength of mice treated with different doses of Erjingwan were examined to evaluate their physiological functions. Hematoxylin-eosin (HE) staining and Masson staining were used to observe the pathological changes and fibrosis in the skeletal muscle of mice. Enzyme-linked immunosorbent assay (ELISA) was adopted to determine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum samples of mice, and biochemical tests were conducted to quantify the levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) in the serum. The protein and mRNA levels of SIRT1, Nrf2, B-cell lymphoma (Bcl-2), and Bcl-2-associated X protein (Bax) were determined by Western blot and Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), respectively. ResultsAfter 4 weeks of drug intervention, the model group exhibited significant reductions in body weight and grip strength (P0.01) compared with the control group. Compared with the model group, all doses of Erjingwan increased the body weight in mice at week 8 (P0.01) and grip strength from week 6 (P0.01). HE staining revealed clear muscle fiber structure in the control group, muscle fiber rupture and atrophy in the model group, and dose-dependent repair of muscle fiber structure in the Erjingwan groups. Masson staining showed minimal collagen fibers and mild fibrosis in the control group, collagen fiber proliferation and severe fibrosis in the model group, and collagen proliferation with dose-dependent inhibition of fibrosis in the Erjingwan groups. ELISA results showed that serum levels of TNF-α and IL-6 were elevated in the model group compared with those in the control group (P0.01). After intervention, the low-dose Erjingwan group exhibited a decreased TNF-α level (P0.05), while the medium and high-dose groups showed decreases in both TNF-α and IL-6 levels (P0.01). Biochemical assays revealed that the model group had decreased SOD and GSH levels (P0.01) and an increased MDA level (P0.01) compared with the control group. The medium and high-dose Erjingwan groups exhibited increases in SOD and GSH levels (P0.01) and decreases in MDA level (P0.01), compared with the model group. WB and Real-time PCR results showed that compared with the control group, the model group presented down-regulated protein and mRNA levels of SIRT1, Nrf2, HO-1, and Bcl-2 in the muscle tissue (P0.01) and up-regulated protein and mRNA levels of Bax (P0.01). Compared with the model group, Erjingwan at different doses up-regulated the protein levels of SIRT1, Nrf2, HO-1, and Bcl-2 (P0.01) and down-regulated the protein and mRNA levels of Bax (P0.01) in the muscle tissue. Low-dose Erjingwan elevated the mRNA levels of Nrf2 and HO-1 (P0.05, P0.01), and medium and high-dose Erjingwan up-regulated the mRNA levels of SIRT1, Nrf2, HO-1, and Bcl-2 (P0.01). ConclusionErjingwan reduced the content of inflammatory factors in skeletal muscle cells, improved the antioxidant capacity, and attenuated pathological changes and fibrosis in the muscle of the mouse model of sarcopenia by regulating the SIRT1/Nrf2/HO-1 pathway, inflammatory response, and apoptosis network.
3.Mechanism study of SIRT3 alleviating oxidative-stress injury in renal tubular cells by promoting mitochondrial biogenesis via regulating mitochondrial redox balance
Yaojun LIU ; Jun ZHOU ; Jing LIU ; Yunfei SHAN ; Huhai ZHANG ; Pan XIE ; Liying ZOU ; Lingyu RAN ; Huanping LONG ; Lunli XIANG ; Hong HUANG ; Hongwen ZHAO
Organ Transplantation 2026;17(1):86-94
Objective To elucidate the molecular mechanism of sirtuin-3 (SIRT3) in regulating mitochondrial biogenesis in human renal tubular epithelial cells. Methods Cells were stimulated with different concentrations of H2O2 and divided into four groups: control (NC), 50 μmol/L H2O2, 110 μmol/L H2O2 and 150 μmol/L H2O2. SIRT3 protein expression was then measured. SIRT3 was knocked down with siRNA, and cells were further assigned to five groups: control (NC), negative-control siRNA (NCsi), SIRT3-siRNA (siSIRT3), NCsi+H2O2, and siSIRT3+H2O2. After 24 h, cellular adenosine triphosphate (ATP) and mitochondrial superoxide anion (O2•−) levels were determined, together with mitochondrial expression of SIRT3, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), superoxide dismutase 2 (SOD2), acetylated-SOD2 and adenosine monophosphate activated protein kinase α1 (AMPKα1). Results The 110 and 150 μmol/L H2O2 decreased SIRT3 protein (both P<0.05). ATP and mitochondrial O2•− did not differ between NC and NCsi groups (both P>0.05). Compared to the NCsi group, the siSIRT3 group exhibited elevated O2•− level, decreased SIRT3 protein and increased expression levels of SOD2 and acetylated SOD2 protein (all P<0.05). Compared to the NCsi group, the NCsi+H2O2 group exhibited decreased cellular ATP levels, elevated mitochondrial O2•− levels, and reduced protein expression levels of SIRT3, SOD2, TFAM, AMPKα1, PGC-1α and NRF1 (all P<0.05). Compared with the siSIRT3 group, the siSIRT3+H2O2 group showed a decrease in cellular ATP levels, an increase in mitochondrial O2•− levels, a decrease in SIRT3, SOD2, TFAM, AMPKα1, PGC-1α and NRF1 protein expression levels and a decrease in acetylated SOD2 protein expression levels (all P<0.05). Compared with the NCsi+H2O2 group, the siSIRT3+H2O2 group showed a decrease in cellular ATP levels, an increase in mitochondrial O2•− levels, a decrease in SIRT3, AMPKα1, PGC-1α and NRF1, TFAM protein expression levels, and an increase in SOD2 and acetylated SOD2 protein expression levels (all P<0.05). Conclusions SIRT3 promotes mitochondrial biogenesis in tubular epithelial cells via the AMPK/PGC-1α/NRF1/TFAM axis, representing a key mechanism through which SIRT3 ameliorates oxidative stress-induced mitochondrial dysfunction.
4.Quality evaluation of Qingwen hufei granules based on fingerprints combined with multi-component content determination
Huiying ZHOU ; Yuan WANG ; Yani WANG ; Yun YANG ; Bo WANG ; Shuanzhu YANG ; Liping CAO ; Hong ZHANG ; Kaihua LONG
China Pharmacy 2026;37(3):338-343
OBJECTIVE To provide a scientific basis for the quality evaluation and clinical application of Qingwen hufei granules. METHODS Fourteen batches of Qingwen hufei granules were used as samples to establish high-performance liquid chromatography (HPLC) fingerprints using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition). The chromatographic peaks were identified and the similarity was evaluated. Cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to conduct chemical pattern recognition analysis on the 14 batches of samples. Meanwhile, the contents of neochlorogenic acid (NGA), chlorogenic acid (CHA), cryptochlorogenic acid (CGA), forsythoside A (FTA), 3,5-O-dicaffeoylquinic acid (3,5-O- DA), 4,5-O-dicaffeoylquinic acid (4,5-O-DA), and angoroside C (AGC) in the samples were determined by HPLC. RESULTS The methodological investigation results of both the fingerprint and the content determination complied with the relevant requirements. Fourteen common peaks were indicated in the HPLC fingerprints of the 14 batches of samples, and 7 of them were identified [NGA (peak 2), CHA (peak 3), CGA (peak 5), FTA (peak 11), 3,5-O-DA (peak 12), 4,5-O-DA (peak 13), and AGC (peak 14)]; the similarity of each sample was greater than 0.94. The results of CA and PCA showed that the samples could be classified into 3 categories; the results of OPLS-DA indicated that peak 4 (unknown), peak 11 (FTA), peak 8 (unknown), peak 9 (unknown), and peak 1 (unknown) were the differential components. The content ranges of NGA, CHA, CGA, 3,5-O-DA, FTA, 4,5-O-DA and AGC in the 14 batches of samples were 0.210 4-0.458 7, 0.269 1-0.506 3, 0.228 1-0.461 1, 0.443 9-1.044 6, 0.066 7-0.155 7, 0.062 8-0.143 8, and 0.057 4-0.105 7 mg/g, respectively. CONCLUSIONS The HPLC fingerprint and multi-component content determination methods established in this study are efficient and reliable, and can be used for the quality evaluation of Qingwen hufei granules.
5.Influencing Factors of Depression in Patients with Postoperative Ovarian Cancer
Jialiang YAO ; Long ZHANG ; Jianhui TIAN ; Ze LIU ; Yun YANG ; Yiyang ZHOU ; Minghua LI ; Wang YAO ; Wenfei SHI ; Xinyi LU ; Pan YU ; Enchao CONG
Cancer Research on Prevention and Treatment 2026;53(5):349-359
Objective To explore the prevalence of depressive symptoms in postoperative patients with ovarian cancer and to analyze its influencing factors from multiple dimensions, including clinical characteristics, psychological factors, and laboratory indicators. Methods A cross-sectional study was conducted, which enrolled 235 postoperative patients with ovarian cancer. Depressive status was assessed using the patient health questionnaire, and the demographic, pathological, and medical record data of the patients were collected using the generalized anxiety disorder scale, Pittsburgh sleep quality index, European organization for research and treatment of cancer quality of life questionnaire core 30, and ECOG performance status score. Peripheral blood tumor marker (CA125), routine blood test, lymphocyte subsets, and serum cytokine levels were measured. Univariate and multivariate binary logistic regression analysis were used for statistical analysis. Results The prevalence of depression in postoperative patients with ovarian cancer was 39.15% (92/235). Univariate analysis showed that ECOG score ≥ 2 points, pain, anxiety, poor sleep quality, low quality of life, low life satisfaction, tumor recurrence, six or more cycles of chemotherapy, as well as higher levels of CA125, NLR, and NAR, and lower hemoglobin levels were significantly associated with depression (all P<0.05). Multivariate binary Logistic regression analysis showed that anxiety (OR=1.975, 95%CI: 1.231-3.170), sleep efficiency (OR=4.181, 95%CI: 1.211-14.43), sleep latency (OR=34.806, 95%CI: 4.258-284.542), ECOG performance status score, cognitive function (OR=0.918, 95%CI: 0.868-0.97), and life satisfaction were independent risk factors for depression (all P<0.05). Laboratory indicators were not independent influencing factors in the multivariate Logistic regression model. Conclusion Depression in postoperative patients with ovarian cancer is influenced by physiological, psychological, and social factors. Clinical management should focus on patients with anxiety, sleep disorders, poor physical condition, and low life satisfaction, and a comprehensive prevention and treatment strategy centered on psychological intervention and taking into account symptom management and social support should be implemented.
6.Study on the mechanism of hyperoside regulating hepatic lipid synthesis to ameliorate non-alcoholic fatty liver disease in mice
Changrui LONG ; Shijian XIANG ; Zhenhua ZHANG ; Huixing WU ; Benjie ZHOU ; Chengyu LU
China Pharmacy 2025;36(6):668-673
OBJECTIVE To investigate the improvement mechanism of hyperoside (HYP) on non-alcoholic fatty liver disease (NAFLD). METHODS Male C57BL/6 mice were randomly divided into normal (NFD) group, model (HFD) group and HYP group, with 8 mice in each group. Except for NFD group, the mice in other groups were fed with HF60 high-fat diet to establish NAFLD model; HYP group was simultaneously given HYP 100 mg/kg intragastrically every day, for 16 consecutive weeks. The body weight and liver weight of mice in each group were recorded 16 h after the last medication; the histopathological changes and lipid accumulation in the liver were observed, and the contents of triglyceride (TAG) in liver tissue and serum contents of TAG, aspartate transaminase (AST) and alanine transaminase (ALT) were measured; LC-MS/MS method was adopted to detect lipid changes in the liver tissue of mice for lipidomics analysis, and protein expressions of lipid synthesis-associated proteins peroxisome proliferator-activated receptor α (PPARα) were also tested. Human hepatocellular carcinoma cell line HepG2 was divided into normal control group, model group, HYP low-concentration group (50 μmol/L), HYP high-concentration group (100 μmol/L), HYP low-concentration+GW6471 (PPARαinhibitor) group, and HYP high-concentration+GW6471 group. Except for normal control group, the remaining cells were induced with oleic acid and palmitic acid to establish a high-fat cell model. The accumulation of lipid droplets in each group of cells was observed, and the TAG content was detected. RESULTS Compared with HFD group, HYP group exhibited significant reductions in liver fat vacuoles, lipid accumulation, liver weight, and TAG content in liver tissue, as well as serum contents of ALT, AST and TAG (P<0.05). Additionally, the expression of PPARα protein in liver tissue was significantly increased (P<0.05), and the pathological morphological changes associated with NAFLD were alleviated. Lipidomic analysis revealed that HYP significantly reduced the levels of TAG, diacylglycerol and other lipids in the liver. Compared with model group, cellular lipid droplet accumulation and TAG content decreased significantly in HYP low- and high-concentration groups (P<0.05); GW6471 could significantly reverse the improvement effect of HYP on above indicators (P<0.05). CONCLUSIONS HYP can effectively ameliorate NAFLD induced by a high-fat diet in mice, and the mechanism may be related to the activation of PPARα to regulate hepatic lipid synthesis.
7.Effect of Kuanxiong Aerosol on Perioperative Coronary Microcirculation in Patients with Unstable Angina Undergoing Elective PCI: A Pilot Randomized Controlled Trial.
Zi-Hao LIU ; Wen-Long XING ; Hong-Xu LIU ; Ju-Ju SHANG ; Ai-Yong LI ; Qi ZHOU ; Zhen-Min ZHANG ; Zhi-Bao LI ; Ke-Ji CHEN
Chinese journal of integrative medicine 2025;31(3):206-214
OBJECTIVE:
To evaluate the immediate effect of Kuanxiong Aerosol (KXA) on perioperative coronary microcirculation in patients with unstable angina (UA) suffering from elective percutaneous coronary intervention (PCI).
METHODS:
From February 2021 to July 2023, UA inpatients who underwent PCI alone in the left anterior descending (LAD) branch were included. Random numbers were generated to divide patients into the trial group and the control group at a ratio of 1:1. The index of coronary microcirculation resistance (IMR) was measured before PCI, and the trial group was given two sprays of KXA, while the control group was not given. IMR was measured again after PCI, cardiac troponin I (cTnI) and creatine kinase isoenzyme-MB (CK-MB) were detected before and 24 h after surgery, and major cardiovascular adverse events (MACEs) were recorded for 30 days. The data statistics and analysis personnel were blinded.
RESULTS:
Totally 859 patients were screened, and 62 of them were involved into this study. Finally, 1 patient in the trial group failed to complete the post-PCI IMR and was excluded, 30 patients were included for data analysis, while 31 patients in the control group were enrolled in data analysis. There was no significant difference in baseline data (age, gender, risk factors, previous history, biochemical index, and drug therapy, etc.) between the two groups. In addition, differences in IMR, cTnI and CK-MB were not statistically significant between the two groups before surgery. After PCI, the IMR level of the trial group was significantly lower than that of the control group (19.56 ± 14.37 vs. 27.15 ± 15.03, P=0.048). Besides, the incidence of perioperative myocardial injury (PMI) was lower in the trial group, but the difference was not statistically significant (6.67% vs. 16.13%, P=0.425). No MACEs were reported in either group.
CONCLUSIONS
KXA has the potential of improving coronary microvascular dysfunction. This study provides reference for the application of KXA in UA patients undergoing elective PCI. (Registration No. ChiCTR2300069831).
Humans
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Percutaneous Coronary Intervention
;
Male
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Microcirculation/drug effects*
;
Female
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Angina, Unstable/physiopathology*
;
Pilot Projects
;
Middle Aged
;
Aged
;
Drugs, Chinese Herbal/pharmacology*
;
Aerosols
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Troponin I/blood*
;
Coronary Circulation/drug effects*
;
Elective Surgical Procedures
8.Assessment of perioperative pulmonary fluid volume using remote dielectric sensing (ReDSTM) non-invasive lung fluid measurement technology in transcatheter tricuspid valve-in-valve implantation: The first case report
Yuliang LONG ; Yuan ZHANG ; Xiaochun ZHANG ; Peng WANG ; Xiaotong CUI ; Wenzhi PAN ; Daxin ZHOU ; Junbo GE
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2025;32(04):571-574
One of its primary surgical treatments of tricuspid regurgitation is tricuspid valve biological valve replacement. Catheter tricuspid valve-in-valve implantation is a novel interventional alternative for biological valve failure. The non-invasive lung fluid measuring device remote dielectric sensing (ReDSTM) has been increasingly incorporated into clinical practice as a means of monitoring chronic heart failure in recent years. This report describes the process and outcomes of the first instance of perioperative lung fluid volume evaluation following transcatheter tricuspid valve implantation utilizing ReDSTM technology. The patient has a short-term, substantial increase in postoperative lung fluid volume as compared to baseline.
9.INVESTIGATION OF PATHOGENS CARRIED BY RODENTS AT YUNNAN HEKOU PORT
Xiao-Long ZHANG ; Dong WANG ; Jing ZHAO ; Zhi-Liang ZHAO ; Ling-Rui YUAN ; Run-Yi ZHOU ; Jie TIAN ; Hui-Lin GUO ; Chun-Yan GAO
Acta Parasitologica et Medica Entomologica Sinica 2025;32(1):27-33,51
Objective This research was performed to identify rodent-borne pathogens in Hekou Port,Yunnan Province.Methods Rodents were captured using cages and dissected to collect their lungs,liver,spleen,and other viscera.Eight pathogens,including Yersinia pestis,Leptospira,Bartonella,and Anaplasmataceae,were identified using polymerase chain reaction amplification.Amplified pathogen sequences from positive samples were sequenced,and BLAST homology searches were conducted using GenBank to confirm pathogen identities.A phylogenetic tree of the identified pathogens was constructed using the neighbor joining method.Results The total of 31 rodents,identified as Rattus tanezumi,R.norvegicus,and Mus musculus,were captured.Among these,R.tanezumi was the dominant species,accounting for 64.52%of the total.Two pathogens,Leptospira interrogans and Neoehrlichia mikurensis,were detected,with positivity rates of 9.68%and 29.03%,respectively.No other pathogens were detected.The overall positivity rate for rodent-borne pathogens was 35.48%.Conclusions The single 16S rRNA gene fragment is insufficient for the molecular identification of all Neoehrlichia species.Accurate species identification should be based on a combined analysis of multiple genes.The prevalence of rodent-borne pathogens in Hekou Port indicates the necessity for enhanced surveillance of rodent-borne diseases and implementation of additional prevention and control measures in border ports.
10.Molecular Identification of Cremastra appendiculata and Other Original Medicinal Materials of Cremastrae Pseudobulbus and Confusing Products Based on ITS Sequences in DNA Barcodes
Jinling ZHANG ; Qiuxiao XIAO ; Qian WANG ; Sijia ZHOU ; Yali LONG ; Yong HUANG ; Lin ZHENG ; Zipeng GONG ; Yang JIN ; Yueting LI
Chinese Journal of Information on Traditional Chinese Medicine 2025;32(6):142-146
Objective To distinguish Cremastra appendiculata(D.Don)Makino,Pleione yunnanensis Rolfe and Pleione bulbocodioides,and its easily confusing products Oreorchis patens and Iphigenia indica Kunth using the ITS sequence in DNA barcodes;To explore the genetic diversity of Cremastra appendiculata germplasm resources.Methods Three different original Cremastra appendiculata,Pleione yunnanensis Rolfe and Pleione bulbocodioides,and their easily confusing products Cremastrae Pseudobulbus of Oreorchis patens and Iphigenia indica Kunth were selected as the research objects,and the genomic DNA of the above samples were extracted by the modified CTAB method,and then the ITS sequences were amplified,sequenced and spliced by PCR technology.The Kimura 2-Parameter(K2P)model was used to calculate the genetic distance,and the phylogenetic tree was constructed with the help of neighbour joining method(NJ)for genetic relationship analysis.Results Except for the Iphigenia indica Kunth species that were not found during the BLAST search,the BLAST comparison results of the other samples were higher than 95%.At the same time,the results of phylogenetic tree showed that Cremastra appendiculata,Pleione yunnanensis Rolfe and Pleione bulbocodioides were clustered into one branch,respectively,and the easily confusing products were also respectively clustered into one branch.Conclusion The ITS sequence in DNA barcodes can be used to accurately distinguish Cremastra appendiculata,Pleione yunnanensis Rolfe and Pleione bulbocodioides,and its easily confusing products Oreorchis patens and Iphigenia indica Kunth.

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