1.Liuwei Dihuangwan Promote Mitophagy to Modulate Neuroinflammation and Behavioral Impairments in Rat Model of Autism Spectrum Disorder (ASD)
Pengjue HUANG ; Mingyue JIANG ; Ji WU ; Niya YIN ; Lei OUYANG ; Qinquan ZHU ; Di ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):52-60
ObjectiveTo observe the effect of Liuwei Dihuangwan on behavioral impairments in the rat model of autism spectrum disorder (ASD) and explore the mechanism of action. MethodsTwelve SD pregnant rats were intraperitoneally injected with valproic acid (VPA) (10 rats) or normal saline (2 rats), and male offspring were selected to establish the model of ASD and the control rats. Rats were randomly assigned into model, low-dose (0.75 g·kg-1) and high-dose (1.5 g·kg-1) Liuwei Dihuangwan, vitamin D (positive drug, 3.7×10-5 g·kg-1), and blank groups. Each group was administrated with the corresponding concentration of drugs or the same volume of normal saline by gavage for 2 weeks. After the intervention, the three-chamber social test was conducted to evaluate social interaction and social preference. The open field test was carried out to observe spontaneous behavior and anxiety state. Hematoxylin-eosin staining (HE) was used to observe the pathological changes of the prefrontal tissue. Transmission electron microscopy was employed to observe the ultrastructure of mitochondria in prefrontal neurons. Immunofluorescence was used to detect the expression of ionized calcium-binding adapter molecule-1 (Iba-1) in the prefrontal tissue. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Western blot was employed to assess the expression differences of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), adenosine monophosphate-activated protein kinase (AMPK), phosphorylated Unc-51-like autophagy-activating kinase 1 (p-ULK1), Unc-51-like autophagy-activating kinase 1 (ULK1), and FUN14 domain-containing protein 1 (FUNDC1). ResultsCompared with the blank group, the model group spent less time sniffing stranger 1 and stranger 2 in the three-chamber social test (P<0.01) and showed reductions in the total distance traveled, average speed, distance traveled in the central area, and time spent in the central area in the open field test (P<0.01). In addition, the model group showed extensive apoptosis of neurons, with shrunken nuclei and red-stained cytoplasm, and extensive necrosis of neurons in the prefrontal tissue, mitochondrial swelling, decreased matrix density, disrupted cristae, and autophagic lysosomes in neurons, increases in the rate of Iba-1 positive cells in the prefrontal area (P<0.01) and the levels of TNF-α and IL-6 (P<0.01), and down-regulation in the expression of p-AMPK/AMPK, p-ULK1/ULK1, and FUNDC1 (P<0.01). Compared with the model group, low-dose and high-dose Liuwei Dihuangwan and the vitamin D prolonged the time spent sniffing stranger 1 and stranger 2 in the three-chamber social test (P<0.05, P<0.01), increased the total distance traveled, average speed, distance traveled in the central area, and time spent in the central area in the open field test (P<0.05, P<0.01), restored the morphology of neurons in the prefrontal tissue, decreased the number of apoptotic cells, alleviated the swelling of mitochondria in neurons, increased the matrix density, mitigated the fragmentation and disorder of cristae, and increased the number of autophagosomes. Moreover, the drugs decreased the rate of Iba-1 positive cells in the prefrontal area (P<0.01), lowered the levels of TNF-α and IL-6 (P<0.01), and up-regulated the expression of p-AMPK/AMPK, p-ULK1/ULK1, and FUNDC1 (P<0.01). ConclusionLiuwei Dihuangwan ameliorate autism-like behaviors and reduce neuronal apoptosis and neuroinflammatory damage in the rat model of ASD by promoting mitophagy mediated by the AMPK/ULK1/FUNDC1 pathway.
2.Effect and mechanism of Prunus mume against hepatic fibrosis
Feng HAO ; Ji LI ; Jing DU ; Yuchen OUYANG ; Yichun CUI ; Shuang WEI
China Pharmacy 2025;36(2):172-178
OBJECTIVE To explore the effect and mechanism of Prunus mume against hepatic fibrosis (HF). METHODS Male SD rats were randomly divided into normal control group (n=10) and modeling group (n=50). The modeling group established HF model using carbon tetrachloride. The modeled rats were randomly divided into model group (normal saline), positive control group [colchicine, 0.09 mg/(kg·d)], and P. mume low-dose, medium-dose and high-dose groups [1.35, 2.70, 5.40 g/(kg·d)], with 9 rats in each group. They were given the corresponding drug/normal saline intragastrically, once a day, for 8 consecutive weeks. After the last medication, the liver index was calculated, while liver function indexes, liver fiber indexes, oxidative stress indicators and inflammatory factors of rats were measured. HE staining was used to observe the pathological changes in liver tissue of rats; Masson staining was used to observe the degree of HF in liver tissue of rats; transmission electron microscopy was used to observe the ultrastructure of liver tissue in rats; TUNEL staining was used to detect liver cell apoptosis in each group of rats. Western blot method was used to detect the protein expressions of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF) in liver tissue of rats. RESULTS Compared with normal control group, the levels of alanine transaminase, alkaline phosphatase, aspartate transaminase, total bilirubin, malondialdehyde, procollagen type Ⅲ protein, Ⅳ-type pre collagenase, laminin, hyaluronic acid, interleukin-6, tumor necrosis factor-α, as well as the protein expressions of TGF-β1 and PDGF in model group were increased significantly, while the levels of superoxide dismutase and glutathione peroxidase were significantly reduced (P<0.01); the HE, Masson staining and transmission electron microscopy observation results showed obvious HF characteristics in rats of model group. Compared with model group, varying degrees of improvement in above indexes were observed in P. mume groups, and the above 2021BSZR011) indicators of rats in P. mume medium-dose and high-dose groups were reversed significantly (P<0.05 or P<0.01). CONCLUSIONS P. mume has an anti-HF effect, which may be achieved through mechanisms such as antioxidation, anti-inflammation, reduction of collagen production, inhibition of PDGF protein expression, and regulation of TGF- β1 signaling pathway.
3.Effect and mechanism of Prunus mume against hepatic fibrosis
Feng HAO ; Ji LI ; Jing DU ; Yuchen OUYANG ; Yichun CUI ; Shuang WEI
China Pharmacy 2025;36(2):172-178
OBJECTIVE To explore the effect and mechanism of Prunus mume against hepatic fibrosis (HF). METHODS Male SD rats were randomly divided into normal control group (n=10) and modeling group (n=50). The modeling group established HF model using carbon tetrachloride. The modeled rats were randomly divided into model group (normal saline), positive control group [colchicine, 0.09 mg/(kg·d)], and P. mume low-dose, medium-dose and high-dose groups [1.35, 2.70, 5.40 g/(kg·d)], with 9 rats in each group. They were given the corresponding drug/normal saline intragastrically, once a day, for 8 consecutive weeks. After the last medication, the liver index was calculated, while liver function indexes, liver fiber indexes, oxidative stress indicators and inflammatory factors of rats were measured. HE staining was used to observe the pathological changes in liver tissue of rats; Masson staining was used to observe the degree of HF in liver tissue of rats; transmission electron microscopy was used to observe the ultrastructure of liver tissue in rats; TUNEL staining was used to detect liver cell apoptosis in each group of rats. Western blot method was used to detect the protein expressions of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF) in liver tissue of rats. RESULTS Compared with normal control group, the levels of alanine transaminase, alkaline phosphatase, aspartate transaminase, total bilirubin, malondialdehyde, procollagen type Ⅲ protein, Ⅳ-type pre collagenase, laminin, hyaluronic acid, interleukin-6, tumor necrosis factor-α, as well as the protein expressions of TGF-β1 and PDGF in model group were increased significantly, while the levels of superoxide dismutase and glutathione peroxidase were significantly reduced (P<0.01); the HE, Masson staining and transmission electron microscopy observation results showed obvious HF characteristics in rats of model group. Compared with model group, varying degrees of improvement in above indexes were observed in P. mume groups, and the above 2021BSZR011) indicators of rats in P. mume medium-dose and high-dose groups were reversed significantly (P<0.05 or P<0.01). CONCLUSIONS P. mume has an anti-HF effect, which may be achieved through mechanisms such as antioxidation, anti-inflammation, reduction of collagen production, inhibition of PDGF protein expression, and regulation of TGF- β1 signaling pathway.
4.Shenfu Injection Improve Chronic Heart Failure by Regulates Glycolytic Pathway Mediated by HIF-1α/PFKFB3 Pathway
Ji OUYANG ; Kun LIAN ; Xiaoqian LIAO ; Lichong MENG ; Lin LI ; Zhenyu ZHAO ; Zhixi HU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(16):136-145
ObjectiveThis study aims to explore the mechanism and targets of Shenfu Injection in regulating glycolysis to intervene in myocardial fibrosis in chronic heart failure based on the hypoxia-inducible factor-1α (HIF-1α)/ 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) signaling pathway. MethodsA rat model of chronic heart failure was established by subcutaneous injection of isoproterenol (ISO). After successful modeling, the rats were randomly divided into the Sham group, Model group, Shenfu injection (SFI, 6 mL·kg-1) group, and inhibitor (3PO, 35 mg·kg-1) group, according to a random number table, and they were treated for 15 days. Cardiac function was evaluated by echocardiography, and serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were detected by enzyme-linked immunosorbent assay (ELISA). Fasting body weight and heart weight were measured, and the heart index (HI) was calculated. Pathological changes in myocardial tissue were observed by hematoxylin-eosin (HE) and Masson staining, and the fibrosis rate was calculated. Biochemical assays were used to determine serum levels of glucose (GLU), lactic acid (LA), and pyruvic acid (PA). Western blot was used to analyze the expression of proteins related to the HIF-1α/PFKFB3 signaling pathway (HIF-1α and PFKFB3), glycolysis-related proteins (HK1, HK2, PKM2, and LDHA), and fibrosis-related proteins [transforming growth factor (TGF)-β1, α-smooth muscle actin (α-SMA), and Collagen type Ⅰ α1 (ColⅠA1)]. Real-time PCR was used to detect the mRNA expression of HIF-1α and PFKFB3 in myocardial tissue. ResultsCompared with the Sham group, the Model group showed significantly decreased left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), interventricular septal thickness (IVSd), and interventricular septal strain (IVSs) (P<0.05), while left ventricular internal dimension at end-diastole (LVDd) and end-systole (LVIDs) were increased (P<0.05). Serum NT-proBNP levels were significantly increased (P<0.01), and body weight was decreased. Heart weight was increased, and the HIT index was increased (P<0.05). Myocardial tissue exhibited inflammatory cell infiltration and collagen fiber deposition, and the fibrosis rate was significantly increased (P<0.05). Serum GLU was decreased (P<0.05), while LA and PA levels were increased (P<0.05). Protein expressions of HIF-1α, PFKFB3, HK1, HK2, PKM2, LDHA, TGF-β1, α-SMA, and ColⅠA1, as well as the mRNA expression of HIF-1α and PFKFB3 were increased (P<0.05). Compared with the Model group, both the SFI group and 3PO groups showed significant improvements in LVEF, LVFS, IVSd, and IVSs (P<0.05) and decreases in LVDd, LVIDs, and NT-proBNP levels (P<0.05). Body weight was significantly increased. Heart weight was significantly decreased, and the HIT index was significantly decreased (P<0.05). Inflammatory cell infiltration, collagen fiber deposition, and the fibrosis rate were significantly decreased (P<0.05). Serum GLU levels were significantly increased (P<0.05), while LA and PA levels were decreased (P<0.05). Expressions of glycolysis-related proteins, fibrosis-related proteins, and HIF-1α/PFKFB3 pathway-related proteins and mRNAs were significantly suppressed (P<0.05). ConclusionSFI improves cardiac function in chronic heart failure by downregulating the expression of HIF-1α/PFKFB3 signaling pathway-related proteins, regulating glycolysis, and inhibiting myocardial fibrosis.
5.Exosomes in obstructive sleep apnea-related diseases.
Zhifeng CHEN ; Yulin SHANG ; Yanru OU ; Subo GONG ; Xudong XIANG ; Xiaoying JI ; Yating PENG ; Ruoyun OUYANG
Chinese Medical Journal 2025;138(20):2540-2551
Obstructive sleep apnea (OSA) is a global public health concern characterized by repeated upper airway collapse during sleep. Research indicates that OSA is a risk factor for the development of various diseases, including cardiovascular disease, metabolic disorders, respiratory diseases, neurodegenerative diseases, and cancer. Exosomes, extracellular vesicles released by most cell types, play a key role in intercellular communication by transporting their contents-such as microRNA, messenger RNA, DNA, proteins, and lipids-to target cells. Intermittent hypoxia associated with OSA alters circulating exosomes and promotes a range of cellular structural and functional disturbances involved in the pathogenesis of OSA-related diseases. This review discusses the potential roles of exosomes and exosome-derived molecules in the onset and progression of OSA-associated diseases, explores the possible underlying mechanisms, and highlights novel strategies for developing exosome-based therapies for these conditions.
Humans
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Exosomes/physiology*
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Sleep Apnea, Obstructive/metabolism*
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Animals
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MicroRNAs/metabolism*
6.Research progress on CD8+T cell dysfunction in chronic hepatitis B virus infection.
Nan ZHANG ; Chuanhai LI ; Rongjie ZHAO ; Liwen ZHANG ; Qing OUYANG ; Liyun ZOU ; Ji ZHANG
Chinese Journal of Cellular and Molecular Immunology 2025;41(5):456-460
Hepatitis B virus (HBV)-specific CD8+ T cells play a central role in controlling HBV infection; however, their function is impaired during chronic HBV infection, manifesting as a state of dysfunction. Recent studies have revealed that CD8+ T cell dysfunction in chronic HBV infection differs from the classical exhaustion observed in other viral infections or tumors. In 2024, several pivotal studies further elucidated novel mechanisms underlying CD8+ T cell dysfunction in chronic HBV infection and identified new therapeutic targets, including 4-1BB and transforming growth factor-beta (TGF-β). This review, while elucidating the dysfunction of CD8+ T cells in chronic HBV infection and its underlying mechanisms, focuses on summarizing the key findings from these latest studies and explores their translational value and clinical significance.
Humans
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Hepatitis B, Chronic/virology*
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CD8-Positive T-Lymphocytes/immunology*
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Hepatitis B virus/physiology*
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Animals
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Transforming Growth Factor beta/immunology*
7.Analysis of pollen sensitization characteristics of artemisia allergic rhinitis in three urban and rural areas of Inner Mongolia
Chunying LI ; Xiaojia LIU ; Haixia XU ; Qiang FU ; Dongyue XU ; Xiaobo CUI ; Ji LIU ; Bolong SONG ; Ming ZHENG ; Yuhui OUYANG ; Xiangdong WANG ; Xiaoling LIU
Chinese Journal of Preventive Medicine 2024;58(6):806-814
Objective:To investigate the distribution rules of artemisia pollen and the clinical sensitization characteristics of allergic rhinitis (AR) induced by artemisia pollen in three urban and rural areas of Inner Mongolia.Methods:From March to October 2019, in 3 central cities (Chifeng, Hohhot, Ordos) and rural areas of Inner Mongolia, an epidemiological investigation method combining multi-stage stratified random sampling and face-to-face questionnaire survey was adopted to screen suspected AR patients, and skin prick test (SPT) was applied for diagnosis. At the same time, pollen monitoring was carried out in 3 areas to analyze the distribution and clinical sensitization characteristics of artemisia pollen.SPSS26.0 statistical software was used to process all the data. Chi-square test was used to compare rates among different age, sex, region and nationality, Spearman test was used to describe correlation analysis, and pairwise comparison of positive rates among multiple samples was used Bonferroni method.Results:Among the 6 393 subjects, 1 093 cases were diagnosed with AR, and the prevalence of AR was 17.10% (1 093/6 393). Among them, pollen-induced allergic rhinitis, the prevalence of PiAR was 10.97% (701/6 393), accounting for 64.14%(701/1 093).The highest incidence was in the youth group (20-39 years old), accounting for 46.94% (329/701).The diagnosed prevalence was higher in females than in males (11.35% vs. 10.64%, χ2 value 12.304, P<0.001).The prevalence rate of ethnic minority was higher than that of Han nationality (13.01% vs. 10.65%, χ2 value 6.296, P=0.008).The prevalence in urban areas was also significantly higher than that in rural areas (18.40% vs. 5.50%, χ2 value 10.497, P<0.001).There was significant difference in prevalence rate among the three regions in Inner Mongolia (6.06% in Chifeng, 13.46% in Hohhot, 16.39% in Ordos, χ2 value 70.054, P<0.001).The main clinical symptoms of artemisia PiAR were sneezing (95.58%), nasal congestion (91.73%) and nasal itching (89.30%).Allergic conjunctivitis accounted for 79.60% (558/701), chronic sinusitis for 55.63% (390/701), asthma for 23.25% (163/701).The pattern of artemisia pollen sensitization was mainly multiple sensitization, and the frequency of clinical symptoms and clinical diseases induced by hypersensitization with other allergens accounted for more than that caused by single artemisia pollen. The spread period of Artemisia pollen in the three regions was from June to October, and the peak state was in August in summer. The peak time of clinical symptoms in artemisia PiAR patients was about 2 weeks earlier than the peak time of pollen concentration, and the two were significantly positively correlated ( R=0.7671, P<0.001). Conclusion:Artemisia pollens are the dominant pollens in late summer and early autumn in Inner Mongolia, and the prevalence of artemisia PiAR is high. Controlling the spread of Artemisia pollens is of great significance for the prevention and treatment of AR.
8.Analysis of pollen sensitization characteristics of artemisia allergic rhinitis in three urban and rural areas of Inner Mongolia
Chunying LI ; Xiaojia LIU ; Haixia XU ; Qiang FU ; Dongyue XU ; Xiaobo CUI ; Ji LIU ; Bolong SONG ; Ming ZHENG ; Yuhui OUYANG ; Xiangdong WANG ; Xiaoling LIU
Chinese Journal of Preventive Medicine 2024;58(6):806-814
Objective:To investigate the distribution rules of artemisia pollen and the clinical sensitization characteristics of allergic rhinitis (AR) induced by artemisia pollen in three urban and rural areas of Inner Mongolia.Methods:From March to October 2019, in 3 central cities (Chifeng, Hohhot, Ordos) and rural areas of Inner Mongolia, an epidemiological investigation method combining multi-stage stratified random sampling and face-to-face questionnaire survey was adopted to screen suspected AR patients, and skin prick test (SPT) was applied for diagnosis. At the same time, pollen monitoring was carried out in 3 areas to analyze the distribution and clinical sensitization characteristics of artemisia pollen.SPSS26.0 statistical software was used to process all the data. Chi-square test was used to compare rates among different age, sex, region and nationality, Spearman test was used to describe correlation analysis, and pairwise comparison of positive rates among multiple samples was used Bonferroni method.Results:Among the 6 393 subjects, 1 093 cases were diagnosed with AR, and the prevalence of AR was 17.10% (1 093/6 393). Among them, pollen-induced allergic rhinitis, the prevalence of PiAR was 10.97% (701/6 393), accounting for 64.14%(701/1 093).The highest incidence was in the youth group (20-39 years old), accounting for 46.94% (329/701).The diagnosed prevalence was higher in females than in males (11.35% vs. 10.64%, χ2 value 12.304, P<0.001).The prevalence rate of ethnic minority was higher than that of Han nationality (13.01% vs. 10.65%, χ2 value 6.296, P=0.008).The prevalence in urban areas was also significantly higher than that in rural areas (18.40% vs. 5.50%, χ2 value 10.497, P<0.001).There was significant difference in prevalence rate among the three regions in Inner Mongolia (6.06% in Chifeng, 13.46% in Hohhot, 16.39% in Ordos, χ2 value 70.054, P<0.001).The main clinical symptoms of artemisia PiAR were sneezing (95.58%), nasal congestion (91.73%) and nasal itching (89.30%).Allergic conjunctivitis accounted for 79.60% (558/701), chronic sinusitis for 55.63% (390/701), asthma for 23.25% (163/701).The pattern of artemisia pollen sensitization was mainly multiple sensitization, and the frequency of clinical symptoms and clinical diseases induced by hypersensitization with other allergens accounted for more than that caused by single artemisia pollen. The spread period of Artemisia pollen in the three regions was from June to October, and the peak state was in August in summer. The peak time of clinical symptoms in artemisia PiAR patients was about 2 weeks earlier than the peak time of pollen concentration, and the two were significantly positively correlated ( R=0.7671, P<0.001). Conclusion:Artemisia pollens are the dominant pollens in late summer and early autumn in Inner Mongolia, and the prevalence of artemisia PiAR is high. Controlling the spread of Artemisia pollens is of great significance for the prevention and treatment of AR.
9.Visualization Analysis on Literature of Ligustri Lucidi Fructus from 1994 to 2023
Kun LIAN ; Ji OUYANG ; Yuehang XU ; Lin LI ; Zhixi HU
Chinese Journal of Information on Traditional Chinese Medicine 2024;31(12):70-76
Objective To analyze the research situation and trend of Ligustri Lucidi Fructus through bibliometric method;To provide reference for related research.Methods The literature on Ligustri Lucidi Fructus included in CNKI,VIP,Wanfang Data,CBM,Web of Science and PubMed from January 1,1994 to August 13,2023 was retrieved.NoteExpress 3.6.0.9155 software was used to manage and screen the literature.The publication time trend.CiteSpace 6.2.R4 and VOSviewer 1.6.18.0 software was used to analyze the authors,source journals,research institutions and keywords,and the knowledge map was drawn.Results A total of 795 Chinese papers and 175 English papers were included,and the annual number of published papers fluctuated.Capital Medical University and Hong Kong Polytechnic University published more papers in Chinese and English respectively.The authors with more papers were Liu Renhui and Wong Mansau respectively.The most published journals were Chinese Journal of Experimental Traditional Medical Formulae,Phytotherapy Research,Journal of Ethnopharmacology.The high frequency keywords included oleanolic acid,specnuezhenide,processing,antioxidant,extraction technology and rat.24 clusters and 44 emergent keywords were generated.Conclusion The research focus in this field includes the extraction and purification of oleanolic acid and specnuezhenide,the compatibility of Ligustri Lucidi Fructus with Chinese herbs such as Epimedii Folium and Polygoni Cuspidati Rhizoma et Radix,the effects and mechanism of delaying aging and treating cancer and other diseases,and cultivation and quality control.
10.Intraoperative frozen section pathology of vaginal margin in radical hysterectomy on the prognosis and quality of life for patients with IB2–IIA2cervical cancer: study protocol for a multicenter randomized controlled trial
Yu LIU ; Weijuan XIN ; Ping WANG ; Mei JI ; Xiaoqing GUO ; Yunyan OUYANG ; Dong ZHAO ; Keqin HUA
Journal of Gynecologic Oncology 2024;35(3):e81-
Background:
Several risk factors have been identified that compromise the treatment outcome in patients with early-to-mid-stage cervical cancer (CC) who are primarily treated with radical surgery. However, there is no report on the impact of intraoperative frozen pathology examination of vaginal margins on the prognosis of patients with CC. This study aimed to conduct a randomized controlled trial (RCT) to determine whether selective vaginal resection can reduce the incidence of operative complications and the risk of postoperative radiotherapy. The impact of the length of the vagina removed in radical hysterectomy (RH) on prognosis and quality of life (QoL) for IB2–IIA2 CC patients will be investigated.
Methods
A multicenter, non-inferiority, RCT at 7 institutions in China is designed to investigate the effect of intraoperative frozen pathology exam of vaginal margin in RH on the survival outcomes for patients with IB2–IIA2 CC. Eligible patients aged 18–70 years will be randomly assigned online by one-to-one random allocation to receive intraoperative frozen pathology exam of vaginal margin or not. If frozen pathology indicates positive margin, continue resection of 1 centimeter of vaginal tissue until negative margin is achieved. The primary end point is 2-year disease-free survival (DFS). Adverse events (AEs) caused by further vagina resection, 5-year DFS, 2-year overall survival (OS), 5-year OS and AEs caused by radiotherapy and QoL are secondary end points. A total of 310 patients will be enrolled from 7 tertiary hospitals in China within 3-year period and followed up for 5 years.Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR2000035668

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