1.GAO Shuzhong's Experience in Treating Idiopathic Tinnitus with Combination of Acupuncture and Chinese Materia Medica
Pengfei WANG ; Yiyang SUN ; Xiaoyan LI ; Wenli YAN ; Ningning MENG ; Guirong YANG ; Yuxia MA
Journal of Traditional Chinese Medicine 2025;66(3):233-237
To summarize Professor GAO Shuzhong's clinical experience in treating idiopathic tinnitus with a combination of acupuncture and Chinese meteria medica. It is believed that idiopathic tinnitus is mostly caused by weak lungs and spleen, kidney essence deficiency, liver constraint transforming into fire, and binding constraint of heart qi. Treatment advocates the combination of acupuncture and Chinese meteria medica in clinical practice. Acupuncture treatment mainly focus on the method of opening the orifices by syndrome identification in combination with Ermen (TE 21), Tinggong (SI 19), Tinghui (GB 2), Shenmai (BL 62) to regulate qi and blood, and supporting with Baihui (GV 20), Yintang (EX-HN 3), Taichong (LR 3), and Yanglingquan (GB 34) to soothe the liver, resolve constraint, and calm the mind. Oral administration of Chinese medicinal prescription usually includes modified Yiqi Congming Decoction (益气聪明汤) and Tongqi Powder (通气散), and the external administration of Chinese medicinal prescription can apply self-prescribed Wenqing Powder (温清散) to navel moxibustion.
2.Effect of Shenshu Fujian Decoction on PDGF/NKD2/Wnt Signaling Pathway in Rats with Chronic Renal Failure
Peng DENG ; Xuekuan HUANG ; Hongyu LUO ; Yuxia JIN ; Dandan WANG ; Xin CHEN ; Shuxian YANG ; Honglin WANG ; Munan WANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(15):79-86
ObjectiveTo observe the effect of Shenshu Fujian decoction on platelet-derived growth factor (PDGF)/naked cuticle homolog 2 (NKD2) /Wnt signaling pathway in rats with chronic renal failure (CRF). MethodsSixty male SD rats were randomly divided into normal group, model group, Niaoduqing group (5 g·kg-1), low-dose Shenshu Fujian decoction group (5.5 g·kg-1), medium-dose Shenshu Fujian decoction group (11 g·kg-1), and high-dose Shenshu Fujian decoction group (22 g·kg-1), with 10 rats in each group. A CRF rat model was established by feeding a 0.5% adenine diet for 21 days. After successful modeling, intragastric administration was given once daily for 28 consecutive days. After treatment, the renal morphology of rats was observed. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were detected. Hematoxylin-eosin (HE) staining and Masson staining were used to detect renal histopathological changes, and collagen volume fraction (CVF) was calculated. Serum levels of inflammatory markers interleukin (IL)-1β and IL-6 were measured using enzyme-linked immunosorbent assay (ELISA). The expressions of fibronectin 1 (FN1), type Ⅰ collagen (ColⅠ), α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-β (PDGFR-β), NKD2, dishevelled protein 2 (DVL2) and β-catenin in renal tissue were detected by immunohistochemistry and Western blot. ResultsCompared with the normal group, the model group showed significant renal pathological changes, a markedly increased kidney weight/body weight ratio (P<0.01), significantly elevated CVF (P<0.01), and notably increased serum levels of SCr, BUN, IL-1β, and IL-6 (P<0.01). Expression levels of FN1, ColⅠ, α-SMA, PDGFR-β, NKD2, DVL2, and β-catenin in renal tissue were also significantly increased (P<0.01). Compared with the model group, all treatment groups showed significantly decreased kidney weight/body weight ratios and CVF (P<0.01), as well as markedly decreased serum SCr, BUN, IL-1β, and IL-6 levels. Protein expression levels of FN1, ColⅠ, α-SMA, PDGFR-β, NKD2, DVL2, and β-catenin in renal tissue were decreased, with more pronounced effects observed in the Niaoduqing, medium-dose, and high-dose Shenshu Fujian decoction groups (P<0.05, P<0.01). ConclusionShenshu Fujian decoction improves renal function, reduces inflammation, and reverses renal fibrosis in CRF rats, possibly by downregulating the expression of PDGF/NKD2/Wnt signaling pathway-related proteins.
3.Quality evaluation of Sanzi powder based on quantitative analysis of multi-component combined with chemical pattern recognition and entropy weight-TOPSIS method
Rongjie LI ; Qian ZHANG ; Wei ZHANG ; Xinkui LI ; Yuxia HU ; Mengdi ZHANG ; Jing LIU ; Fang WANG ; Fengye ZHOU ; Jun LI
China Pharmacy 2025;36(15):1846-1851
OBJECTIVE To comprehensively evaluate the quality of Sanzi powder from different batches based on 12 components quantitative analysis combined with chemical pattern recognition and entropy weight-TOPSIS method. METHODS The contents of 12 components in 15 batches of Sanzi powder (No. S1-S15) were determined by HPLC-MS/MS, such as ethyl gallate, gallic acid, ferulic acid, corilagin, genipin-1-O-β-D-gentiobioside, toosendanin, geniposide, caffeic acid, methyl deacetylated coumarinate, tannic acid, rutin, quercetin. Cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) were conducted on the assay results. Using variable importance projection (VIP) value>1 and P<0.05 as the evaluation criteria, the quality differential markers in Sanzi powder were screened. The entropy weight method was used to calculate the weight value, and TOPSIS method was used to rank the quality of 15 batches of Sanzi powder from superior to inferior. RESULTS The contents of the 12 components were 13.494-24.292, 2 069.608-3 188.100, 1.410-3.616, 1 065.030-2 630.584, 1 404.704-1 838.078, 101.640-354.268, 9 193.720-14 777.854, 1.240-5.060, 148.028-5 541.990, 4 261.422-5 607.438, 107.560- 195.512, 2.226-4.192 μg/g, respectively. The results of CA, PCA and OPLS-DA indicated that 15 batches of Sanzi powder could be clustered into two groups. Specifically, batches S3, S7, S10 and S15 were grouped into one category, and remaining batches were grouped into one category. VIP values of geniposide, quercetin, caffeic acid, and methyl deacetylated coumarinate were all greater than 1, with corresponding P-values less than 0.05. The results of the entropy weight-TOPSIS analysis revealed that methyl deacetylate exhibited the smallest information entropy and the highest weight. The relative closeness degrees of samples S3, S7, S10 and S15 ranged from 0.789 to 0.973, while the remaining samples ranged from 0.054 to 0.172. CONCLUSIONS The contents of 12 components in Sanzi powder could be determined accurately by using HPLC-MS/MS technology. Methyl deacetylated coumarinate, geniposide, quercetin and caffeic acid were identified as the quality differential markers. It was found that the overall quality of samples S3, S7, S10 and S15 were superior to that of other batches. Notably, the quality of Gardeniae Fructus decoction pieces emerges as a critical factor in ensuring the consistency of the preparation’s quality.
4.Successive trigger needling combined with conventional acupuncture for 40 cases of cervical spondylosis with qi stagnation and blood stasis.
Hai XU ; Xuanjie WANG ; Yuxia HE ; Haoran CHU
Chinese Acupuncture & Moxibustion 2025;45(2):159-161
OBJECTIVE:
To observe the efficacy of successive trigger needling combined with conventional acupuncture for cervical spondylosis of qi stagnation and blood stasis.
METHODS:
A total of 40 patients with cervical spondylosis of qi stagnation and blood stasis were selected, and successive trigger needling at ashi points combined with conventional acupuncture at Baihui (GV20) and bilateral C3-C7 Jiaji (EX-B2), Jiquan (HT1), etc. were delivered, once a day, 5 times a week as one course, and 2 courses were required totally. Before treatment and after 1, 2 weeks of treatment, the pain visual analogue scale (VAS) score was observed, and the clinical efficacy was evaluated after treatment.
RESULTS:
After 1, 2 weeks of treatment, the VAS scores were decreased compared with that before treatment (P<0.001). After treatment, the total effective rate was 97.5% (39/40).
CONCLUSION
Successive trigger needling combined with conventional acupuncture can effectively treat the cervical spondylosis of qi stagnation and blood stasis, reduce pain and improve the clinical symptoms and signs.
Humans
;
Spondylosis/physiopathology*
;
Male
;
Acupuncture Therapy
;
Female
;
Middle Aged
;
Adult
;
Qi
;
Aged
;
Acupuncture Points
;
Treatment Outcome
;
Combined Modality Therapy
5.Analgesic effect of "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique of acupuncture on primary dysmenorrhea based on NOD1/RIP2/NF-κB signaling pathway in the rats.
Xu JIN ; Yanlin ZHANG ; Boya CHANG ; Jia REN ; Jianheng HAO ; Yuxia CAO ; Haijun WANG ; Laixi JI
Chinese Acupuncture & Moxibustion 2025;45(2):209-216
OBJECTIVE:
To observe the effect of "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique on the relative protein expression of the signaling pathway of nucleotide-binding oligomerization domain-containing protein 1 (NOD1)/ receptor-interacting protein 2 (RIP2)/nuclear factor kappa-B (NF-κB) and the expression of proinflammatory cytokines in the rats with primary dysmenorrhea (PD), so as to explore the underlying mechanism of this acupuncture technique for pain alleviation in PD.
METHODS:
Thirty female SD rats of SPF grade with normal estrous cycle were randomized into a blank group, a model group and an acupuncture group, 10 rats in each one. Using the intraperitoneal injection with estradiol benzoate combined with oxytocin, PD model was prepared in the model group and the acupuncture group. In the acupuncture group, during model preparation, the intervention with "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique was delivered simultaneously, 20 min each time, once daily for consecutive 10 days. On day 11, within 30 min after the intraperitoneal injection with oxytocin, the writhing reaction (latency, frequency and score) was recorded; the morphology of uterine tissue was observed with HE staining, the contents of prostaglandin E2 (PGE2), prostaglandin F2α (PGF2α), interleukin (IL)-1β, IL-18, cyclooxygenase-2 (COX-2), and tumor necrosis factor-α(TNF-α) in the serum were detected using ELISA method; the relative protein expression of NOD1, RIP2, NF-κB p65, phosphorylation-NF-κB p65 (p-NF-κB p65) was detected in the uterine tissue using Western blot method; and the mRNA expression of NOD1, RIP2 and NF-κB p65 was detected with the quantitative real-time PCR employed.
RESULTS:
Compared with the blank group, in the model group, the writhing latency was prolonged (P<0.01), the writhing frequency and score increased (P<0.01) in the rats; the endometrial epithelial cells showed massive degeneration and necrosis, with severe endometrial edema and widespread shedding, combined with neutrophil infiltration; the serum PGE2 content was dropped (P<0.01), while those of PGF2α, IL-1β, IL-18, COX-2, and TNF-α elevated (P<0.01); the protein expression of NOD1, RIP2, NF-κB p65 and p-NF-κB p65, and the mRNA expression of NOD1, RIP2 and NF-κB p65 in uterine tissue increased (P<0.01). In comparison with the model group, in the acupuncture group, the writhing latency was prolonged (P<0.01), the writhing frequency and score were reduced (P<0.01) in the rats; there was less degeneration and necrosis of endometrial epithelial cells, with mild endometrial edema and very little neutrophil infiltration; the serum PGE2 content increased (P<0.01), while those of PGF2α, IL-1β, IL-18, COX-2, and TNF-α decreased (P<0.01); the protein expression of NOD1, RIP2, NF-κB p65 and p-NF-κB p65 and the mRNA expression of NOD1, RIP2 and NF-κB p65 in uterine tissue were dropped (P<0.05, P<0.01).
CONCLUSION
"Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique can alleviate the pain symptom of PD rats, and its action mechanism may be related to inhibiting the active expression of NOD1/RIP2/NF-κB signaling pathway in the uterine tissue, thereby reducing the inflammatory response.
Animals
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Female
;
Rats, Sprague-Dawley
;
Rats
;
Signal Transduction
;
Dysmenorrhea/metabolism*
;
NF-kappa B/metabolism*
;
Acupuncture Points
;
Humans
;
Acupuncture Analgesia
;
Nod1 Signaling Adaptor Protein/metabolism*
;
Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism*
;
Acupuncture Therapy
6.Effect of "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture on gut microbiota in mice with poor ovarian response.
Boya CHANG ; Jia REN ; Xu JIN ; Jianheng HAO ; Zhen GAO ; Yuxia CAO ; Haijun WANG
Chinese Acupuncture & Moxibustion 2025;45(6):770-780
OBJECTIVE:
To explore the possible mechanism by which the "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture improves ovarian function in mice with poor ovarian response (POR) by observing its effect on gut microbiota.
METHODS:
A total of 35 SPF-grade C57BL/6 female mice were screened for normal estrous cycles using vaginal smears, and 30 mice were selected. Ten mice were assigned to the blank group, while the remaining mice were used to establish the POR model by intragastric administration of tripterygium wilfordii suspension. The successfully modeled mice were randomly divided into a model group and an acupuncture group, with 10 mice in each group. After modeling, the acupuncture group received the "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture method once daily for 20 minutes per session. Ovulation induction began the day after the intervention, and samples were collected after ovulation induction. Vaginal cytology was used to observe estrous cycle changes, and the number of oocytes obtained, ovarian wet weight, and ovarian index were recorded. Serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and anti-Müllerian hormone (AMH) were detected using ELISA. HE staining was used to observe ovarian histology. Gut microbiota was analyzed using 16S rRNA gene sequencing technology. Western blot was used to detect the relative protein expression levels of Occludin and zonula occludens-1 (ZO-1) in colonic tissue. Correlation analysis was conducted among serum hormone indexes, the number of oocytes obtained, ovarian index and gut microbiota.
RESULTS:
Compared with the blank group, the model group showed a higher estrous cycle disorder rate (P<0.01), increased serum FSH and LH levels, and a higher LH/FSH ratio (P<0.01), while the number of oocytes obtained, ovarian wet weight, ovarian index, and serum E2 and AMH levels were significantly reduced (P<0.01). Compared with the model group, the acupuncture group showed a lower estrous cycle disorder rate (P<0.01), decreased serum FSH and LH levels, and a lower LH/FSH ratio (P<0.01), along with an increased number of oocytes obtained, higher ovarian wet weight, ovarian index, and elevated serum AMH and E2 levels (P<0.01, P<0.05). The blank group had a large number of well-developed primordial follicles, with abundant and closely arranged follicles at various stages. In the model group, there was a significant increase in the number of atretic follicles, a reduction in the number of follicles at various stages, and loosely arranged ovarian tissue. Compared with the blank group, the model group showed a significant decrease in the number of normal follicles (P<0.01) and an increase in the number of atretic follicles (P<0.01). The acupuncture group showed a reduction in atretic follicles and an increase in the number of follicles at various stages compared with the model group, with a significant increase in normal follicles (P<0.01) and a decrease in atretic follicles (P<0.01). Compared with the blank group, the model group exhibited reduced gut microbiota diversity and richness, with significantly lower Chao1 and Shannon indices (P<0.01), and a greater clustering distance from the blank group. The model group also showed an increase in the relative abundance of Firmicutes_D, Verrucomicrobiota, Paramuribaculum, Dubosiella, and Muribaculum (P<0.01, P<0.05), while the relative abundance of Firmicutes_A and the relative protein expression of Occludin and ZO-1 in colonic tissue were decreased (P<0.01). Compared with the model group, the acupuncture group showed improved gut microbiota diversity and richness, with increased Chao1 and Shannon indices (P<0.05), and a clustering distance closer to the blank group. The acupuncture group exhibited reduced relative abundance of Firmicutes_D, Verrucomicrobiota, and Muribaculum (P<0.05, P<0.01), while the relative abundance of Firmicutes_A and the relative protein expression of Occludin and ZO-1 were significantly increased (P<0.01, P<0.05). Correlation analysis indicated a relationship between gut microbiota and serum hormone indicators, as well as the ovarian index. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the metabolic pathways of the intersecting species were related to amino acid biosynthesis and nucleotide metabolism.
CONCLUSION
The "Zhibian" (BL54) toward "Shuidao" (ST28) acupuncture method improves ovarian function in POR mice, and its mechanism may be related to regulating gut microbiota structure and maintaining intestinal barrier homeostasis.
Animals
;
Female
;
Gastrointestinal Microbiome
;
Mice
;
Acupuncture Therapy
;
Mice, Inbred C57BL
;
Humans
;
Ovary/physiopathology*
;
Acupuncture Points
;
Follicle Stimulating Hormone/metabolism*
;
Luteinizing Hormone/metabolism*
;
Estrous Cycle
;
Anti-Mullerian Hormone/blood*
7.Expert consensus on clinical protocol for treating herpes zoster with fire needling.
Xiaodong WU ; Bin LI ; Baoyan LIU ; Lin HE ; Zhishun LIU ; Shixi HUANG ; Keyi HUI ; Hongxia LIU ; Yuxia CAO ; Shuxin WANG ; Zhe XU ; Cang ZHANG ; Jingsheng ZHAO ; Yali LIU ; Nanqi ZHAO ; Nan DING ; Jing HU
Chinese Acupuncture & Moxibustion 2025;45(12):1825-1832
The expert consensus on the clinical treatment of herpes zoster with fire needling was developed, and the commonly used fire needling treatment scheme verified by clinical research was selected to form a standardized diagnosis and treatment scheme for acute herpes zoster and postherpetic neuralgia (PHN), so as to answer the core problems in clinical application. The consensus focuses on patients with herpes zoster, and forms recommendations for 9 key clinical issues, covering simple fire needling and TCM comprehensive therapy based on fire needling, including fire needling combined with cupping, fire needling combined with Chinese herb, fire needling combined with cupping and Chinese herb, fire needling combined with filiform needling, fire needling combined with moxibustion, and provides specific recommendations and operational guidelines for various therapies.
Humans
;
Herpes Zoster/therapy*
;
Acupuncture Therapy/instrumentation*
;
Consensus
;
Clinical Protocols
8.Role of sphingolipid metabolism signaling in a novel mouse model of renal osteodystrophy based on transcriptomic approach.
Yujia WANG ; Yan DI ; Yongqi LI ; Jing LU ; Bofan JI ; Yuxia ZHANG ; Zhiqing CHEN ; Sijie CHEN ; Bicheng LIU ; Rining TANG
Chinese Medical Journal 2025;138(1):68-78
BACKGROUND:
Renal osteodystrophy (ROD) is a skeletal pathology associated with chronic kidney disease-mineral and bone disorder (CKD-MBD) that is characterized by aberrant bone mineralization and remodeling. ROD increases the risk of fracture and mortality in CKD patients. The underlying mechanisms of ROD remain elusive, partially due to the absence of an appropriate animal model. To address this gap, we established a stable mouse model of ROD using an optimized adenine-enriched diet and conducted exploratory analyses through ribonucleic acid sequencing (RNA-seq).
METHODS:
Eight-week-old male C57BL/6J mice were randomly allocated into three groups: control group ( n = 5), adenine and high-phosphate (HP) diet group ( n = 20), and the optimized adenine-containing diet group ( n = 20) for 12 weeks. We assessed the skeletal characteristics of model mice through blood biochemistry, microcomputed tomography (micro-CT), and bone histomorphometry. RNA-seq was utilized to profile gene expression changes of ROD. We elucidated the functions of differentially expressed genes (DEGs) using gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA). DEGs were validated via quantitative real-time polymerase chain reaction (qRT-PCR).
RESULTS:
By the fifth week, adenine followed by an HP diet induced rapid weight loss and high mortality rates in the mouse group, precluding further model development. Mice with optimized adenine diet-induced ROD displayed significant abnormalities in serum creatinine and blood urea nitrogen levels, accompanied by pronounced hyperparathyroidism and hyperphosphatemia. The femur bone mineral density (BMD) of the model mice was lower than that of control mice, with substantial bone loss and cortical porosity. ROD mice exhibited substantial bone turnover with an increase in osteoblast and osteoclast markers. Transcriptomic profiling revealed 1907 genes with upregulated expression and 723 genes with downregulated expression in the femurs of ROD mice relative to those of control mice. Pathway analyses indicated significant enrichment of upregulated genes in the sphingolipid metabolism pathway. The significant upregulation of alkaline ceramidase 1 ( Acer1 ), alkaline ceramidase 2 ( Acer2 ), prosaposin-like 1 ( Psapl1 ), adenosine A1 receptor ( Adora1 ), and sphingosine-1-phosphate receptor 5 ( S1pr5 ) were successfully validated in mouse femurs by qRT-PCR.
CONCLUSIONS
Optimized adenine diet mouse model may be a valuable proxy for studying ROD. RNA-seq analysis revealed that the sphingolipid metabolism pathway is likely a key player in ROD pathogenesis, thereby providing new avenues for therapeutic intervention.
Animals
;
Mice
;
Chronic Kidney Disease-Mineral and Bone Disorder/genetics*
;
Male
;
Disease Models, Animal
;
Mice, Inbred C57BL
;
Sphingolipids/metabolism*
;
Transcriptome/genetics*
;
Signal Transduction/genetics*
;
X-Ray Microtomography
;
Adenine
9.Expert consensus on orthodontic treatment of protrusive facial deformities.
Jie PAN ; Yun LU ; Anqi LIU ; Xuedong WANG ; Yu WANG ; Shiqiang GONG ; Bing FANG ; Hong HE ; Yuxing BAI ; Lin WANG ; Zuolin JIN ; Weiran LI ; Lili CHEN ; Min HU ; Jinlin SONG ; Yang CAO ; Jun WANG ; Jin FANG ; Jiejun SHI ; Yuxia HOU ; Xudong WANG ; Jing MAO ; Chenchen ZHOU ; Yan LIU ; Yuehua LIU
International Journal of Oral Science 2025;17(1):5-5
Protrusive facial deformities, characterized by the forward displacement of the teeth and/or jaws beyond the normal range, affect a considerable portion of the population. The manifestations and morphological mechanisms of protrusive facial deformities are complex and diverse, requiring orthodontists to possess a high level of theoretical knowledge and practical experience in the relevant orthodontic field. To further optimize the correction of protrusive facial deformities, this consensus proposes that the morphological mechanisms and diagnosis of protrusive facial deformities should be analyzed and judged from multiple dimensions and factors to accurately formulate treatment plans. It emphasizes the use of orthodontic strategies, including jaw growth modification, tooth extraction or non-extraction for anterior teeth retraction, and maxillofacial vertical control. These strategies aim to reduce anterior teeth and lip protrusion, increase chin prominence, harmonize nasolabial and chin-lip relationships, and improve the facial profile of patients with protrusive facial deformities. For severe skeletal protrusive facial deformities, orthodontic-orthognathic combined treatment may be suggested. This consensus summarizes the theoretical knowledge and clinical experience of numerous renowned oral experts nationwide, offering reference strategies for the correction of protrusive facial deformities.
Humans
;
Orthodontics, Corrective/methods*
;
Consensus
;
Malocclusion/therapy*
;
Patient Care Planning
;
Cephalometry
10.Effects of Harmine Inducing Apoptosis on Mitochondrial Fusion and Division in PC 12 Cells
Yuehong GONG ; Meiling ZHAO ; Ruijia MA ; Yuxia LIN ; Jun ZHAO ; Jianhua WANG
Herald of Medicine 2024;43(2):174-183
Objective To investigate the effects of harmine(HM)on the expression level of mitochondrion fusion related proteins and mitochondrial function injury in PC 12 cells.Methods PC 12 cells were divided into cell control group,HM group,mitochondrion mitosis inhibitor Mdivi-1 group,HM+Mdivi-1 group,mitochondrion fission agonist WY14643 group,HM+WY14643 group,with drug concentrations of 1,10,25,50,100 μmol·L-1.After 24 h treatment,the MTT method was used to detect the cell survival rate,and a microscope was used to observe the cell morphology,MitoTracker Red probe staining was used to observe the mitochondrial morphology and the length ratio of vertical and horizontal axes,JC-1 staining was used to detect the mitochondrial membrane potential,and a kit was used to detect ATP level and lactate dehydrogenase(LDH)activity.Immunofluorescence staining and Western blotting were used to assess the expression levels of caspase-3,apoptosis-promoting protein(Bax)cytochrome C(cyt-c),mitochondrial fusion protein(Mfn2)and mitochondrial mitotic protein(Drp-1).The interference sequence of Drp1 was transfected by electroporation,and the siRNA sequence with good transfection effect was screened.The related indicators were detected by fluorescence method,MTT method,and immunoblotting method in cooperation with drug intervention.Results MTT results showed that compared with the cell control group,the survival rate of HM group,Mdivi-1 group,HM+Mdivi-1 group,WY14643 group and HM+WY14643 group decreased significantly(P<0.01),and the EC50 were(11.48±2.32),(12.35±1.67),(14.88±2.07),(39.14±3.25),(20.09±1.97),respectively.According to this,subsequent experiments selected 20 μmol·L-1for HM,WY 14643 and HM+WY14643 as working concentrations to construct PC 12 cell model.Microscopic observation and MitoTracker Red probe staining showed that the cell density in the drug group decreased in varying degrees,and a transition from branched to round morphology in the drug-treated groups was observed.The morphology of mitochondria tended to be round,and the ratio of the length of the longitudinal axis to transverse axis was(3.33±0.72)in the cell control group,(2.19±0.58)in the HM group,(2.45±0.44)in Mdivi-1 group,and(1.43±0.62)in HM+Mdivi-1 group,respectively.The results of JC-1 staining showed that compared with the cell control group,the mitochondrial mode potential of the HM group significantly decreased(P<0.01).ROS significantly increased(P<0.01)and ATP levels decreased(P<0.01),and LDH enzyme activity increased(P<0.01).Immunofluorescence staining and Western blotting results showed that compared with the cell control group,the expression levels of proapoptotic proteins Bax,cytochrome C,and caspase-3 in the HM group were significantly increased(all P<0.01).Compared with the cell control group,the expression level of mitochondrial fission related protein Drp1 in HM group was significantly higher(P<0.01).The expression level of mitochondrial fusion related protein Mfn2 significantly decreased(P<0.01).After specific interference with Drp1 and synergistic intervention with HM,the survival rate of PC 12 cells in each interference group decreased compared to each drug intervention group.The expression of Drp1 and Mfn2 was downregulated,and the differences were statistically significant(P<0.05 or P<0.01).Conclusion HM can reduce the mitochoudrial membrane potential and ATP levels by accumulating ROS,there by activating the caspase-3 apoptosis pathway and promoting cell apoptosis.Mitochondrial fusion division may be involved in the damage of PC12 cells caused by HM,initiating apoptosis through the mitochondrial pathway.

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