1.Tracing Development of LIU Wansu's Theory of ''Fire-heat Inducing Stroke''
Xin LAN ; Changxiang LI ; Haojia ZHANG ; Jialin CHENG ; Zijin SUN ; Liyang DONG ; Zilin REN ; Xueqian WANG ; Fafeng CHENG ; Qingguo WANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(8):32-41
LIU Wansu, as the foremost of the four great masters of the Jin-Yuan period, established the "theory of fire-heat'' and extended the fire-heat pathogenesis framework to the field of stroke, thereby forming the theory of ''fire-heat inducing stroke''. This achieved a paradigmatic shift in stroke etiology from ''exogenous wind inducing stroke'' to ''fire-heat inducing stroke''. This paper systematically reviews the developmental trajectory of LIU Wansu's ''fire-heat inducing stroke'' theory and explores the social background, academic origins, and core connotations of its theoretical construction. The study found that, based on the ''Nineteen Pathomechanisms'' in the Huangdi's Internal Classic (Huang Di Nei Jing) and combined with clinical practice, LIU Wansu proposed that fire-heat is the fundamental cause of stroke, and that the Six Climatic Factors and the Five Zhi-Emotions can all transform into fire. He further constructed a stratified syndrome differentiation and therapeutic system centered on clearing heat and purging fire, emphasizing differentiated treatment of exterior and interior syndromes, Six Meridians syndrome differentiation, and seasonally adjusted medication. This theory not only resolved the diagnostic and therapeutic dilemmas of febrile epidemic diseases during the Jin-Yuan period, but also exerted a profound influence on later physicians such as ZHANG Zihe and ZHU Danxi, thereby promoting the pluralistic development of stroke theory in traditional Chinese medicine (TCM). Modern pharmacological research provides solid scientific evidence, confirming that the ''fire-heat'' pathological state is highly associated with key mechanisms such as excessive inflammatory responses, oxidative stress, and excitatory amino acid toxicity following cerebral ischemia. Heat-clearing and fire-purging prescriptions and agents, such as Huanglian Jiedu Tang and baicalin, can exert multi-target neuroprotective effects by regulating inflammatory signaling, enhancing antioxidant enzyme activity, and balancing neurotransmitters. This not only verifies the scientific basis of the ''fire-heat inducing stroke'' theory from a modern biological perspective but also provides conclusive evidence for the clinical application of heat-clearing and fire-purging therapy. LIU Wansu's ''fire-heat inducing stroke'' theory represents a major milestone in the historical understanding of stroke pathogenesis, and its academically transitional insights continue to hold core guiding value for the pattern identification and treatment of ischemic stroke today.
2.Herbal Textual Research on Piperis Longi Fructus in Famous Classical Formulas
Haihua WANG ; Xiaoqi JING ; Juan LI ; Dabang REN ; Fusheng ZHANG ; Zhilai ZHAN
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(8):210-219
This article systematically analyzes the historical evolution of the name, origin, medicinal parts, producing area, harvesting and processing, nature, flavor and efficacy of Piperis Longi Fructus by referring to the materia medica, medical books, and prescription books of past dynasties, combined with the relevant modern literature, in order to provide a basis for the development and utilization of famous classical formulas containing this herb. According to the herbal textual research, the name of Piper longum first appeared in Nanfang Caomuzhuang, and it also has other aliases such as Biboli, Halou, and Hujiaohua. Historically, the origin of Piperis Longi Fructus has been P. longum of the Piperaceae family. In ancient times, both the fruit and root were used as medicine, and since the Republic of China, the fruit has been mainly used as medicine. The medicinal part is the dried, nearly ripe or ripe fruit spikes. Piperis Longi Fructus is native to India and has been introduced into China since the Tang dynasty. In the Ming dynasty, Bencao Pinhui Jingyao clearly stated that the genuine producing area was "Duanzhou", present-day Zhaoqing in Guangdong province. Nowadays, it is planted in Guangdong, Guangxi, Hainan, Yunnan and other regions. Historically and currently, harvesting occurs in autumn. The ancient processing method uniformly involved removing the stems, soaking in the sourest vinegar overnight, baking, and scraping off the peels and grains with a knife until clean. In modern times, impurities are removed, and it is dried in the sun and crushed when used. The properties, functions and applications of P. longum are basically the same in ancient and modern times. It tastes pungent, is warm in nature, and non-toxic. It has the effects of warming the middle-jiao to dispel cold, lowering Qi and relieving pain, and is used for cold pain in the epigastrium and abdomen, vomiting, diarrhea, chest pain, headache, and toothache. Based on the research results, it is recommended that when developing famous classical formulas containing Piperis Longi Fructus, the dried nearly ripe or ripe fruit spikes of P. longum should be used. If there are no clear processing requirements, it is recommended to use the raw products for medicinal use, and the specific processing methods can refer to the relevant requirements under Piperis Longi Fructus in the 2025 edition of the Pharmacopoeia of the People's Republic of China. If processing requirements such as soaking in vinegar and peeling are clearly specified, it is recommended to follow the ancient methods.
3.Analysis of knowledge related to human papillomavirus and vaccination willingness among college students in Guizhou Province
REN Li, WU Yuqian, ZHU Liwan,LI Fenxiang, FAN Shujun, GAN Jianzhe, DONG Shuwen, LU Yong
Chinese Journal of School Health 2026;47(2):212-216
Objective:
To explore human papillomavirus (HPV) and vaccination related knowledge and vaccination willingness of college students in Guizhou Province and their related factors, so as to provide a basis for formulating targeted intervention strategies.
Methods:
From May to June 2025, by applying convenience sampling method,4 567 college students were selected from 8 universities in Guizhou Province to conduct a questionnaire survey. Awareness of HPV and vaccination related knowledge, vaccination willingness as well as related factors among college students were also analyzed. The t test and Chi square test were used for comparison between groups, and multifactor Logistic regression was employed to analyze the related factors of HPV vaccination willingness among college students.
Results:
The HPV and vaccine knowledge score of college students in Guizhou Province was ( 10.50 ±2.09), and the score of girls (10.81±1.82) was higher than that of boys (10.19±2.30) ( t=10.09, P <0.01). The HPV vaccination willingness rate of college students was 65.6%, and the rate was higher in girls than in boys (67.1%,64.1%, χ 2=4.75, P <0.05). Multi factor Logistic regression analysis showed that ethnicity and HPV testing were related factors that affected college students willingness to vaccinate (minority: OR boy =1.23, OR girl =1.35; previous HPV testing: OR boy =0.56, OR girl =0.59); boys willingness to vaccinate was related to the number of sexual partners ( OR =0.60), family history of cancer ( OR =0.65), and sexual behavior related HPV knowledge scores ( OR =0.89), while girls willingness to vaccinate was related to bisexual sexual orientation ( OR =0.59), previous HIV testing ( OR =0.60), and HPV and vaccine basic knowledge scores ( OR =0.86) (all P <0.05).
Conclusions
College students in Guizhou Province have higher HPV vaccine related knowledge scores and are more willing to vaccinate, and those above are higher in girls than in boys. Health education content should be optimized based on gender differences, and promote the willingness and behavior of HPV vaccination among college students.
4.Analysis of the disease burden of hypertensive heart disease among individuals aged≥60 years globally and in China from 1990 to 2021
Jiali LI ; Chunzhen REN ; Fan LIU ; Keyan WANG ; Zhijiang BI ; Xiaoxiao ZHAO ; Lixin KE ; Haibo WANG ; Wenxi PENG ; Zhifei WANG ; Qiang ZHANG ; Peng XU ; Yingdong LI ; Xiuxiu DENG ; Xinke ZHAO ; Cuncun LU
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(02):281-290
Objective To systematically analyze the characteristics of the disease burden of hypertensive heart disease (HHD) in the elderly (≥60 years) globally and in China from 1990 to 2021, and to predict its future trends from 2022 to 2040, with the aim of providing data support for optimizing comprehensive prevention and control strategies for HHD. Methods Based on the Global Burden of Disease (GBD) 2021 database, the number of prevalent cases and disability-adjusted life years (DALYs) of HHD in the elderly were extracted for the world, China, and five regions categorized by sociodemographic index (SDI). Joinpoint regression was used to analyze the temporal trends of age-standardized prevalence rate and age-standardized DALYs rate of HHD in the elderly. A three-factor decomposition method was applied to evaluate the relative contributions of aging, population growth, and epidemiological changes to the variations in the elderly HHD burden. Additionally, a Bayesian age-period-cohort model was used to predict the elderly HHD burden from 2022 to 2040. Results In 2021, the number of prevalent elderly HHD cases reached 10 283 000 globally and 3 412 400 in China, representing increases of 179.20% and 159.20% respectively, compared with 1990. The DALYs of elderly HHD were 18 812 700 person-years globally and 4 731 400 person-years in China, rising by 76.08% and 29.45% respectively from 1990. Meanwhile, the growth rates of the number of prevalent cases and DALYs of elderly HHD varied across different SDI regions. From 1990 to 2021, the age-standardized prevalence rate of elderly HHD in China, as well as the age-standardized DALYs rate of elderly HHD both globally and in China, showed significant downward trends (all average annual percentage changes<0, all P<0.001). In 2021, the 70-74 years age group accounted for the highest proportion of prevalent cases and DALYs of elderly HHD, both globally and in China. Decomposition analysis revealed that population growth was the dominant factor driving the increase in the elderly HHD burden across all regions. The prediction model results indicated that the number of prevalent cases and DALYs of elderly HHD would continue to rise globally and in China from 2022 to 2040, with the growth rate of the elderly HHD burden in China between 2021 and 2040 expected to exceed the global average. Conclusion Over the past 32 years, although the age-standardized disease rates of elderly HHD have mainly shown a downward trend globally and in China, the absolute number of the disease burden has increased substantially. The projection model indicates a continued upward trajectory, with the growth rate in China higher than the global average. Therefore, there is an urgent need to implement precise prevention and control strategies to effectively mitigate the disease burden of elderly HHD.
5.Longitudinal cohort study on pubertal development trajectories of testicular and breast development among children
Chinese Journal of School Health 2026;47(3):408-412
Objective:
To characterize longitudinal trajectories of testicular development in boys and breast development in girls, so as to provide reference data for understanding patterns of pubertal sexual maturation.
Methods:
Based on the Shanghai Pudong New Area Cohort Study on Growth, Development and Health in Children and Adolescents, a baseline survey was conducted in 2020 using a mult stage cluster random sampling method. A total of 2 184 children who completed all follow ups during the primary school period from 13 elementary schools in Pudong New Area,Shanghai,with annual follow ups during 2021-2025. Testicular volume and Tanner stage of breast development were assessed by professional physicians using standardized visual inspection and palpation. The age distribution of testicular volume and breast development was fitted by using cumulative link mixed models and Turnbull s nonparametric maximum likelihood estimation method.
Results:
Median ages for testicular volumes of 2, 3, 4 and 5 mL in boys were 7.07, 9.24, 10.29, and 11.57 years old, respectively. Median ages for Tanner breast stages Ⅱ, Ⅲ, Ⅳ, and Ⅴ in girls were 8.55 , 10.17, 11.18, and 13.78 years old, respectively. Based on overweight and obesity, stratified analysis showed that earlier pubertal onset among overweight/obesity children, and the key milestones for pubertal initiation were testicular volume reaching 4 mL in boys and breast Tanner II in girls for 10.29, 10.83; 8.18, 9.00 years.
Conclusion
Overweight and obesity are associated with earlier pubertal initiation,but there are certain gender and developmental stage specific patterns.
6.A systematic review of application value of machine learning to prognostic prediction models for patients with lumbar disc herniation
Zhipeng WANG ; Xiaogang ZHANG ; Hongwei ZHANG ; Xiyun ZHAO ; Yuanzhen LI ; Chenglong GUO ; Daping QIN ; Zhen REN
Chinese Journal of Tissue Engineering Research 2026;30(3):740-748
OBJECTIVE:Based on different algorithms of machine learning,the prediction model of lumbar disc herniation has become a trend and hot spot in the development of precision medicine.However,there is limited evidence on the reporting quality and methodological quality of prediction models of lumbar disc herniation outcomes using machine learning.This article is aimed to explore the performance of machine learning algorithms in predicting the prognosis of lumbar disc herniation by comprehensively analyzing the report quality and risk of bias of previous studies that developed and validated prognosis prediction models based on machine learning through a comprehensive literature search,in order to explore the performance of machine learning algorithms in predicting the prognosis of lumbar disc herniation.METHODS:The databases of CNKI,WanFang,VIP,SinOMED,PubMed,Web of Science,Embase,and The Cochrane Library were searched by computer.Studies on the use of machine learning to develop(and/or validate)prognostic prediction models for lumbar disc herniation were collected from the inception of the database to December 31,2023.Two researchers independently screened the literature,extracted data,and assessed the risk of bias of the included studies.The reporting quality and risk of bias of the included studies were assessed by the Multivariable Transparent Reporting of Predictive Models(TRIPOD)statement and the Predictive Model Risk of Bias Assessment Tool(PROBAST).The results of the evaluation were analyzed using descriptive statistics and visual charts.RESULTS:(1)A total of 23 articles were included,and the TRIPOD compliance of each study ranged from 11%to 87%,with a median compliance of 54%.The quality of reporting of titles,detailed descriptions of treatment measures,blinding of predictors,handling of missing data,details of risk stratification,specific procedures for enrollment,model interpretation,and model performance was mostly poor,with TRIPOD adherence rates ranging from 4%to 35%.(2)Of all included studies,61%had a high risk of bias and 39%had an unclear overall risk of bias.The area under the curve,accuracy,sensitivity and specificity were used to evaluate the performance of the model.The areas under the curve of 20 models were reported,ranging from 0.561 to 0.999.Three models reported the accuracy of the model,ranging from 82.07%to 89.65%.(3)Among all included studies,the statistical analysis domain was most often assessed as having a high risk of bias,mainly due to the small number of valid samples,the selection of predictors based on univariate analysis and the lack of calibration and discrimination assessment of the model in the study.CONCLUSION:These results indicate that machine learning can achieve good predictive ability in the development and validation of prognostic models for lumbar disc herniation.The commonly used algorithms include regression algorithm,support vector machine,decision tree,random forest,artificial neural network,naive Bayes and other algorithms.Reasonable algorithms combined with clinical practice can improve the accuracy of prognosis prediction of lumbar disc herniation.However,the reporting and methodological quality of prognosis prediction models based on machine learning are poor,the prediction performance of different models varies greatly,and the generalization and extrapolation of research models are unclear.There is an urgent need to improve the design,implementation and reporting of such studies.To promote the application of machine learning in the clinical practice of lumbar disc herniation prediction models,it is necessary to comprehensively consider various predictors related to the prognosis of the disease before modeling,and strictly follow the relevant standards of PROBAST tool during modeling.
7.Comparative Study on Effect of Jingui Shenqiwan and Liuwei Dihuangwan on Reproductive Ability and Brain Function of Normal Mice
Hong SUN ; Fan LEI ; Chenggong LI ; Rui LUO ; Shixian HU ; Bin REN ; Juan HAO ; Yi DING ; Lijun DU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):1-14
ObjectiveTo explore the effects of Jingui Shenqiwan (JSW) and Liuwei Dihuangwan (LDW) on the reproductive ability and brain function of normal mice and compare the actions of the two medications. MethodsSeven groups of female and male mice were divided at a ratio of 2∶1. Except for the control group, the other six groups were as follows: a group of both males and females receiving JSW (3.0 g·kg-1), a group of both males and females receiving LDW (4.5 g·kg-1), a group of males receiving water and females receiving JSW, a group of males receiving water while females receiving LDW, a group of females receiving water while males receiving JSW, and a group of females receiving water while males receiving LDW. Each group was administered the drug for 14 days and then caged together at a 2∶1 (female∶male) ratio to detect the number of pregnant mice and calculate the pregnancy rate. Pregnant mice continued receiving the drug until they naturally gave birth, which was followed by the observation of newborn mice, calculation of their average number, and the measurement of the offspring's preference for sugar water and neonatal recognition index. At the end of the experiment, the weights of the thymus and spleen were measured to calculate the organ coefficients, and mRNA or protein expression was analyzed in the brain and testes or ovaries. A 1% sucrose solution was used to examine the euphoria of their brain reward systems, while novel object recognition test (NOR) was applied to assess their memory capabilities. mRNA expression was detected using real-time quantitative polymerase chain reaction (Real-time PCR) assay, and protein expression was analyzed with Western blot. ResultsCompared with the control group, oral administration of JSW to both male and female mice for 14 days significantly increased the pregnancy rate of female mice on day 2 after being caged together (P<0.05), while LDW showed a trend but no statistical significance. Additionally, compared with the control group, JSW could upregulate the gene expression of gonadotropin-releasing hormone (GnRH) in the thalamus, as well as reproductive stem cell factor (SCF) and tyrosine kinase receptor (c-Kit) in the testes and reproductive stem cell marker mouse vasa homologue (MVH) in the ovaries, upregulate the expression of proteins influencing neuronal functional activity, such as brain-derived neurotrophic factor (BDNF), in hippocampal neurons (P<0.05), and enhance sucrose preference in male mice (P<0.05). Compared with the control group, JSW significantly increased sucrose preference and novel object recognition index in offspring mice (P<0.05), which was related to the upregulation of hippocampal dopamine D1 receptor (D1R) and N-methyl-D-aspartate receptor (Nmdar) gene expression. Compared with the control group, both JSW and LDW could upregulate the protein expression of glucocorticoid receptor (GR), BDNF, and tyrosine kinase receptor B (TrkB) in the hippocampus of offspring mice (P<0.05). ConclusionJSW significantly enhances the reproductive ability of normal mice, which is not only related to the release of gonadotropin but also associated with its regulation of brain function. Additionally, JSW has a certain regulatory effect on the brain function of the offspring mice.
8.Zuoguiwan Mitigates Oxidative Stress in Rat Model of Hyperthyroidism Due to Kidney-Yin Deficiency via DRD4/NOX4 Pathway
Ling LIN ; Qianming LIANG ; Changsheng DENG ; Li RU ; Zhiyong XU ; Chao LI ; Mingshun SHEN ; Yueming YUAN ; Muzi LI ; Lei YANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):43-51
ObjectiveTo decipher the mechanism by which Zuoguiwan (ZGW) treat hyperthyroidism in rats with kidney-Yin deficiency based on the dopamine receptor D4 (DRD4)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) signaling pathway. MethodsThe rat model of kidney-Yin deficiency was induced by unilateral intramuscular injection of dexamethasone (0.35 mg·kg-1). After successful modeling, the rats were randomized into model, methimazole (positive control, 5 mg·kg-1), low-, medium-, and high-dose (1.85, 3.70, 7.40 g·kg-1, respectively) ZGW, and normal control groups. After 21 days of continuous gavage, the behavioral indexes and body weight changes of rats were evaluated. The pathological changes of the renal tissue were observed by hematoxylin-eosin staining. The serum levels of thyroid hormones [triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)], renal function indexes [serum creatine (Scr) and blood urea nitrogen (BUN)], energy metabolism markers [cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)], and oxidative stress-related factors [superoxide dismutase (SOD), malondialdehyde (MDA), and NADPH)] were measured by enzyme-linked immunosorbent assay (ELISA). Western blot was employed to analyze the expression of DRD4, NOX4, mitochondrial respiratory chain complex proteins [NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4) and cytochrome C oxidase subunit 4 (COX4)], and inflammation-related protein [tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), p38 mitogen-activated protein kinase (MAPK)] pathway in the renal tissue. ResultsCompared with the normal group, the model group showed mental malaise, body weight decreases (P<0.01), inflammatory cell infiltration in the renal tissue, a few residual parotid glands in the thyroid, elevations in serum levels of T3, T4, Scr, BUN, cAMP, cAMP/cGMP, MDA, and NADPH (P<0.01), down-regulation in protein levels of TSH, SOD, and DRD4 (P<0.05, P<0.01), and up-regulation in expression of NOX4, p-p38 MAPK/p38 MAPK, and inflammatory factors (P<0.01). Compared with the model group, ZGW increased the body weight (P<0.05, P<0.01), reduced the infiltration of renal interstitial inflammatory cells, restored the thyroid structure and follicle size, lowered the serum levels of T3, T4, Scr, BUN, cAMP, cAMP/cGMP, MDA and NADPH (P<0.05, P<0.01), up-regulated the expression of TSH, SOD and DRD4 (P<0.05, P<0.01), and down-regulated the expression of NOX4, p-p38 MAPK/p38 MAPK, and inflammatory factors (P<0.05, P<0.01). Moreover, high-dose ZGW outperformed methimazole (P<0.05). ConclusionBy activating DRD4, ZGW can inhibit the expression of NOX4 mediated by the p38 MAPK pathway, reduce oxidative stress and inflammatory response, thereby ameliorating the pathological state of hyperthyroidism due to kidney-Yin deficiency. This study provides new molecular mechanism support for the clinical application of ZGW.
9.Xiaozheng Zhitong Paste Alleviates Bone Cancer Pain by Regulating PD-1/PD-L1-induced Osteoclast Formation
Lu SHANG ; Juanxia REN ; Guangda ZHENG ; Linghan MENG ; Lingyun WANG ; Changlin LI ; Dongtao LI ; Yaohua CHEN ; Guiping YANG ; Yanju BAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(5):72-79
ObjectiveThis study aims to investigate the action mechanism by which Xiaozheng Zhitong paste (XZP) alleviates bone cancer pain (BCP) by regulating programmed death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway-induced osteoclast formation. MethodsThirty female C57BL/6 mice were randomly allocated into the following groups (n=6 per group): normal control group, model group, low‑dose XZP group (31.5 g·kg-1), high‑dose XZP group (63 g·kg-1), and PD‑1 inhibitor (Niv) group. A bone cancer pain (BCP) model was established by injecting Lewis lung carcinoma cells. Mice in the normal control and model groups received topical application of a blank paste matrix at the wound site. Mice in the low‑ and high‑dose XZP groups were treated with XZP applied topically twice daily. Mice in the Niv group were topically administered the blank paste matrix and additionally received Niv via tail‑vein injection every two days. All interventions were continued for 21 days. During this period, behavioral tests were performed to assess mechanical, motor, and thermal nociceptive sensitivities. After 21 days, all mice were euthanized, and bone tissue from the operated side was collected for sectioning and preservation. Tartrate‑resistant acid phosphatase (TRAP) staining was used to evaluate osteoclast expression in the lesioned bone tissue. Immunohistochemistry was performed to detect the expression of Runt‑related transcription factor 2 (Runx2) in the lesioned bone tissue. Immunofluorescence was employed to assess the expression of PD‑1 and PD‑L1 in the lesioned bone tissue. ResultsCompared with the normal group, the model group showed significantly decreased limb mechanical withdrawal threshold, spontaneous paw flinching, and thermal withdrawal latency (P<0.01), increased number of osteoclasts in the lesioned bone tissue (P<0.01), and reduced expression of Runx2 (P<0.01). Compared with the model group, the BCP mice in the XZP low-dose group, XZP high-dose group, and Niv group exhibited increased limb mechanical withdrawal threshold, movement scores, and thermal withdrawal latency (P<0.01). The XZP low-dose group showed no significant changes in osteoclast number or Runx2 expression, while the XZP high-dose group and Niv group demonstrated significantly reduced osteoclast numbers (P<0.01) and significantly increased Runx2 expression (P<0.01). In the lesioned bone tissue of BCP mice, the XZP low-dose group showed no significant decrease in the percentage of PD-1 expression, but a decrease in the percentage of PD-L1 expression (P<0.05). In contrast, both the XZP high-dose group and the Niv group exhibited significant reductions in the percentages of PD-1 and PD-L1 expression (P<0.01). ConclusionXZP alleviates the pain of mice with BCP by blocking the PD-1/PD-L1 pathway to inhibit osteoclastogenesis.
10.Mechanism of Xiaozheng Zhitong Paste in Alleviating Bone Cancer Pain by Regulating Microglial Pyroptosis Based on PINK1/Parkin/NLRP3 Signaling Pathway
Lingyun WANG ; Guangda ZHENG ; Lu SHANG ; Juanxia REN ; Changlin LI ; Dongtao LI ; Haixiao LIU ; Yaohua CHEN ; Guiping YANG ; Yanju BAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(5):80-90
ObjectiveThe paper aims to investigate the mechanism by which Xiaozheng Zhitong paste (XZP) alleviates bone cancer pain (BCP) through regulating the PTEN-induced putative kinase 1 (PINK1)/Parkin-mediated mitophagy-NOD-like receptor protein 3 (NLRP3) inflammasome pathway to suppress microglial pyroptosis. MethodsLipopolysaccharide (LPS) and LPS-adenosine triphosphate (ATP) were used to establish an inflammation and pyroptosis model in microglial cells. The cells were randomly divided into the following groups: control group, LPS group, LPS+low-dose XZP group, LPS+high-dose XZP group, LPS-ATP group, LPS-ATP+low-dose XZP group, LPS-ATP+high-dose XZP group, LPS-ATP+XZP group, and LPS-ATP+XZP+CsA group. Techniques including terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, enzyme-linked immunosorbent assay (ELISA), Western blot, and confocal fluorescence staining were employed to assess the effects of XZP on microglial apoptosis, inflammatory cytokine release, inflammasome activation, pyroptosis, and mitophagy. ResultsIn vitro experiments showed that compared with the blank group, the LPS group exhibited significantly increased levels of microglial apoptosis and pro-inflammatory factors interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)(P<0.01), along with significantly upregulated protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and phosphorylated nuclear factor-κB p65 (p-NF-κB p65) (P<0.01). Compared with the LPS group, the high-dose LPS-XZP group significantly reduced the level of apoptosis (P<0.01) and the content of the aforementioned pro-inflammatory factors (P<0.01). Both the low- and high-dose LPS-XZP groups dose-dependently downregulated the protein expression of iNOS, COX-2, and p-NF-κB p65 (P<0.05, P<0.01). Compared with the blank group, the LPS-ATP group showed significantly upregulated expression of pyroptosis-related proteins, including Caspase-1/pro-Caspase-1, N-terminal fragment of gasdermin D (GSDMD-N)/full-length gasdermin D (GSDMD-F), NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), IL-1β precursor (pro-IL-1β), and mature IL-1β (P<0.01). The levels of pyroptotic factors IL-1β and IL-18 were significantly elevated (P<0.01), and membrane pore formation and intracellular reactive oxygen species (ROS) levels were significantly increased (P<0.01). Compared with the LPS-ATP group, both the low- and high-dose LPS-ATP+XZP groups dose-dependently downregulated the expression of the aforementioned pyroptosis-related proteins (P<0.05, P<0.01). The low-dose LPS-ATP+XZP group reduced IL-1β levels (P<0.01), while the high-dose group reduced both IL-1β and IL-18 levels (P<0.01) Both the low- and high-dose LPS-ATP+XZP groups dose-dependently reduced membrane pore formation and intracellular ROS production (P<0.01). Compared with the blank group, the LPS-ATP group showed significantly reduced expression of mitophagy-related proteins PINK1 and Parkin, and a decreased ratio of microtubule-associated protein 1 light chain 3Ⅱ(LC3Ⅱ) to LC3Ⅰ(P<0.01), while p62 expression was significantly increased (P<0.01). Mitochondrial ROS levels were significantly enhanced (P<0.01). Compared with the LPS-ATP group, both the low- and high-dose LPS-ATP+XZP groups dose-dependently reversed the expression of these proteins (P<0.05, P<0.01) and reduced mitochondrial ROS levels (P<0.01). After treatment with the mitophagy inhibitor cyclosporin A (CsA), the beneficial effects of XZP on mitochondrial function and its inhibitory effects on pyroptosis-related protein expression were significantly reversed (P<0.05, P<0.01). ConclusionXZP reduces ROS levels by activating PINK1/Parkin-mediated mitophagy, thereby inhibiting NLRP3 inflammasome activation and microglial pyroptosis, which provides new molecular evidence for the mechanism by which XZP alleviates BCP.


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