1.Action Mechanism of Huamoyan Granules in Treatment of Knee Osteoarthritis Based on TRPV1/p38 MAPK Pathway
Jin ZHANG ; Lili YANG ; Canwen ZHENG ; Jing KANG ; Yanlei MA ; Yue SHI ; Lei LI ; Hongxu MENG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):79-89
ObjectiveThis paper aims to observe the protective effect of Huamoyan granules on knee osteoarthritis (KOA) and explore whether its protective effect is oriented toward an anti-inflammatory direction by regulation of macrophage polarization, which can effectively inhibit the progression of pathological inflammatory response, reduce the release of inflammatory pain mediators, and downregulate the protein expression level of transient receptor potential vanilloid 1 (TRPV1), so as to provide experimental evidence for its clinical application and investigate its action mechanism. MethodsAfter adaptive feeding, Sprague-Dawley (SD) rats were randomly divided into six groups: sham group, model group, celecoxib group, and high, medium, and low-dose synovitis granule groups (9.6, 4.8, 2.4 g·kg-1). The administration dose of celecoxib capsules was 20 mg·kg-1. There were 10 rats in the sham group and 12 rats in the model group and each administration group. A KOA animal model was established by means of intra-articular injection of sodium iodoacetate into the knee joint. From the 10th day of the experiment, each administration group was given intragastric administration at a dose of 10 mL·kg-1 for 4 weeks. General conditions of rats in each group were assessed daily. The pressure pain threshold (PPT) to mechanical stimulation and joint diameter were recorded. X-ray examination was performed on the right knee joints of rats for imaging analysis. Enzyme linked immunosorbent assay (ELISA) was performed to detect the tumor necrosis factor-α (TNF-α), serum interleukin-1β (IL-1β), and other pro-inflammatory cytokines in rat serum samples, as well as the expression levels of neurogenic inflammatory mediators such as nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP). Histopathological changes in the knee joint synovial tissues were examined by hematoxylineosin (HE) staining. Safranin O-fast green staining was performed to observe and evaluate the degree of knee cartilage lesions. Western blot was employed to quantitatively analyze TRPV1, p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated (p)-p38 MAPK in rat knee synovial tissues. Immunofluorescence (IF) was used to measure and assess M1/M2 macrophage polarization. ResultsCompared with those in the sham group, the circumference and joint diameter of the right knee were markedly enlarged in the model group (P<0.01), while PPTs of rats showed a significant reduction (P<0.01). The contents of IL-1β, TNF-α, CGRP, and NGF in rats' serum were significantly elevated (P<0.01), and the synovial Krenn score was increased (P<0.01). The Mankin score of cartilage tissue was increased (P<0.01), and the protein expressions of TRPV1 and p-p38 MAPK/p38 MAPK were significantly upregulated (P<0.01). The experimental intervention significantly reduced the proportion of pro-inflammatory M1 macrophages in the total macrophage population (P<0.01), and the percentage of M2 macrophages was decreased (P<0.01). The M1/M2 macrophage ratio was significantly elevated (P<0.01). Knee joint diameters of all dose groups of Huamoyan granules and the celecoxib group were reduced (P<0.01) compared with those of the model group, and the PPT recovery speeds in the high and medium-dose groups of Huamoyan granules were more obvious (P<0.05). The contents of IL-1β, CGRP, and NGF in the rats' serum in all administration groups were significantly reduced (P<0.05, P<0.01), and the content of TNF-α in rats' serum was significantly reduced (P<0.01). All dose groups of Huamoyan granules demonstrated significant reductions in both synovial Krenn score (P<0.05, P<0.01) and protein expression of TRPV1 and p-p38 MAPK/p38 MAPK in rats' synovial tissues (P<0.01). The percentage of M1 macrophages in the synovial tissues of the celecoxib group and all dose groups of Huamoyan granules was decreased (P<0.01). The percentage of M2 macrophages was increased (P<0.05), and the M1/M2 ratio was decreased (P<0.01). ConclusionHuamoyan granules can alleviate the inflammatory response of KOA, reduce the release of inflammatory pain mediators, and downregulate TRPV1 protein expression by regulating macrophage polarization. Its mechanism may be related to the TRPV1/p38 MAPK signaling pathway, thereby achieving the effect of improving peripheral pain hypersensitivity in KOA.
2.Mechanisms of Jiangtang No. 3 Prescription in Alleviating Adipose Tissue Insulin Resistance in Diabetic Rats via TLR4/NF-κB/NLRP3 Signaling Pathway-mediated Inflammation
Tongxun WANG ; Lantian LIU ; Runqi LI ; Haoxiang LI ; Yi ZHAO ; Tian TIAN ; Rufeng MA ; Sihua GAO ; Dandan ZHAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):109-117
ObjectiveTo observe the effects of Jiangtang No. 3 prescription on inflammatory pathways and insulin resistance-related indicators in rats with type 2 diabetes mellitus (T2DM), and to elucidate its molecular mechanism in combating diabetes. MethodsA T2DM rat model was established using a high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). Successfully modeled rats were randomly assigned to the model group, metformin group, and low-, medium-, and high-dose Jiangtang No. 3 prescription groups, and a normal group was also set. Daily gavage was administered for 8 weeks as follows: metformin at 0.1 g·kg-1·d-1, Jiangtang No. 3 prescription granules at 1.62, 3.24, 6.48 g·kg-1·d-1 for the respective dose groups, and sterile water for the normal and model groups. Rat body weight, fasting blood glucose (FBG), oral glucose tolerance test (OGTT), and insulin tolerance test (ITT) were measured. After drug intervention, enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), non-esterified fatty acids (NEFA), interleukin (IL)-1β, IL-18, and insulin (INS). Hematoxylin-eosin (HE) staining was used to observe morphological changes in adipose tissue. Real-time quantitative PCR was used to detect the mRNA expression of Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), NOD-like receptor protein 3 (NLRP3), Caspase-1, IL-1β, IL-18, and gasdermin D (GSDMD) in adipose tissue. Western blot was used to measure the corresponding protein expression levels. ResultsCompared with the model group, Jiangtang No. 3 prescription groups exhibited significantly increased body weight (P<0.05, P<0.01), significantly reduced FBG (P<0.05, P<0.01), significant reductions in TC, TG, NEFA, and LDL (P<0.05, P<0.01), and a significant increase in HDL (P<0.01). Serum levels of inflammatory mediators IL-1β and IL-18 were significantly decreased (P<0.01), the homeostatic model assessment of insulin resistance (HOMA-IR) index was significantly reduced (P<0.05, P<0.01), and adipose tissue pathology was improved. The protein expression levels of TLR4, NF-κB, NLRP3, Caspase-1, IL-1β, IL-18, and GSDMD were markedly decreased (P<0.05, P<0.01), and the mRNA expression levels of these indicators were also significantly downregulated (P<0.05, P<0.01). Some effects were superior to those of the positive control drug metformin, and certain indicators exhibited dose-dependent improvements. ConclusionT2DM rats display significant inflammatory responses, disordered glucose and lipid metabolism, and insulin resistance. Jiangtang No. 3 prescription effectively suppresses inflammatory mediators, improves glucose and lipid metabolism and insulin resistance, and ameliorates pathological changes in adipose tissue. Its mechanism may be related to the regulation of the TLR4/NF-κB/NLRP3 signaling pathway in visceral adipose tissue, thereby influencing downstream inflammatory mediators.
3.Expert Consensus on Clinical Application of Qidong Yixin Oral Liquid
Changkuan FU ; Xiaochang MA ; Mingjun ZHU ; Yue DENG ; Hongxu LIU ; Mingxue ZHANG ; Ying CHEN ; Yan ZHOU ; Ling ZHANG ; Jianhua FU ; Wei YANG ; Yu'er HU ; Ming CHEN ; Yanming XIE ; Yuanyuan LI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):147-158
The prescription of Qidong Yixin oral liquid is derived from the experience of national medical master Ren Jixue in treating viral myocarditis (VMC). It has the functions of tonifying Qi, nourishing the heart,calming the mind, and relieving palpitations. It is used to treat VMC and angina pectoris of coronary heart disease caused by deficiency of both Qi and Yin. However,the understanding of its efficacy evidence, advantageous aspects, dosage and administration, and medication safety remains insufficient in clinical practice. Therefore,the development of the Expert Consensus on the Clinical Application of Qidong Yixin Oral Liquid (hereinafter referred to as consensus) was initiated. Consensus strictly followed the process and methods of the expert consensus on the clinical application of Chinese patent medicines of the China Association of Chinese Medicine,successively completing multiple tasks such as the consensus project initiation,determination of clinical problems,evidence search and evaluation,formation of recommendation opinions and consensus suggestions,solicitation of opinions,peer review, submission for review and release, and so on. Consensus formed a total of 10 recommendation opinions and 12 consensus suggestions,clarifying the clinical positioning,efficacy advantages,syndrome differentiation,dosage and administration,combination therapy,timing of medication,adverse reactions,contraindications, and precautions of Qidong Yixin oral liquid,indicating that it has good clinical advantages and safety in the treatment of VMC and angina pectoris of coronary heart disease,providing norms and references for physicians to safely and rationally apply Qidong Yixin oral liquid. Consensus was reviewed and approved for release by the Standardization Office of the China Association of Chinese Medicine on December 23, 2024. Standard number:GSCACM-376-2024.
4.Ancient and Modern Documentation of Classic Formula Sangjuyin
Xiaofang WANG ; Lyuyuan LIANG ; Jialei CAO ; Ziming XU ; Wangju ZHOU ; Yiping WANG ; Yujie CHANG ; Ruiting SU ; Yihan LI ; Jingwen LI ; Bingqi WEI ; Bingxiang MA ; Wenli SHI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):200-207
Sangjuyin, as a pungent and cooling agent with precise therapeutic effect, is a classic pungent formula for cooling relief of the epidermis, which is highly respected by medical practitioners. This formula is from the Wenbing Tiaobian written by WU Jutong in the Qing dynasty, on the basis of which subsequent medical practitioners have made additions and subtractions to apply it. The authors used the bibliometric method to systematically organize the medical books from the Qing dynasty and the Republic of China and modern literature to analyze the composition, concoction, decoction, efficacy, and previous and modern application of Sangjuyin. After examination, the drug base of this formula is basically clear. Armeniacae Semen Amarum is the dried mature seeds of Armeniaca vulgaris, family Rosaceae. Forsythiae Fructus is the dried fruit of Forsythia suspensa, family Mulleinaceae. Menthae Haplocalycis Herba is the dried above-ground part of Mentha haplocalyx, family Labiatae. Mori Folium is the dried leaves of Morus alba, family Moraceae. Chrysanthemi Flos is the dried head of Chrysanthemum morifolium, family Asteraceae. Platycodonis Radix is the dried root of Eryngium grandiflorum, family Eryngium. Glycyrrhizae Radix et Rhizoma is the dried root and rhizome of Glycyrrhiza uralensis of the Leguminosae family, and Phragmitis Rhizoma is the fresh or dried rhizome of Phragmites communis of the Gramineae family. It is recommended that the eight drugs be used in raw form as medicine. The dosage and method of decoction were converted into a modern single dosage of 7.46 g Armeniacae Semen Amarum, 5.60 g Forsythiae Fructus, 2.98 g Menthae Haplocalycis Herba, 9.33 g Mori Folium, 3.73 g Chrysanthemi Flos, 7.46 g Platycodonis Radix, 2.98 g Glycyrrhizae Radix et Rhizoma, and 11.19 g Phragmitis Rhizoma, with 400 mL water added, and the solution was boiled to obtain 200 mL, taken twice a day. Sangjuyin has the efficacy of dispersing wind and clearing heat, promoting lung and relieving cough, and it is used for treating the initial onset of wind-warmth and the evidence of evil spirits in the lungs and collaterals. Modern research has shown that Sangjuyin is often used in the treatment of cough, pneumonia, rhinitis, and other respiratory diseases, and the results of this study provide a reference for the later development of Sangjuyin.
5.Effects of total flavonoids from Carthamus tinctorius L. on hepatic stellate cell activation based on miRNA-204/NUAK1/Hippo signaling axis
Mingqi LI ; Xiaolu ZHAO ; Chenlu ZHANG ; Yinghe WANG ; Yuehong MA
China Pharmacy 2026;37(3):311-316
OBJECTIVE To investigate the effects of total flavonoids from Carthamus tinctorius L. (TFCTL) on hepatic stellate cell (HSC) activation based on the microRNA (miRNA)-204/NUAK family SNF1-like kinase 1 (NUAK1)/Hippo signaling axis, thereby elucidating the potential mechanism underlying their antifibrotic effects. METHODS The HSC-T6 cells were divided into control group, model group, TFCTL low-concentration group (20 μg/mL), TFCTL medium-concentration group (40 μg/mL), and TFCTL high-concentration group (60 μg/mL). Except for control group, the remaining groups were treated with 5 ng/mL of transforming growth factor-β to induce the activation of hepatic stellate cells, followed by the addition of corresponding drug solutions/culture medium and incubation for 24 hours. Cell apoptosis was assessed, the expression levels of α-smooth muscle actin (α-SMA), type Ⅰ collagen (Collagen Ⅰ) and proteins associated with the Hippo/Yes-associated protein (YAP) pathway [YAP, large tumor suppressor kinase 1 (LATS1), and mammalian STE20-like kinase 1 (MST1)] were detected. Additionally, cell transfection was used to investigate the activity of the miRNA-204/NUAK1/Hippo signaling axis at both the genetic and protein levels. RESULTS After intervention with TFCTL, the apoptosis rate of HSC-T6 cells and the protein expressions of MST1 (except for the TFCTL high-concentration group) and LATS1 were significantly increased (P<0.05), while the protein expressions of α-SMA, CollagenⅠ, and YAP (except for the TFCTL medium-concentration group) were significantly decreased (P<0.05). Further results from cell transfection experiments revealed that after transfection with miRNA-204 mimics, the mRNA it’s protein expressions of α-SMA, CollagenⅠ, NUAK1, and YAP in HSC-T6 cells were significantly decreased (P<0.05), while the mRNA and protein expressions of LATS1 and the mRNA expression of MST1 were significantly increased (P<0.05). Conversely, the results were opposite following transfection with miRNA-204 inhibitors. CONCLUSIONS TFCTL can exert anti-hepatic fibrosis effects by up-regulating the expression of miRNA-204, thereby down- regulating the expressions of NUAK1, inactivating the Hippo/YAP pathway, which in turn suppresses the activation of HSC and promotes their apoptosis.
6.Current Status,Strategies and Prospects of Traditional Chinese Medicine Diagnosis and Treatment for Irritable Bowel Syndrome
Yandong WEN ; Zhi YANG ; Shaogang HUANG ; Zhongyu LI ; Xiangxue MA ; Qing XU ; Liqing DU ; Bochao YUAN ; Yibing TIAN ; Wentong GE ; Xiaofan ZHAO ; Chang LIU ; Xudong TANG
Journal of Traditional Chinese Medicine 2026;67(4):404-409
Irritable bowel syndrome (IBS) is a functional bowel disorder characterized primarily by abdominal pain and altered defecation habits. In recent years, traditional Chinese medicine (TCM) has made progress in multiple aspects of IBS research and treatment, including syndrome distribution, development of TCM formulas, clinical efficacy evaluation, external therapies, and psychosocial regulation. However, it still faces challenges such as over-reliance on symptomatic manifestations rather than biomarkers for diagnostic criteria, and the lack of high-quality evidence-based data supporting the efficacy of TCM formulas in treating IBS. This paper proposed that TCM diagnosis and treatment of IBS should adhere to the strategy of integrating the holistic concept with syndrome differentiation and treatment, combining TCM external therapies such as acupuncture, moxibustion and acupoint application), and emphasizing individualized diagnosis and treatment for psychosomatic abnormalities. Future research should integrate multi-omics technologies, artificial intelligence and other methods to deepen the understanding of the pathogenesis of IBS and the mechanisms of TCM formulas, so as to promote the standardization and internationalization of TCM in the diagnosis and treatment of IBS.
7.Body image disturbance status in AS patients and analysis of its influencing factors
Min NIU ; Jingman YUAN ; Liya MA ; Hao XU ; Jun LI ; Meixi YAN ; Xinru DU ; Hanhui MA ; Xichao YANG
Journal of Public Health and Preventive Medicine 2026;37(1):158-162
Objective To understand the status of body image disturbance and its influencing factors in patients with ankylosing spondylitis (AS), so as to provide a scientific basis for the clinical management of AS. Methods A total of 353 AS patients admitted from January 2022 to December 2024 were selected as research subjects. Chinese version of Body Image Disturbance Questionnaire (BIDQ) was used to investigate the body image disturbance in AS patients. Single factor analysis was performed by t test and analysis of variance, and multiple factors were analyzed by multivariate linear regression. Results The total score of BIDQ in 342 AS patients was (25.01±4.22). Multivariate linear regression analysis results showed that self-paid medical expense, nighttime VAS score and negative emotion PANAS score could positively predict body image disturbance in AS patients (standardized regression coefficient=0.413, 0.413, 0.460, P<0.05), and PSSS score, positive emotion PANAS score and exercise management CDSSM score could negatively predict body image disturbance (standardized regression coefficient=-0.245, -0.134, -0.247, P<0.05). Conclusion The body image disturbance in AS patients is worthy of clinical attention. Nighttime pain, negative emotion and self-paid medical treatment can increase the risk of body image disturbance. Positive emotion, social support and high self-management level of exercise behavior can reduce the formation of body image disturbance, which can provide new ideas for clinical management of AS patients.
8.Ameliorating Effect of Yifei Tongluo Prescription on Bleomycin-induced Pulmonary Fibrosis in Rats via Regulating NLRP3/Caspase-1/GSDMD Signaling Pathway and Epithelial-mesenchymal Transition
Bowen ZHOU ; Zefeng LI ; Xian MA ; Xuannian LI ; Jingwen WANG ; Fei XU ; Huaman LIU ; Xinhua JIA
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(1):150-159
ObjectiveTo observe the effects of Yifei Tongluo prescription on the NOD-like receptor protein 3 (NLRP3)/Caspase-1/gasdermin D (GSDMD) pathway and epithelial-mesenchymal transition (EMT) in rats with pulmonary fibrosis. MethodsTracheal instillation of bleomycin was conducted to establish a rat model of pulmonary fibrosis. Thirty Sprague-Dawley (SD) rats were randomly divided into a blank group, a model group, a prednisone acetate group (1.17 mg·kg-1), and low- and high-dose Yifei Tongluo prescription groups (10.62 and 21.24 g·kg-1, respectively). Administration started on the 7th day after modeling, once a day for 28 consecutive days. The lung coefficient of each group was calculated. The pathological changes of lung tissues in each group were observed by hematoxylin-eosin (HE) staining and Masson staining. The expression of α-smooth muscle actin (α-SMA) and vimentin in rat lung tissues was detected by immunohistochemistry. The expression of NLRP3 inflammasome, E-cadherin (E-cad), and typeⅠ collagen (ColⅠ) in lung tissues was detected by immunofluorescence. The content of hydroxyproline (HYP), tumor necrosis factor (TNF)-α, interleukin (IL)-18, and IL-1β in rat serum was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), IL-1β, and transforming growth factor (TGF)-β1 in rat lung tissues were determined by real-time quantitative polymerase chain reaction (Real-time PCR). The protein expression levels of NLRP3, GSDMD, ASC, and Caspase-1 in rat lung tissues were determined by Western blot. ResultsCompared with the blank group, the model group exhibited a significantly increased lung coefficient (P<0.01) and significantly increased range of pulmonary interstitial inflammation and collagen deposition. In addition, the levels of α-SMA, Vimentin, E-cad, and ColⅠ in lung tissues were significantly increased (P<0.01). The levels of fibrosis- and inflammation-related factors HYP, TNF-α, IL-18, and IL-1β in serum were significantly upregulated (P<0.01). The levels of factors related to the activation of NLRP3 inflammasome in lung tissues, including NLRP3, GSDMD, ASC, Caspase-1, IL-1β, and TGF-β1, were significantly upregulated (P<0.01). Compared with the model group, the Yifei Tongluo prescription groups showed improved lung coefficients. Additionally, the extent of lung inflammation and collagen deposition was significantly reduced. The expression of α-SMA, Vimentin, E-cad, and ColⅠ in lung tissue was significantly decreased (P<0.01). The levels of HYP, TNF-α, IL-18, and IL-1β in serum were significantly reduced (P<0.01). The expression levels of NLRP3, GSDMD, ASC, Caspase-1, IL-1β, and TGF-β1 in lung tissue were also significantly decreased (P<0.01). ConclusionYifei Tongluo prescription can regulate the NLRP3/Caspase-1/GSDMD pathway, down-regulate release of pro-inflammatory and pro-fibrotic cytokines, alleviate NLRP3 inflammasome-mediated pyroptosis and EMT, and thereby improve pulmonary fibrosis in rats.
9.Analysis of unexamined items in physical examinations of radiation workers at an occupational health examination institution in Henan Province, China, 2023
Lihong MA ; Fengling ZHAO ; Yuzheng LI ; Han LIU ; Yumin LV
Chinese Journal of Radiological Health 2026;35(1):12-17
Objective To analyze the unexamined items and situations in occupational health examinations of radiation workers, and provide a reference for the revision of occupational health examination standards for radiation workers. Methods A total of 29 630 radiation workers who underwent occupational health examinations at The Third People’s Hospital of Henan Province in 2023 were selected, and the non-examination rates were statistically analyzed according to occupation, gender, and age. Results The overall non-examination rate of non-medical radiation workers was significantly lower than that of the medical radiation workers (P<0.05). The non-examination rate of chest X-rays among medical radiation workers was significantly higher than that of non-medical radiation workers (P<0.05), while no significant differences were found in other items (P>0.05). Gender-stratified analysis showed that the non-examination rate of routine urine tests was higher in females than in males in both medical and non-medical radiation workers (P<0.05). Age-stratified analysis revealed no significant differences in non-examination rates among different age groups in non-medical radiation workers (P>0.05), whereas the chest X-ray non-examination rate was relatively high in medical radiation workers under 30 years old (P<0.05). Conclusion Significant differences were observed in the non-examination rates of occupational health examinations among radiation workers based on occupation, gender, and age. The overall non-examination rate was relatively low in non-medical radiation workers.
10.Investigation of radon activity concentration and dose assessment in subways of Nanning City, China
Xiufang LU ; Yilong MA ; Rongzheng HUANG ; Ziyue LI ; Jiajie LEI ; Lanying FENG ; Zhangfan CHEN ; Xinchun ZHAO
Chinese Journal of Radiological Health 2026;35(1):67-73
Objective To investigate the radon activity concentrations in subways of Nanning City and assess the average annual effective doses for subway staff and passengers due to radon exposure. Methods Sixty-three stations across the subway lines 2, 3, and 5 were selected as study sites. Radon activity concentrations were measured using the scintillation counting method with scintillation vials. Results The radon activity concentrations in subway lines 2, 3, and 5 were 7.9-24.4, 12.0-26.2, and 12.6-18.2 Bq/m3, respectively. The average radon activity concentrations for these three lines were (17.4 ± 4.6), (19.1 ± 4.1), and (14.6 ± 1.7) Bq/m3, respectively. Statistical analysis using SPSS 26.0 software revealed a significant difference in radon activity concentrations among these stations (P<0.01). Considering the data in previous research, the average radon activity concentration across all stations in the subway lines of Nanning City was determined to be 17.4 Bq/m3. The estimated average annual effective dose due to radon exposure was 0.131 mSv for subway staff and 0.033 mSv for passengers. Conclusion The radon activity concentrations in the subway lines of Nanning City were significantly lower than the national standard limit (400 Bq/m3). The annual effective doses from radon exposure for both subway staff and passengers were below the limits specified in the Basic Standards for Protection Against Ionizing Radiation and for the Safety of Radiation Sources (GB18871—2002). The health impact of radon and its progeny on subway staff and passengers in the subway lines of Nanning City was extremely low and can be considered negligible.


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