1.Effect Analysis of Different Interventions to Improve Neuroinflammation in The Treatment of Alzheimer’s Disease
Jiang-Hui SHAN ; Chao-Yang CHU ; Shi-Yu CHEN ; Zhi-Cheng LIN ; Yu-Yu ZHOU ; Tian-Yuan FANG ; Chu-Xia ZHANG ; Biao XIAO ; Kai XIE ; Qing-Juan WANG ; Zhi-Tao LIU ; Li-Ping LI
Progress in Biochemistry and Biophysics 2025;52(2):310-333
Alzheimer’s disease (AD) is a central neurodegenerative disease characterized by progressive cognitive decline and memory impairment in clinical. Currently, there are no effective treatments for AD. In recent years, a variety of therapeutic approaches from different perspectives have been explored to treat AD. Although the drug therapies targeted at the clearance of amyloid β-protein (Aβ) had made a breakthrough in clinical trials, there were associated with adverse events. Neuroinflammation plays a crucial role in the onset and progression of AD. Continuous neuroinflammatory was considered to be the third major pathological feature of AD, which could promote the formation of extracellular amyloid plaques and intracellular neurofibrillary tangles. At the same time, these toxic substances could accelerate the development of neuroinflammation, form a vicious cycle, and exacerbate disease progression. Reducing neuroinflammation could break the feedback loop pattern between neuroinflammation, Aβ plaque deposition and Tau tangles, which might be an effective therapeutic strategy for treating AD. Traditional Chinese herbs such as Polygonum multiflorum and Curcuma were utilized in the treatment of AD due to their ability to mitigate neuroinflammation. Non-steroidal anti-inflammatory drugs such as ibuprofen and indomethacin had been shown to reduce the level of inflammasomes in the body, and taking these drugs was associated with a low incidence of AD. Biosynthetic nanomaterials loaded with oxytocin were demonstrated to have the capability to anti-inflammatory and penetrate the blood-brain barrier effectively, and they played an anti-inflammatory role via sustained-releasing oxytocin in the brain. Transplantation of mesenchymal stem cells could reduce neuroinflammation and inhibit the activation of microglia. The secretion of mesenchymal stem cells could not only improve neuroinflammation, but also exert a multi-target comprehensive therapeutic effect, making it potentially more suitable for the treatment of AD. Enhancing the level of TREM2 in microglial cells using gene editing technologies, or application of TREM2 antibodies such as Ab-T1, hT2AB could improve microglial cell function and reduce the level of neuroinflammation, which might be a potential treatment for AD. Probiotic therapy, fecal flora transplantation, antibiotic therapy, and dietary intervention could reshape the composition of the gut microbiota and alleviate neuroinflammation through the gut-brain axis. However, the drugs of sodium oligomannose remain controversial. Both exercise intervention and electromagnetic intervention had the potential to attenuate neuroinflammation, thereby delaying AD process. This article focuses on the role of drug therapy, gene therapy, stem cell therapy, gut microbiota therapy, exercise intervention, and brain stimulation in improving neuroinflammation in recent years, aiming to provide a novel insight for the treatment of AD by intervening neuroinflammation in the future.
2.Comparison of SEC-RI-MALLS and SEC-RID methods for determining molecular weight and molecular weight distribution of PLGA
WANG Baocheng ; ZHANG Xiaoyan ; ZHOU Xiaohua ; ZHAO Xun ; MA Congyu ; GAO Zhengsong ; SHI Haiwei ; YUAN Yaozuo ; HANG Taijun
Drug Standards of China 2025;26(1):110-116
Objective: To establish a method for determining the molecular weight and molecular weight distribution of Poly(Lactide-co-Glycolide Acid) (PLGA) using Size Exclusion Chromatography-Refractive Index-Multiangle Laser Light Scattering (SEC-RI-MALLS) and Size Exclusion Chromatography-Refractive Index (SEC-RID), and to compare the results obtained from these two methods.
Methods: For SEC-RI-MALLS, tetrahydrofuran was used as the mobile phase, Shodex GPC KF-803L was employed as the chromatographic column with a flow rate of 1 mL·min-1, column temperature at 30 ℃, and an injection volume of 100 μL. For SEC-RID, tetrahydrofuran was also used as the mobile phase, Agilent PLgel 5 μm MIXD-D was used as the chromatographic column with a flow rate of 1 mL·min-1, column temperature at 30 ℃, differential detector temperature at 35 ℃, and an injection volume of 20 μL. The molecular weight and molecular weight distribution were calculated using Agilent’s GPC software. The newly established methods were validated methodologically, and the molecular weight and molecular weight distribution of 13 batches of samples were determined.
Results: The precision, accuracy, stability, and repeatability tests for SEC-RI-MALLS showed RSD values of 1.35%, 1.58%, 1.53%, and 1.26%, respectively. The SEC-RID method exhibited good linearity (r=0.999 9), with RSD values for precision, accuracy, stability, and repeatability tests (n=6) of 2.05%, 1.62%, 1.30%, and 2.97%, respectively. The results obtained from SEC-RI-MALLS were lower than those from SEC-RID, and the molecular weight distribution coefficient was smaller, but the results from the paired T-test performed with the value measured by SEC-RID method and the value measured by SEC-RI-MALLS method multiplied a conversion coefficient of 1.5 showed no significant difference between the two methods.
Conclusion: Both methods are stable and reliable, and can be used for the determination of PLGA molecular weight and molecular weight distribution based on the specific situations.
3.Sufei Pingchuan Formula (肃肺平喘方) for the Treatment of Bronchiectasis Patients Combined with Airflow Limitation of Phlegm-Heat Obstructing the Lung and Lung-Spleen Qi Deficiency Syndrome: A Randomised Controlled Trial
Shasha YUAN ; Haiyan ZHANG ; Xia SHI ; Bing WANG ; Xiaodong CONG ; Qing MIAO
Journal of Traditional Chinese Medicine 2025;66(6):581-587
ObjectiveTo evaluate the effectiveness and safety of Sufei Pingchuan Formula (肃肺平喘方) in the treatment of bronchiectasis with airflow limitation, phlegm-heat obstructing the lung, and lung-spleen qi deficiency syndrome. MethodsA randomized, double-blind, placebo-controlled trial was conducted. A total of 72 patients with stable bronchiectasis with airflow limitation of phlegm-heat obstructing the lung and lung-spleen qi deficiency syndrome were randomly divided into treatment group and control group, with 36 cases in each group. On the basis of regular inhalation of tiotropium bromide inhalation spray, the treatment group was given Sufei Pingchuan Formula granules, and the control group was given Sufei Pingchuan Formula granule simulant. The course of treatment in both groups was 12 weeks. The pulmonary function of both groups before and after treatment was observed, specifically focusing on forced expiratory volume in one second (FEV1); the modified British Medical Research Council (mMRC) dyspnea scale, 24-hour sputum volume, COPD assessment test (CAT), and traditional Chinese medicine (TCM) syndrome scores were assessed before treatment and after 4, 8, and 12 weeks of treatment; acute exacerbations were recorded at weeks 4, 8, and 12; additionally, changes in routine blood tests, urinalysis, liver and kidney function, and adverse events were monitored before and after treatment. ResultsAfter treatment, 4 patients in the treatment group and 6 in the control group dropped out. After 12 weeks of treatment, FEV1 increased in both groups compared to pre-treatment levels (P<0.05), but the difference between groups was not statistically significant (P>0.05). Compared to before treatment, the treatment group showed a reduction in mMRC scores after 12 weeks (P<0.05) and a decrease in 24-hour sputum volume, CAT scores, and TCM syndrome scores at weeks 4, 8, and 12 (P<0.05). In the control group, 24-hour sputum volume decreased after 12 weeks (P<0.05), and TCM syndrome scores decreased at weeks 8 and 12 (P<0.05). Compared to the control group, the treatment group showed a greater reduction in mMRC scores at week 12 (P<0.05), a decrease in 24-hour sputum volume and TCM syndrome scores at weeks 4, 8, and 12 (P<0.05), and lower CAT scores at weeks 8 and 12 (P<0.05). The frequency and number of acute exacerbations in the treatment group were significantly lower than those in the control group at week 12 (P<0.05). No severe adverse events occurred in either group. ConclusionSufei Pingchuan Formula can improve the pulmonary function FEV1, the severity of dyspnea, reduce 24-hour sputum volume and frequent acute exacerbations, and improve the quality of life in patients with bronchiectasis and airflow limitation, with good safety.
4.Luteolin improves myocardial cell death induced by serum from rats with spinal cord injury
Wenwen ZHANG ; Mengru XU ; Yuan TIAN ; Lifei ZHANG ; Shu SHI ; Ning WANG ; Yuan YUAN ; Li WANG ; Haihu HAO
Chinese Journal of Tissue Engineering Research 2025;29(1):38-43
BACKGROUND:Cardiac dysfunction due to spinal cord injury is an important factor of death in patients with spinal cord injury;however,the specific mechanism is still not clear.Therefore,revealing the mechanism of cardiac dysfunction in spinal cord injury patients is of great significance to improve their quality of life and survival rate. OBJECTIVE:To investigate the mechanism of luteolin in improving serum-induced myocardial cell death in spinal cord injury rats. METHODS:Allen's impact instrument was used to damage the spine T9-T11 of male SD rats to establish a spinal cord injury model meanwhile a sham operation group was set as the control group.The serum of rats of each group was collected.H9c2 cells were divided into a blank control group,a sham operated rat serum group,a spinal cord injury rat serum group and a luteolin pretreatment group.The cells in blank control group were only cultured with ordinary culture medium.The cells in the sham operated rat serum group were treated with medium containing 10%serum from sham operated rat.The cells in the spinal cord injury rat serum group were treated with medium containing 10%serum from spinal cord injury rat.The cells in the luteolin pretreatment group were precultured with a final concentration of 20 μmol/L luteolin for 4 hours and then changed to a medium containing 10%rat serum from spinal cord injury rat.After 24 hours of culture,the survival rate of each group of H9c2 cells was measured by CCK-8 assay.Western blot assay was used to detect the expression of autophagy related protein LC3 and p62 in H9c2 cells in each group. RESULTS AND CONCLUSION:Compared with the blank control group,there was no significant change in cell survival rate in the sham operated rat serum group(P>0.05).Compared with the sham operated rat serum group,the cell survival rate(P<0.01)and the expression of LC3 protein(P<0.05)in spinal cord injury rat serum group was significantly reduced,and the expression of p62 protein was significantly increased(P<0.05).Compared with the spinal cord injury rat serum group,the survival rate of cells in the luteolin pretreatment group significantly increased(P<0.000 1);the expression of LC3 protein significantly increased(P<0.05),and the expression of p62 protein significantly decreased(P<0.05).The results indicate that luteolin may improve myocardial cell death induced by serum from rats with spinal cord injury by promoting autophagy.
5.Chemical consitituents and hypoglycemic activity of Qinhuai No. 1 Rehmannia glutinosa
Meng YANG ; Zhi-you HAO ; Xiao-lan WANG ; Chao-yuan XIAO ; Jun-yang ZHANG ; Shi-qi ZHOU ; Xiao-ke ZHENG ; Wei-sheng FENG
Acta Pharmaceutica Sinica 2025;60(1):205-210
Eight compounds were isolated and purified from the ethyl acetate part of 70% acetone extract of
6.Analysis of Differences in Secondary Metabolites Between Dendrobium nobile Bionic Wild Cultivated on Epiphytic Stones and Trees Based on Widely Targeted Metabolomics
Yifan SHI ; Changqing ZHOU ; Jiaojiao WANG ; Lin CHEN ; Hongping CHEN ; Fu WANG ; Yuan HU ; Youping LIU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(10):218-224
ObjectiveTo explore the differences in the accumulation of secondary metabolites of Dendrobium nobile cultivated on epiphytic stones and trees, so as to elucidate the scientific connotation of "only those that grow on stones has superior quality", and provide a direction for the cultivation and quality evaluation of D. nobile. MethodsUltra-performance liquid chromatography-triple quadrupole/linear ion trap mass spectrometry(UPLC-QTRAP-MS/MS)-based widely targeted metabolomics was used to detect the metabolites of D. nobile cultivated on epiphytic stones and trees. And the combination of principal component analysis(PCA), hierarchical cluster analysis(HCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) was performed for multivariate statistical analysis of metabolites. Differential metabolites were screened by variable importance in the projection(VIP) value≥1 and log2fold change(FC)≥1 or ≤-1, and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was conducted. ResultsA total of 1 267 metabolites were identified in the stems of D. nobile from the two cultivation modes, dominated by flavonoids(292), phenolic acids(284), and alkaloids(189). Through OPLS-DA screening, 473 differential metabolites were obtained. Compared to epiphytic tree-cultivated D. nobile, epiphytic stone-cultivated D. nobile exhibited upregulation of flavonoids, phenolic acids, alkaloids, lignans and coumarins, while quinones and terpenoids were down-regulated. The differential metabolites mainly included flavonoid glycosides and alkaloids, and these differential metabolites significantly contributed to characterizing the two cultivation patterns. KEGG enrichment analysis revealed significant enrichment in pathways of flavone and flavonol biosynthesis, flavonoid biosynthesis, tyrosine metabolism, and phenylalanine metabolism in epiphytic stone-cultivated D. nobile. ConclusionEpiphytic stone cultivation is beneficial for the accumulation of phenolic acids, flavonoids, and alkaloids in D. nobile, indicating that the "only those that grow on stones has superior quality" documented in the materia medica has certain scientific basis, and the findings also provide a reference for quality evaluation and discrimination research between epiphytic stone and tree cultivated D. nobile.
7.Process Optimization and Health Risk Assessment of Calcined Haematitum Based on QbD Concept
Yue YANG ; Jingwei ZHOU ; Jialiang ZOU ; Guorong MEI ; Yifan SHI ; Lei ZHONG ; Jiaojiao WANG ; Xuelian GAN ; Dewen ZENG ; Xin CHEN ; Lin CHEN ; Hongping CHEN ; Shilin CHEN ; Yuan HU ; Youping LIU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(13):187-196
ObjectiveTo investigate the processing technology of calcined Haematitum based on the concept of quality by design(QbD) and to assess its health risk. MethodsTaking whole iron content, Fe2+ dissolution content and looseness as critical quality attributes(CQAs), and calcination temperature, calcination time, spreading thickness and particle size as critical process parameters(CPPs) determined by the failure mode and effect analysis(FMEA), the processing technology of calcined Haematitum was optimized by orthogonal test combined with analytic hierarchy process-criteria importance through intercriteria correlation(AHP-CRITIC) hybrid weighting method. The contents of heavy metals and harmful elements were determined by inductively coupled plasma mass spectrometry, and the health risk assessment was carried out by daily exposure(EXP), target hazard quotient(THQ) and lifetime cancer risk(LCR), and the theoretical value of the maximum limit was deduced. ResultsThe optimal processing technology for calcined Haematitum was calcination at 650 ℃, calcination time of 1 h, particle size of 0.2-0.5 cm, spreading thickness of 1 cm, and vinegar quenching for 1 time[Haematitum-vinegar(10:3)]. The contents of 5 heavy metals and harmful elements in 13 batches of calcined Haematitum were all decreased with reductions of up to 5-fold. The cumulative THQ of 2 batches of samples was>1, while the cumulative THQ of all batches of Haematitum was>1. The LCR of As in 1 batches of Haematitum was 1×10-6-1×10-4, and the LCR of the rest was<1×10-6, and the LCRs of calcined Haematitum were all<1×10-6, indicating that the carcinogenic risk of calcined Haematitum was low, but special attention should still be paid to Haematitum medicinal materials. Preliminary theoretical values of the maximum limits of Cu, As, Cd, Pb and Hg were formulated as 1 014, 25, 17, 27, 7 mg·kg-1. ConclusionThe optimized processing technology of calcined Haematitum is stable and feasible, and the contents of heavy metals and harmful elements are reduced after processing. Preliminary theoretical values of the maximum limits of Cu, As, Cd, Pb and Hg are formulated to provide a scientific basis for the formulation of standards for the limits of harmful elements in Haematitum.
8.Efficacy and Mechanism of Shuanghua Drink in Treating Primary Dysmenorrhea Based on COX-2/NF-κB Signaling Pathway
Yuncheng MA ; Yuanyuan SHI ; Zhen LIU ; Yuxi WANG ; Yuan TIAN ; Qian LI ; Xiaozhu WANG ; Cheng HE ; Wenhui XU ; Weiling WANG ; Jian GAO ; Ting WANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(12):72-80
ObjectiveTo evaluate the efficacy of Shuanghua drink in treating primary dysmenorrhea in the rat model and explore its mechanism of action. MethodsAn oxytocin-induced writhing mouse model was established to evaluate the analgesic effect of Shuanghua drink. Forty-eight non-pregnant female institute of cancer research (ICR) mice were randomly divided into six groups, including a blank group, a model group, an ibuprofen group (85.00 mg·kg-1), a low-dose group of Shuanghua drink (7.14 mL·kg-1), a medium-dose group of Shuanghua drink (14.28 mL·kg-1), and a high-dose group of Shuanghua drink (28.57 mL·kg-1). Each group consisted of eight mice. All treatment groups received daily intragastric administration at corresponding doses for 10 consecutive days. One hour after the final administration, 2 U of oxytocin was intraperitoneally injected per mouse. The writhing latency and number of writhing within 20 minutes were recorded. A primary dysmenorrhea rat model was established by using estradiol benzoate and oxytocin to evaluate the inhibitory effect of Shuanghua drink on the contraction of uterine smooth muscle. Forty-eight non-pregnant female Sprague-Dawley (SD) rats were divided into six groups, including a blank group, a model group, an ibuprofen group (51.00 mg·kg-1), a low-dose group of Shuanghua drink (4.28 mL·kg-1), a medium-dose group of Shuanghua drink (8.57 mL·kg-1), and a high-dose group of Shuanghua drink (17.10 mL·kg-1). Each group consisted of eight rats. Rats received subcutaneous injections of estradiol benzoate for 10 consecutive days to enhance uterine sensitivity. On the eleventh day, oxytocin (2 U/rat) was intraperitoneally administered to induce abnormal uterine contractions for establishing the primary dysmenorrhea model. All treatment groups received daily intragastric administration from the second day of modeling for 10 days. The effects of Shuanghua drink were evaluated by using parameters including uterine motility and the variation rate of uterine motility. The mechanism of action was investigated in rats with primary dysmenorrhea. The content of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), thromboxane B2 (TXB2), prostacyclin metabolite (6-keto-PGF1α), and β-endorphin (β-EP) in uterine tissue of rats was detected by using enzyme-linked immunosorbent assay (ELISA). The changes in the content of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) were analyzed via colorimetric assay. Western blot was performed to determine the content of phosphorylated inhibitor of kappa B kinase beta (p-IKKβ)/IKKβ, phosphorylated inhibitor of kappa B alpha (p-IκBα), IκBα, phosphorylated p65 (p-p65), p65, and cyclooxygenase-2 (COX-2) proteins in uterine tissue of rats. ResultsIn the oxytocin-induced writhing mouse model, the model group exhibited significantly shortened writhing latency and increased writhing frequency compared to the control group (P<0.01). Both the ibuprofen group and the high-dose group of Shuanghua drink displayed prolonged writhing latency (P<0.05), while the ibuprofen group and the low-dose, medium-dose, and high-dose groups of Shuanghua drink exhibited reduced writhing frequency (P<0.01). In the primary dysmenorrhea rat model, the uterine motility and its variation rate in the model group were significantly higher than those in the blank group (P<0.01). These parameters were markedly suppressed by ibuprofen and Shuanghua drink at all tested doses (P<0.01). For the mechanism of action, the model group showed significantly increased PGF2α/PGE2, TXB2/6-keto-PGF1α, NO, and iNOS in uterine tissue (P<0.05, P<0.01) and significantly decreased β-EP (P<0.01). These parameters were significantly attenuated in the ibuprofen group and the low-dose, medium-dose, and high-dose groups of Shuanghua drink. The PGF2α/PGE2 (P<0.01), TXB2/6-keto-PGF1α (P<0.01), NO (medium-dose group P<0.05), and iNOS (P<0.01) were reduced, and the β-EP (medium-dose group P<0.05) was up-regulated. Compared to the model group, the ibuprofen group and medium-dose group of Shuanghua drink showed significantly increased content of β-EP in the serum of rats (P<0.05). Compared to the blank group, the model group showed significantly elevated expressions of COX-2, p-IKKβ/IKKβ, p-IκBα/IκBα, and p-p65/p65 proteins (P<0.01) and significantly reduced anti-inflammatory protein IκBα (P<0.05). Compared to the model group, the ibuprofen group and the low-dose, medium-dose, and high-dose groups of Shuanghua drink showed significantly reduced expressions of COX-2 (P<0.01), p-IKKβ/IKKβ (P<0.01), p-IκBα/IκBα (P<0.05, P<0.01), and p-p65/p65(P<0.01) and up-regulated expression of IκBα protein (P<0.05, P<0.01). ConclusionShuanghua drink effectively alleviates primary dysmenorrhea through analgesia and suppression of abnormal contractions of uterine smooth muscle. Its mechanism may be mediated by reduced levels of PGF2α/PGE2, TXB2/6-keto-PGF1α, iNOS, and NO, elevated β-EP level, and inhibited COX-2/NF-κB signaling pathway.
9.Process Optimization and Health Risk Assessment of Calcined Haematitum Based on QbD Concept
Yue YANG ; Jingwei ZHOU ; Jialiang ZOU ; Guorong MEI ; Yifan SHI ; Lei ZHONG ; Jiaojiao WANG ; Xuelian GAN ; Dewen ZENG ; Xin CHEN ; Lin CHEN ; Hongping CHEN ; Shilin CHEN ; Yuan HU ; Youping LIU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(13):187-196
ObjectiveTo investigate the processing technology of calcined Haematitum based on the concept of quality by design(QbD) and to assess its health risk. MethodsTaking whole iron content, Fe2+ dissolution content and looseness as critical quality attributes(CQAs), and calcination temperature, calcination time, spreading thickness and particle size as critical process parameters(CPPs) determined by the failure mode and effect analysis(FMEA), the processing technology of calcined Haematitum was optimized by orthogonal test combined with analytic hierarchy process-criteria importance through intercriteria correlation(AHP-CRITIC) hybrid weighting method. The contents of heavy metals and harmful elements were determined by inductively coupled plasma mass spectrometry, and the health risk assessment was carried out by daily exposure(EXP), target hazard quotient(THQ) and lifetime cancer risk(LCR), and the theoretical value of the maximum limit was deduced. ResultsThe optimal processing technology for calcined Haematitum was calcination at 650 ℃, calcination time of 1 h, particle size of 0.2-0.5 cm, spreading thickness of 1 cm, and vinegar quenching for 1 time[Haematitum-vinegar(10:3)]. The contents of 5 heavy metals and harmful elements in 13 batches of calcined Haematitum were all decreased with reductions of up to 5-fold. The cumulative THQ of 2 batches of samples was>1, while the cumulative THQ of all batches of Haematitum was>1. The LCR of As in 1 batches of Haematitum was 1×10-6-1×10-4, and the LCR of the rest was<1×10-6, and the LCRs of calcined Haematitum were all<1×10-6, indicating that the carcinogenic risk of calcined Haematitum was low, but special attention should still be paid to Haematitum medicinal materials. Preliminary theoretical values of the maximum limits of Cu, As, Cd, Pb and Hg were formulated as 1 014, 25, 17, 27, 7 mg·kg-1. ConclusionThe optimized processing technology of calcined Haematitum is stable and feasible, and the contents of heavy metals and harmful elements are reduced after processing. Preliminary theoretical values of the maximum limits of Cu, As, Cd, Pb and Hg are formulated to provide a scientific basis for the formulation of standards for the limits of harmful elements in Haematitum.
10.Efficacy and Mechanism of Shuanghua Drink in Treating Primary Dysmenorrhea Based on COX-2/NF-κB Signaling Pathway
Yuncheng MA ; Yuanyuan SHI ; Zhen LIU ; Yuxi WANG ; Yuan TIAN ; Qian LI ; Xiaozhu WANG ; Cheng HE ; Wenhui XU ; Weiling WANG ; Jian GAO ; Ting WANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(12):72-80
ObjectiveTo evaluate the efficacy of Shuanghua drink in treating primary dysmenorrhea in the rat model and explore its mechanism of action. MethodsAn oxytocin-induced writhing mouse model was established to evaluate the analgesic effect of Shuanghua drink. Forty-eight non-pregnant female institute of cancer research (ICR) mice were randomly divided into six groups, including a blank group, a model group, an ibuprofen group (85.00 mg·kg-1), a low-dose group of Shuanghua drink (7.14 mL·kg-1), a medium-dose group of Shuanghua drink (14.28 mL·kg-1), and a high-dose group of Shuanghua drink (28.57 mL·kg-1). Each group consisted of eight mice. All treatment groups received daily intragastric administration at corresponding doses for 10 consecutive days. One hour after the final administration, 2 U of oxytocin was intraperitoneally injected per mouse. The writhing latency and number of writhing within 20 minutes were recorded. A primary dysmenorrhea rat model was established by using estradiol benzoate and oxytocin to evaluate the inhibitory effect of Shuanghua drink on the contraction of uterine smooth muscle. Forty-eight non-pregnant female Sprague-Dawley (SD) rats were divided into six groups, including a blank group, a model group, an ibuprofen group (51.00 mg·kg-1), a low-dose group of Shuanghua drink (4.28 mL·kg-1), a medium-dose group of Shuanghua drink (8.57 mL·kg-1), and a high-dose group of Shuanghua drink (17.10 mL·kg-1). Each group consisted of eight rats. Rats received subcutaneous injections of estradiol benzoate for 10 consecutive days to enhance uterine sensitivity. On the eleventh day, oxytocin (2 U/rat) was intraperitoneally administered to induce abnormal uterine contractions for establishing the primary dysmenorrhea model. All treatment groups received daily intragastric administration from the second day of modeling for 10 days. The effects of Shuanghua drink were evaluated by using parameters including uterine motility and the variation rate of uterine motility. The mechanism of action was investigated in rats with primary dysmenorrhea. The content of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), thromboxane B2 (TXB2), prostacyclin metabolite (6-keto-PGF1α), and β-endorphin (β-EP) in uterine tissue of rats was detected by using enzyme-linked immunosorbent assay (ELISA). The changes in the content of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) were analyzed via colorimetric assay. Western blot was performed to determine the content of phosphorylated inhibitor of kappa B kinase beta (p-IKKβ)/IKKβ, phosphorylated inhibitor of kappa B alpha (p-IκBα), IκBα, phosphorylated p65 (p-p65), p65, and cyclooxygenase-2 (COX-2) proteins in uterine tissue of rats. ResultsIn the oxytocin-induced writhing mouse model, the model group exhibited significantly shortened writhing latency and increased writhing frequency compared to the control group (P<0.01). Both the ibuprofen group and the high-dose group of Shuanghua drink displayed prolonged writhing latency (P<0.05), while the ibuprofen group and the low-dose, medium-dose, and high-dose groups of Shuanghua drink exhibited reduced writhing frequency (P<0.01). In the primary dysmenorrhea rat model, the uterine motility and its variation rate in the model group were significantly higher than those in the blank group (P<0.01). These parameters were markedly suppressed by ibuprofen and Shuanghua drink at all tested doses (P<0.01). For the mechanism of action, the model group showed significantly increased PGF2α/PGE2, TXB2/6-keto-PGF1α, NO, and iNOS in uterine tissue (P<0.05, P<0.01) and significantly decreased β-EP (P<0.01). These parameters were significantly attenuated in the ibuprofen group and the low-dose, medium-dose, and high-dose groups of Shuanghua drink. The PGF2α/PGE2 (P<0.01), TXB2/6-keto-PGF1α (P<0.01), NO (medium-dose group P<0.05), and iNOS (P<0.01) were reduced, and the β-EP (medium-dose group P<0.05) was up-regulated. Compared to the model group, the ibuprofen group and medium-dose group of Shuanghua drink showed significantly increased content of β-EP in the serum of rats (P<0.05). Compared to the blank group, the model group showed significantly elevated expressions of COX-2, p-IKKβ/IKKβ, p-IκBα/IκBα, and p-p65/p65 proteins (P<0.01) and significantly reduced anti-inflammatory protein IκBα (P<0.05). Compared to the model group, the ibuprofen group and the low-dose, medium-dose, and high-dose groups of Shuanghua drink showed significantly reduced expressions of COX-2 (P<0.01), p-IKKβ/IKKβ (P<0.01), p-IκBα/IκBα (P<0.05, P<0.01), and p-p65/p65(P<0.01) and up-regulated expression of IκBα protein (P<0.05, P<0.01). ConclusionShuanghua drink effectively alleviates primary dysmenorrhea through analgesia and suppression of abnormal contractions of uterine smooth muscle. Its mechanism may be mediated by reduced levels of PGF2α/PGE2, TXB2/6-keto-PGF1α, iNOS, and NO, elevated β-EP level, and inhibited COX-2/NF-κB signaling pathway.

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