1.Therapeutic Effect of Cranial Painkiller Pills' Extract Powder in Treatment of Trigeminal Neuralgia Induced by Injection of Talci Pulvis into Infraorbital Foramen of Model Rats Based on OTULIN-regulated Neuroinflammation
Shuran LI ; Xinwei WANG ; Jing SUN ; Dan XIE ; Ronghua ZHAO ; Lei BAO ; Zihan GENG ; Qiyue SUN ; Jingsheng ZHANG ; Yaxin WANG ; Xihe CUI ; Xinying LI ; Bing HAN ; Tianjiao LU ; Xiaolan CUI ; Liying LIU ; Shanshan GUO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):21-28
ObjectiveThis paper aims to verify the therapeutic effect of Cranial Painkiller pills' extract powder prepared by the new process on the rat's trigeminal neuralgia model caused by infraorbital injection of Talci Pulvis, evaluate its potential clinical application value, and compare the therapeutic effect with that of Cranial Painkiller granules, so as to provide data support for the application of the Cranial Painkiller pills' extract powder and precise treatment. MethodsThe rat's trigeminal neuralgia model was constructed by infraorbital injection of Talci Pulvis, and the rats were randomly divided into the normal group, model group, carbamazepine group (60 mg·kg-1), Cranial Painkiller granules group (2.70 g·kg-1), and low, medium, and high dosage groups of Cranial Painkiller pills' extract powder (1.35, 2.70, 5.40 g·kg-1) according to the basal mechanical pain thresholds, and there were 10 rats in each group. The drug was administered by gavage to each group 2 h after modeling, and distilled water was given by gavage to the normal and model groups under the same conditions once a day for 10 d. Von Frey brushes were used to measure mechanical pain thresholds in rats. Hematoxylin-eosin (HE) staining was used to detect pathological changes in the trigeminal ganglion, and enzyme-linked immunosorbent assay (ELISA) was used to detect the inflammatory factors interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) levels in rat serum, as well as neuropeptide substance P (SP) and β-endorphin (β-EP) levels in rat brain tissue. Western blot technique was used to detect the levels of NLRP3, ASC, Caspase-1, and OTULIN proteins in rat brain tissue. ResultsCompared with the normal group, the pain threshold of rats in the model group showed a continuous significant decrease (P<0.01). The pathological damage of brain tissue was significant (P<0.01), and the inflammatory levels of IL-1, IL-6, IL-8, and TNF-α in serum were significantly elevated (P<0.01). The level of the SP in the brain tissue was significantly elevated (P<0.01), and the level of β-EP was significantly reduced (P<0.01), while the level of OTULIN was significantly reduced, and NLRP3, ASC, and Caspase-1 protein levels were significantly elevated (P<0.01). After administration of the drug, compared with the model group, the pain threshold of each dose group of the Cranial Painkiller pills' extract powder and the Cranial Painkiller granules group significantly increased (P<0.01). The inflammatory levels of IL-1, IL-6, IL-8, and TNF-α and SP levels significantly decreased (P<0.01), and the β-EP levels were significantly elevated (P<0.01), while the levels of OTULIN protein were significantly elevated (P<0.05, P<0.01), and the levels of NLRP3, ASC proteins were decreased (P<0.01)in high dose Cranial Painkiller pills' extract powder. Meanwhile, compared with those in the model group, the trigeminal ganglion lesions of rats in the Cranial Painkiller pills' extract powder and Cranial Painkiller granules groups showed different degrees of improvement (P<0.05, P<0.01). ConclusionThe Cranial Painkiller pills' extract powder has significant therapeutic effects on the rat model of trigeminal neuralgia induced by infraorbital injection of Talci Pulvis, and its mechanism is related to the improvement of OTULIN-regulated neuroinflammation.
2.Mechanism of Tibetan Medicine Sanwei Doukoutang to Improve Cognitive Dysfunction in 5×FAD Mice Based on Wnt/β-catenin Signaling Pathway
Shuran LI ; Yaxin WANG ; Jing SUN ; Lei BAO ; Zihan GENG ; Dan XIE ; Ronghua ZHAO ; Yanyan BAO ; Qiyue SUN ; Jingsheng ZHANG ; Xinwei WANG ; Xinying LI ; Xihe CUI ; Xiaowei YANG ; LIUXIAN ; Mengyao CUI ; Qingshan LIU ; Shanshan GUO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):54-60
ObjectiveTo explore the effects of the Tibetan medicine Sanwei Doukoutang (SWDK) on cognitive dysfunction in mice suffering from Alzheimer's disease (AD) and its related mechanism. MethodsFifty SPF 5 × FAD mice were randomly divided into model group, total ginsenoside group(0.04 g·kg-1), high-, medium-, and low-dose groups of SWDK (32.60, 16.30, 8.15 g·kg-1), with 10 mice in each group, and ten wild-type mice of the same age were used as the normal group, male and female in 1∶1. Gavage administration was performed once daily for 8 weeks. The Morris water maze test and contextual fear memory experiment were used to observe learning and memory function. Hematoxylin-eosin (HE) staining was utilized to observe the changes in the pathomorphology of brain tissue in mice. The levels of synaptophysin (SYP) and postsynaptic dense substance 95 (PSD95) in mice serum were detected by enzyme-linked immunosorbent assay (ELISA). The positive expression of brain-derived neurotrophic factor(BDNF) in the dentate gyrus (DG) region of mouse brain tissue was observed by immunohistochemistry (IHC). The protein levels of BDNF, Wnt family member 3A(Wnt3a), and β-catenin were detected in the hippocampus of mice by Western blot. ResultsCompared with the normal group of mice, the model group of mice had significantly more complex swimming routes and lower swimming speed (P<0.01), significantly lower percentage of time spent in the target quadrant (P<0.01), and a significantly lower percentage of freezing time (P<0.05). The number of neurons in the hippocampal region of mice was obviously reduced and unevenly arranged. The levels of SYP and PSD95(P<0.01) in the serum of mice were reduced, and the positive expression of BDNF in the DG region of the brain tissue of mice was reduced. The levels of hippocampal BDNF, Wnt3a, and β-catenin proteins in the hippocampus of mice were obviously reduced (P<0.05, P<0.01). Compared with the model group, the mice in the SWDK group and the total ginsenoside group had significantly shorter swimming routes, the high- and medium- dose SWDK groups significantly higher swimming speeds (P<0.01), significantly higher percentage of time spent in the target quadrant (P<0.01), obviously higher percentage of Freezing time (P<0.05), and obviously more neurons in the hippocampal region of the mice with tighter arrangement. The mice had elevated levels of serum SYP (P<0.05, P<0.01), PSD95 (P<0.01), increased BDNF-positive cells in the DG region of brain tissue, and obviously elevated levels of BDNF, Wnt3a, and β-catenin proteins in the hippocampus of mice (P<0.05, P<0.01). ConclusionSWDK can significantly improve the cognitive dysfunction of AD mice, and its mechanism may be related to regulating the Wnt/β-catenin signaling pathway, which promotes BDNF expression and thereby enhances synaptic plasticity, allowing neuronal signaling to be restored.
3.Therapeutic Effect of Cranial Painkiller Pills' Extract Powder in Treatment of Trigeminal Neuralgia Induced by Injection of Talci Pulvis into Infraorbital Foramen of Model Rats Based on OTULIN-regulated Neuroinflammation
Shuran LI ; Xinwei WANG ; Jing SUN ; Dan XIE ; Ronghua ZHAO ; Lei BAO ; Zihan GENG ; Qiyue SUN ; Jingsheng ZHANG ; Yaxin WANG ; Xihe CUI ; Xinying LI ; Bing HAN ; Tianjiao LU ; Xiaolan CUI ; Liying LIU ; Shanshan GUO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):21-28
ObjectiveThis paper aims to verify the therapeutic effect of Cranial Painkiller pills' extract powder prepared by the new process on the rat's trigeminal neuralgia model caused by infraorbital injection of Talci Pulvis, evaluate its potential clinical application value, and compare the therapeutic effect with that of Cranial Painkiller granules, so as to provide data support for the application of the Cranial Painkiller pills' extract powder and precise treatment. MethodsThe rat's trigeminal neuralgia model was constructed by infraorbital injection of Talci Pulvis, and the rats were randomly divided into the normal group, model group, carbamazepine group (60 mg·kg-1), Cranial Painkiller granules group (2.70 g·kg-1), and low, medium, and high dosage groups of Cranial Painkiller pills' extract powder (1.35, 2.70, 5.40 g·kg-1) according to the basal mechanical pain thresholds, and there were 10 rats in each group. The drug was administered by gavage to each group 2 h after modeling, and distilled water was given by gavage to the normal and model groups under the same conditions once a day for 10 d. Von Frey brushes were used to measure mechanical pain thresholds in rats. Hematoxylin-eosin (HE) staining was used to detect pathological changes in the trigeminal ganglion, and enzyme-linked immunosorbent assay (ELISA) was used to detect the inflammatory factors interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) levels in rat serum, as well as neuropeptide substance P (SP) and β-endorphin (β-EP) levels in rat brain tissue. Western blot technique was used to detect the levels of NLRP3, ASC, Caspase-1, and OTULIN proteins in rat brain tissue. ResultsCompared with the normal group, the pain threshold of rats in the model group showed a continuous significant decrease (P<0.01). The pathological damage of brain tissue was significant (P<0.01), and the inflammatory levels of IL-1, IL-6, IL-8, and TNF-α in serum were significantly elevated (P<0.01). The level of the SP in the brain tissue was significantly elevated (P<0.01), and the level of β-EP was significantly reduced (P<0.01), while the level of OTULIN was significantly reduced, and NLRP3, ASC, and Caspase-1 protein levels were significantly elevated (P<0.01). After administration of the drug, compared with the model group, the pain threshold of each dose group of the Cranial Painkiller pills' extract powder and the Cranial Painkiller granules group significantly increased (P<0.01). The inflammatory levels of IL-1, IL-6, IL-8, and TNF-α and SP levels significantly decreased (P<0.01), and the β-EP levels were significantly elevated (P<0.01), while the levels of OTULIN protein were significantly elevated (P<0.05, P<0.01), and the levels of NLRP3, ASC proteins were decreased (P<0.01)in high dose Cranial Painkiller pills' extract powder. Meanwhile, compared with those in the model group, the trigeminal ganglion lesions of rats in the Cranial Painkiller pills' extract powder and Cranial Painkiller granules groups showed different degrees of improvement (P<0.05, P<0.01). ConclusionThe Cranial Painkiller pills' extract powder has significant therapeutic effects on the rat model of trigeminal neuralgia induced by infraorbital injection of Talci Pulvis, and its mechanism is related to the improvement of OTULIN-regulated neuroinflammation.
4.Mechanism of Tibetan Medicine Sanwei Doukoutang to Improve Cognitive Dysfunction in 5×FAD Mice Based on Wnt/β-catenin Signaling Pathway
Shuran LI ; Yaxin WANG ; Jing SUN ; Lei BAO ; Zihan GENG ; Dan XIE ; Ronghua ZHAO ; Yanyan BAO ; Qiyue SUN ; Jingsheng ZHANG ; Xinwei WANG ; Xinying LI ; Xihe CUI ; Xiaowei YANG ; LIUXIAN ; Mengyao CUI ; Qingshan LIU ; Shanshan GUO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):54-60
ObjectiveTo explore the effects of the Tibetan medicine Sanwei Doukoutang (SWDK) on cognitive dysfunction in mice suffering from Alzheimer's disease (AD) and its related mechanism. MethodsFifty SPF 5 × FAD mice were randomly divided into model group, total ginsenoside group(0.04 g·kg-1), high-, medium-, and low-dose groups of SWDK (32.60, 16.30, 8.15 g·kg-1), with 10 mice in each group, and ten wild-type mice of the same age were used as the normal group, male and female in 1∶1. Gavage administration was performed once daily for 8 weeks. The Morris water maze test and contextual fear memory experiment were used to observe learning and memory function. Hematoxylin-eosin (HE) staining was utilized to observe the changes in the pathomorphology of brain tissue in mice. The levels of synaptophysin (SYP) and postsynaptic dense substance 95 (PSD95) in mice serum were detected by enzyme-linked immunosorbent assay (ELISA). The positive expression of brain-derived neurotrophic factor(BDNF) in the dentate gyrus (DG) region of mouse brain tissue was observed by immunohistochemistry (IHC). The protein levels of BDNF, Wnt family member 3A(Wnt3a), and β-catenin were detected in the hippocampus of mice by Western blot. ResultsCompared with the normal group of mice, the model group of mice had significantly more complex swimming routes and lower swimming speed (P<0.01), significantly lower percentage of time spent in the target quadrant (P<0.01), and a significantly lower percentage of freezing time (P<0.05). The number of neurons in the hippocampal region of mice was obviously reduced and unevenly arranged. The levels of SYP and PSD95(P<0.01) in the serum of mice were reduced, and the positive expression of BDNF in the DG region of the brain tissue of mice was reduced. The levels of hippocampal BDNF, Wnt3a, and β-catenin proteins in the hippocampus of mice were obviously reduced (P<0.05, P<0.01). Compared with the model group, the mice in the SWDK group and the total ginsenoside group had significantly shorter swimming routes, the high- and medium- dose SWDK groups significantly higher swimming speeds (P<0.01), significantly higher percentage of time spent in the target quadrant (P<0.01), obviously higher percentage of Freezing time (P<0.05), and obviously more neurons in the hippocampal region of the mice with tighter arrangement. The mice had elevated levels of serum SYP (P<0.05, P<0.01), PSD95 (P<0.01), increased BDNF-positive cells in the DG region of brain tissue, and obviously elevated levels of BDNF, Wnt3a, and β-catenin proteins in the hippocampus of mice (P<0.05, P<0.01). ConclusionSWDK can significantly improve the cognitive dysfunction of AD mice, and its mechanism may be related to regulating the Wnt/β-catenin signaling pathway, which promotes BDNF expression and thereby enhances synaptic plasticity, allowing neuronal signaling to be restored.
5.Transarterial infusion chemotherapy combined with lipiodol chemoembolization for the treatment of advanced colorectal cancer
Xiaolong DING ; Shuai WANG ; Yaozhen MA ; Meipan YIN ; Tao LIU ; Shuiling JIN ; Xiaobing LI ; Chunxia LI ; Xinwei HAN ; Gang WU
Journal of Interventional Radiology 2024;33(2):186-190
Objective To discuss the clinical safety,feasibility and efficacy of transcatheter arterial infusion chemotherapy(TAI)combined with lipiodol chemoembolization in the treatment of advanced colorectal cancer(CRC).Methods The clinical data of 37 patients with advanced CRC,who received TAI combined with lipiodol chemoembolization at the First Affiliated Hospital of Zhengzhou University of China between June 2016 and December 2022,were retrospectively analyzed.The clinical efficacy was evaluated,the progression-free survival(PFS)and the serious complications were recorded.Results A total of 55 times of TAI combined with lipiodol chemoembolization procedures were successfully accomplished in the 37 patients.The mean used amount of lipiodol emulsion was 2.9 mL(0.8-10 mL).No serious complications such as bleeding and intestinal perforation occurred.The median follow-up time was 24 months(range of 3-48 months).The postoperative one-month,3-month,6-month and 12-month objective remission rates(ORR)were 67.6%(25/37),67.6%(25/37),64.9%(24/37)and 56.8%(21/37)respectively,and the postoperative one-month,3-month,6-month and 12-month disease control rates(DCR)were 91.9%(34/37),91.9%(34/37),89.2%(33/37)and 81.1%(30/37)respectively.The median PFS was 16 months(range of 2-47 months).As of the last follow-up,22 patients survived and 15 patients died of terminal stage of tumor.Conclusion Preliminary results of this study indicate that TAI combined with lipiodol chemoembolization is clinically safe and effective for advanced CRC,and it provide a new therapeutic method for patients with advanced CRC.
6.Advances in the study of outer membrane vesicles of gram-negative bacteria in infection and immune regulation
Yifan LI ; Xinwei LIU ; Yongwei LI
Chinese Journal of Microbiology and Immunology 2024;44(1):39-43
Outer membrane vesicle (OMV), originating from the outermost membrane of cells, is the extracellular vesicles released by gram-negative bacteria, containing bacterial outer membrane components such as phospholipids, lipopolysaccharide (LPS), outer membrane protein, and bacterial-specific antigens. OMV plays an important role in bacterial physiology and pathogenesis, involving in biofilm formation, horizontal gene transfer, stress and inflammatory responses, and delivery of toxins and other biomolecules. It also plays a vital role in immune regulation and the establishment and maintenance of balanced intestinal microflora. This article provides an overview of the roles of OMV in bacterial infections and immune regulation and the potential application value of OMV in tumor-targeted therapy and new vaccine preparation in the hope to provide new ideas for the prevention and treatment of bacterial infections.
7.Dihydroartemisinin promotes radiotherapy sensitivity of nasopharyngeal carcinoma CNE-2Z cells by activating chloride channels
Shiqing LIU ; Congran ZHOU ; Xinwei TANG ; Hanfen ZHOU ; XueKe LI ; Xi-Uying HOU ; Haifeng YANG ; Linyan ZHU
Chinese Journal of Pathophysiology 2024;40(2):255-264
AIM:To investigate the role of ClC-3 chloride channel in the promotion of radio sensitization of na-sopharyngeal carcinoma CNE-2Z cells by dihydroartemisinin(DHA).METHODS:MTT was used to detect the inhibito-ry effect of DHA on the viability of CNE-2Z cells and normal nasopharyngeal epithelial NP69-SV40T cells,the radio sensi-tization effect of DHA on CNE-2Z cells was detected by cloning assay,the expression of ClC-3 protein was detected by Western blot,the expression of ClC-3 protein was down-regulated by siRNA technology,and the chlorine current of cells was recorded by whole cell patch-clamp technology.RESULTS:(1)Compared with NP69-SV40T cells,DHA selective-ly inhibited the proliferation of CNE-2Z cells,with IC10 values of(13.020±4.831)μmol/L and(5.244±1.050)μmol/L,respectively(P<0.01).(2)The results of clonal formation experiments showed that DHA had a radio sensitizing effect on CNE-2Z cells,with a radio sensitization ratio of 1.9.(3)DHA could activate the chlorine channel of CNE-2Z cells and produce an outward chlorine current,but had no effect on the chlorine channel of NP69-SV40T cells.(4)DHA promoted the expression of ClC-3 chloric channel protein in CNE-2Z cells(P<0.01).(5)Chlorine channel blocker NPPB could in-hibit the radio sensitizing effect of DHA on CNE-2Z cells by 1.84 times,and also inhibited the chlorine current activated by DHA.(6)the down-regulation of CNE-2Z ClC-3 protein could inhibit the radio sensitization effect of DHA on CNE-2Z cells by 4.19 times,and the activation of chlorine current by DHA on CNE-2Z cells was no longer produced.CONCLU-SION:DHA has a radio sensitizing effect on nasopharyngeal carcinoma CNE-2Z cells,which is likely to be related to the activation of ClC-3 chloride channel.
8.Value of modified ROX index in predicting the prognosis of acute respiratory distress syndrome treated with high-flow nasal cannula oxygen therapy
Caixia ZHANG ; Xinwei WANG ; Xinnian LIU ; Chuan DU
Chinese Journal of Primary Medicine and Pharmacy 2024;31(3):392-398
Objective:To evaluate the value of the ROX index [blood oxygen saturation (SpO 2)/fraction of inspiration O 2 (FiO 2)/respiratory rate (RR)], ROX-heart rate (HR) index (ROX index/HR × 100), modified ROX (mROX) index [partial pressure of oxygen in the blood (PaO 2)/FiO 2/RR], and mROX-HR index (mROX index/HR × 100) in predicting prognosis for patients with acute respiratory distress syndrome (ARDS) treated with high-flow nasal cannula oxygen therapy (HFNC). Methods:The clinical data of 100 patients with ARDS who received HFNC between January 2018 and December 2022 at The Third People's Hospital of Hubei Province, Jianghan University, were retrospectively analyzed. These patients were divided into two groups based on whether HFNC treatment was successful or not: a success group with 65 patients and a failure group with 35 patients. The differences in the ROX index, ROX-HR index, mROX index, and mROX-HR index in the observation group were observed at the designated time points: 2, 12, and 24 hours after HFNC treatment. Receiver operating characteristic (ROC) curves were utilized to evaluate the value of ROX index, ROX-HR index, mROX index, and mROX-HR index in predicting the success or failure of HFNC treatment at 2, 12, and 24 hours. Cutoff values were determined.Results:There were no significant differences in age, gender, body mass index, Acute Physiology and Chronic Health Evaluation (APACHE II) score, Sequential Organ Failure Assessment score, or the proportions of underlying diseases and pulmonary causes between the success and failure groups (all P > 0.05). Furthermore, there were no significant differences in baseline HR, RR, FiO 2, SpO 2, partial pressure of carbon dioxide (PaCO 2), PaO 2, pH, lactate, oxygenation index, ROX index, mROX index, ROX-HR index, or mROX-HR index between the two groups (all P > 0.05). The ROX index in the success group at 2, 12, and 24 hours after HFNC treatment was 6.86 ± 1.09, 6.31 ± 1.61, and 8.24 ± 2.29, respectively. These values were significantly higher than those in the failure group (6.36 ± 0.67, 5.65 ± 1.44, and 5.41 ± 0.84) at the corresponding time points ( F = 5.97, 4.04, 49.40, all P < 0.05). At 2, 12, and 24 hours after HFNC treatment, the mROX index in the success group was 5.94 ± 1.28, 5.74 ± 1.23, and 8.51 ± 2.64, respectively. These values were significantly higher than those in the failure group (5.26 ± 0.74, 4.80 ± 0.97, 4.81 ± 1.17) at the corresponding time points ( F = 8.23, 15.38, 61.79, all P < 0.05). At 2, 12, and 24 hours after HFNC treatment, the ROX-HR index in the success group was 6.53 ± 1.32, 6.85 ± 1.44, and 7.57 ± 1.47, respectively. These values were significantly higher than those in the failure group (5.79 ± 1.04, 5.87 ± 1.03, 5.57 ± 0.63) at the corresponding time points ( F = 8.28, 12.61, 58.34, all P < 0.05). At 2, 12, and 24 hours after HFNC treatment, the mROX-HR index in the success group was 6.11 ± 1.30, 6.86 ± 1.13, and 7.79 ± 1.79, respectively. These values were significantly higher than those in the failure group (5.20 ± 1.06, 5.66 ± 1.46, 4.92 ± 0.90) at the corresponding time points ( F = 12.60, 20.87, 78.56, all P < 0.05). The receiver operating characteristic curve analysis revealed that the optimal thresholds were 6.56, 6.02, 6.24, and 5.25 for the ROX index, mROX index, ROX-HR index, and mROX-HR index, respectively. The area under the curve (AUC) values were 0.63, 0.66, 0.68, and 0.72, with sensitivity of 55.4%, 47.7%, 56.9%, and 76.9%, and specificity of 71.4%, 91.4%, 77.1%, and 62.9%, respectively. At 12 hours after treatment, the optimal thresholds were 6.09, 5.53, 6.52, and 5.99, with AUC values of 0.62, 0.70, 0.67, and 0.80, sensitivity of 55.4%, 53.8%, 61.5%, and 80.0%, and specificity of 74.3%, 77.1%, 71.4%, and 74.3%, respectively. At 24 hours after treatment, the optimal thresholds were 6.23, 6.4, 5.99, and 6.22, with AUC values of 0.88, 0.90, 0.91, and 0.93, sensitivity of 81.5%, 80.0%, 87.7%, and 83.1%, and specificity of 91.4%, 94.3%, 80.0%, and 91.4%, respectively. Conclusion:The use of the ROX index, mROX index, ROX-HR index, and mROX-HR index can aid in predicting the prognosis of ARDS patients. The predictive value of these indices increases as treatment time progresses. The mROX-HR index offers marked advantages during the initial stages of treatment and could serve as a reliable early predictor.
9.Quercetin protects THP-1 macrophages against cellular stress response induced by Pseudomonas aeruginosa protease LasB
Yanying REN ; Xinwei LIU ; Rui ZHU ; Xiaojuan YOU ; Dengzhou LI ; Zhiqiang HE ; Chunxia WANG ; Ximing YANG ; Yongwei LI
Chinese Journal of Preventive Medicine 2024;58(3):337-346
Objective:To investigate the protective effect of quercetin against LasB-induced apoptosis, inflammation, and oxidative stress in THP-1 macrophages, providing valuable insights into the use of quercetin as a virulence inhibitor for Pseudomonas aeruginosa infection treatment. Methods:This was an experimental study. The experimental strain was the standard strain. The LasB protein was obtained utilizing protein recombination technology, while the enzyme activity of LasB was assessed through both the Elastin Congo red assay and fluorescently labelled elastin assay. The LasB-induced THP-1 macrophage infection model was established, and quercetin was utilized for intervention. Cell viability was evaluated via CCK-8 assay, while cell morphology was observed under an inverted microscope. Apoptosis detection involved employing both TUNEL and Annexin V/PI staining. The mRNA expression and protein levels of inflammatory cytokines and COX-2 were determined by RT-qPCR and ELISA respectively. Intracellular ROS levels were quantified using the DCFH-DA fluorescent probe. One-way ANOVA was used for statistical analysis, and Tukey test was used for multiple comparisons. Results:The pLasB with a molecular weight of 33 000 and acceptable enzymatic activity (purity>90%), was successfully obtained. THP-1 macrophages treated with pLasB at a concentration of 100 μg/ml presented significantly decreased viability and integrity rate when compared with the normal control group. Additionally, pLasB promoted apoptosis, up-regulated the levels of inflammatory cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12, and TNF-α, increased intracellular ROS fluorescence intensity, and elevated COX-2 mRNA expression level. Furthermore, the viability of THP-1 macrophages was significantly enhanced under quercetin intervention at concentrations of 2.5 μmol/L, 5 μmol/L and 10 μmol/L. The apoptosis rate exhibited a significant reduction from 18.32%±0.17% to 13.17%±0.20%, 11.43%±0.06% and 7.74%±0.04%, respectively ( F=1 679, P<0.05). There was a notable down-regulation of pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-12 and TNF-α while the anti-inflammatory cytokine IL-10 showed a significant up-regulation. Both intracellular ROS fluorescence intensity ( F=86.92, P<0.05) and COX-2 level ( F=24.62, P<0.05) demonstrated a substantial decrease. Conclusion:Quercetin demonstrates significant efficacy in inhibiting LasB-induced apoptosis, inflammation, and oxidative stress in THP-1 macrophages, which highlights immense potential as a potent virulence inhibitor of Pseudomonas aeruginosa.
10.Quercetin protects THP-1 macrophages against cellular stress response induced by Pseudomonas aeruginosa protease LasB
Yanying REN ; Xinwei LIU ; Rui ZHU ; Xiaojuan YOU ; Dengzhou LI ; Zhiqiang HE ; Chunxia WANG ; Ximing YANG ; Yongwei LI
Chinese Journal of Preventive Medicine 2024;58(3):337-346
Objective:To investigate the protective effect of quercetin against LasB-induced apoptosis, inflammation, and oxidative stress in THP-1 macrophages, providing valuable insights into the use of quercetin as a virulence inhibitor for Pseudomonas aeruginosa infection treatment. Methods:This was an experimental study. The experimental strain was the standard strain. The LasB protein was obtained utilizing protein recombination technology, while the enzyme activity of LasB was assessed through both the Elastin Congo red assay and fluorescently labelled elastin assay. The LasB-induced THP-1 macrophage infection model was established, and quercetin was utilized for intervention. Cell viability was evaluated via CCK-8 assay, while cell morphology was observed under an inverted microscope. Apoptosis detection involved employing both TUNEL and Annexin V/PI staining. The mRNA expression and protein levels of inflammatory cytokines and COX-2 were determined by RT-qPCR and ELISA respectively. Intracellular ROS levels were quantified using the DCFH-DA fluorescent probe. One-way ANOVA was used for statistical analysis, and Tukey test was used for multiple comparisons. Results:The pLasB with a molecular weight of 33 000 and acceptable enzymatic activity (purity>90%), was successfully obtained. THP-1 macrophages treated with pLasB at a concentration of 100 μg/ml presented significantly decreased viability and integrity rate when compared with the normal control group. Additionally, pLasB promoted apoptosis, up-regulated the levels of inflammatory cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12, and TNF-α, increased intracellular ROS fluorescence intensity, and elevated COX-2 mRNA expression level. Furthermore, the viability of THP-1 macrophages was significantly enhanced under quercetin intervention at concentrations of 2.5 μmol/L, 5 μmol/L and 10 μmol/L. The apoptosis rate exhibited a significant reduction from 18.32%±0.17% to 13.17%±0.20%, 11.43%±0.06% and 7.74%±0.04%, respectively ( F=1 679, P<0.05). There was a notable down-regulation of pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-12 and TNF-α while the anti-inflammatory cytokine IL-10 showed a significant up-regulation. Both intracellular ROS fluorescence intensity ( F=86.92, P<0.05) and COX-2 level ( F=24.62, P<0.05) demonstrated a substantial decrease. Conclusion:Quercetin demonstrates significant efficacy in inhibiting LasB-induced apoptosis, inflammation, and oxidative stress in THP-1 macrophages, which highlights immense potential as a potent virulence inhibitor of Pseudomonas aeruginosa.

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