1.Rapid characterization and identification of non-volatile components in Rhododendron tomentosum by UHPLC-Q-TOF-MS method.
Su-Ping XIAO ; Long-Mei LI ; Bin XIE ; Hong LIANG ; Qiong YIN ; Jian-Hui LI ; Jie DU ; Ji-Yong WANG ; Run-Huai ZHAO ; Yan-Qin XU ; Yun-Bo SUN ; Zong-Yuan LU ; Peng-Fei TU
China Journal of Chinese Materia Medica 2025;50(11):3054-3069
This study aimed to characterize and identify the non-volatile components in aqueous and ethanolic extracts of the stems and leaves of Rhododendron tomentosum by using sensitive and efficient ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry(UHPLC-Q-TOF-MS) combined with a self-built information database. By comparing with reference compounds, analyzing fragment ion information, searching relevant literature, and using a self-built information database, 118 compounds were identified from the aqueous and ethanolic extracts of R. tomentosum, including 35 flavonoid glycosides, 15 phenolic glycosides, 12 flavonoids, 7 phenolic acids, 7 phenylethanol glycosides, 6 tannins, 6 phospholipids, 5 coumarins, 5 monoterpene glycosides, 6 triterpenes, 3 fatty acids, and 11 other types of compounds. Among them, 102 compounds were reported in R. tomentosum for the first time, and 36 compounds were identified by comparing them with reference compounds. The chemical components in the ethanolic and aqueous extracts of R. tomentosum leaves and stems showed slight differences, with 84 common chemical components accounting for 71.2% of the total 118 compounds. This study systematically characterized and identified the non-volatile chemical components in the ethanolic and aqueous extracts of R. tomentosum for the first time. The findings provide a reference for active ingredient research, quality control, and product development of R. tomentosum.
Rhododendron/chemistry*
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Chromatography, High Pressure Liquid/methods*
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Drugs, Chinese Herbal/chemistry*
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Mass Spectrometry/methods*
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Plant Leaves/chemistry*
2.Bendamustine improves brain edema after cerebral ischemia-reperfusion in mice by inhibiting NETs formation
Yang GENG ; Ningning ZONG ; Lixuan YANG ; Yun XU
Chinese Journal of Neuromedicine 2025;24(10):986-996
Objective:To explore how benzydamine (BA) improves brain edema in mice after cerebral ischemia-reperfusion.Methods:(1) One hundred and twenty 8 week-old male C57BL/6 mice were randomly divided into a sham-operated group, a middle cerebral artery occlusion (MCAO) group, a MCAO+low-dose BA group (L-BA group), and a MCAO+high-dose BA group (H-BA group), with 30 mice in each group. MCAO models in mice of the later 3 groups were established by suture method, while mice in the sham-operated group underwent the same surgical procedure without MCAO. At 6 hours after modeling, mice in the L-BA group and H-BA group were intraperitoneally injected with 5 mg/kg or 10 mg/kg BA, respectively, once daily for 3 days, while mice in the shamoperated group and MCAO group were intraperitoneally injected with same volume of normal saline instead. Dynamics of cerebral perfusion were monitored by laser speckle imaging in MCAO model mice at baseline, during occlusion, and following reperfusion. Three days after modeling, neurological deficits were assessed by modified neurological severity score (mNSS), neurological function was evaluated by forelimb grip strength and rotarod tests; cerebral infarct size was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and neuronal death was assessed by Fluoro-Jade B staining; cerebral edema was quantified by dry-wet weight method, blood-brain barrier (BBB) permeability was assessed by Evans blue dye extravasation, and expressions of tight junction proteins (Claudin-5, zonula occludens-1 [ZO-1]) were detected by immunofluorescent staining; expressions of neutrophil extracellular traps (NETs)-related proteins (citrullinated histone H3 [citH3], myeloperoxidase [MPO] and matrix metalloproteinase 9 [MMP-9]) were determined by Western blotting. (2) Bone marrow neutrophils were extracted from male C57BL/6 mice and randomly divided into a control group, a phorbol 12-myristate 13-acetate (PMA) group, and a PMA+BA group; neutrophils in the PMA group were stimulated with PMA (50 nmol/L), while neutrophils in the PMA+BA group were co-treated with 50 nmol/L PMA and 50 μmol/L BA; and those in the control group were given an equal amount of dimethyl sulfoxide. Sytox Green staining was used to detect the NETs proportion in neutrophils.Results:(1) Baseline cerebral perfusion was robust (1 237.75±98.16 PU), which was markedly reduced during occlusion (297.36±77.63 PU) in the ipsilateral middle cerebral artery territory, and improved following reperfusion (939.21±73.63 PU). Compared with the MCAO group, mice in the L-BA group and H-BA group had lower mNSS score, increased paw grip strength, prolonged rotarod retention time, reduced infarct size, fewer neuronal death, lower brain tissue water content, reduced blood-brain barrier permeability, increased fluorescent intensities of Claudin-5 (0.51±0.11, 0.71±0.04, and 0.83±0.05) and ZO-1 (0.43±0.09, 0.65±0.05, and 0.81±0.03), and decreased protein expressions of citH3 (2.33±0.15, 1.92±0.03, and 1.42±0.04), MPO (2.14±0.08, 1.71±0.06, and 1.37±0.03) and MMP-9 (2.62±0.09, 1.83±0.06, and 1.41±0.05), with significant differences ( P<0.05). All the above changes in the H-BA group were more significant than those in the L-BA group ( P<0.05). (2) Compared with that in the control group (10.00%±8.00%), the proportion of NETs formation per field in both PMA group (85.33%±2.08%) and PMA+BA group (58.46%±5.29%) was significantly increased ( P<0.05); the PMA+BA group showed a significant reduction in NETs formation compared with the PMA group ( P<0.05). Conclusion:BA can alleviate cerebral edema in mice after cerebral ischemia-reperfusion, and its mechanism may be involved in inhibiting NETs formation.
3.Nefazodone improves neurological function impairment in MCAO mice by inhibiting excessive activation of microglia
Lixuan YANG ; Ningning ZONG ; Yang GENG ; Jian CHEN ; Yun XU
Chinese Journal of Neuromedicine 2025;24(10):1013-1024
Objective:To investigate the effect of nefazodone on excessive activation of microglia and its regulatory mechanism, as well as its effect on neurological injury in mice subjected to middle cerebral artery occlusion (MCAO).Methods:(1) BV2 cell line was routinely cultured in vitro, and primary microglia from the cortex of neonatal C57BL/6J mice were cultured. Cell counting kit-8 (CCK-8) assay was employed to assess the effects of nefazodone (0, 10, 20, 30, 50 μmol/L) on viability of BV2 cells and primary microglia to determine the working concentration. BV2 cells and primary microglia were divided into a normal control group, a lipopolysaccharide (LPS) group, and a nefazodone group; cells in the nefazodone group were pretreated with 20 μmol/L nefazodone for 2 h; cells in the LPS group and nefazodone group were stimulated with LPS (0.5 μg/mL for BV2 cells and 0.1 μg/mL for primary microglia) for 24 h; cells in the normal control group received an equivalent volume of solvent. Immunofluorescent staining was used to detect the expressions of ionized calcium-binding adapter molecule 1 (Iba1) and CD68. Reverse transcription quantitative PCR (RT-qPCR) was performed to measure interleukin ( IL) -1β, IL-6, tumor necrosis factor-α ( TNF-α), nitric oxide synthase 2 ( Nos2), C-C motif chemokine ligand 2 ( CCL2), and β-hexosaminidase subunit β ( Hexb) mRNA expressions. ELISA was used to quantify the concentrations of supernatant IL-1β, IL-6, and TNF-α in BV2 cells. Western blotting was applied to detect the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 cells. Griess reagent assay was used to measure supernatant nitric oxide (NO) level in BV2 cells. Western blotting was also used to assess the protein expressions of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38, nuclear factor kappa B p65 and p-p65 in BV2 cells. (2) Thirty male C57BL/6J mice were randomly divided into a normal control group, a MCAO group, and a nefazodone group, with 10 mice in each group. MCAO model in the MCAO group and nefazodone group was established using suture method; the nefazodone group received an intraperitoneal injection of nefazodone (15 mg/kg) 30 min after modeling, while the normal control group received an equivalent volume of solvent. Three days after modeling, neurological deficits were evaluated using modified neurological severity score (mNSS), and behavioral changes were evaluated by paw grip strength test and foot-fault test. Cerebral infarction volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Iba1 protein expression in the ischemic penumbra was detected by Western blotting. Results:(1) CCK-8 assay showed no significant difference in viability of BV2 cells between the normal control group and 10 or 20 μmol/L nefazodone groups ( P>0.05), and viability of BV2 cells in 30 and 50 μmol/L nefazodone groups was statistically lower than that of normal control group ( P<0.05). Immunofluorescent staining revealed that compared with the normal control group, the LPS group had significantly increased fluorescent intensities of CD68 and Iba1; compared with the LPS group, the nefazodone group had significantly decreased fluorescent intensities of CD68 and Iba1 ( P<0.05). RT-qPCR results indicated that compared with those in the normal control group, the Nos2, CCL2, IL-1β, IL-6, and TNF-α mRNA expressions in both BV2 cells and primary microglia of the LPS group were significantly increased; compared with the LPS group, the nefazodone group had significantly decreased CCL2, IL-1β, and IL-6 mRNA expressions in BV2 cells, and significantly decreased Nos2, CCL2, IL-1β, IL-6, and TNF-α mRNA expressions in primary microglia ( P<0.05). ELISA showed that compared with those in the normal control group, the supernatant IL-1β, IL-6, and TNF-α levels significantly increased in the BV2 cells of LPS group; compared with those in the LPS group, supernatant IL-1β, IL-6 and TNF-α levels statistically decreased in the nefazodone group ( P< 0.05). Western blotting demonstrated that compared with those in the normal control group, the iNOS and COX-2 protein expressions significantly increased in BV2 cells of the LPS group ( P<0.05); compared with those in the LPS group, the iNOS and COX-2 protein expressions in BV2 cells of the nefazodone group statistically decreased ( P<0.05). Griess assay showed that compared with the normal control group, BV2 cells in the LPS group had significantly increased supernatant NO concentration ( P <0.05); compared with the LPS group, BV2 cells in the nefazodone group had significantly decreased supernatant NO concentration ( P<0.05). Western blotting revealed that compared with those in the normal control group, the p-ERK/ERK and p-JNK/JNK ratios significantly increased in BV2 cells of the LPS group ( P<0.05); compared with the LPS group, the p-p65/p65, p-ERK/ERK and p-JNK/JNK ratios significantly decreased in BV2 cells of the nefazodone group ( P<0.05). (2) Behavioral tests showed that compared with the normal control group, the MCAO group had significantly decreased forelimb grip strength and increased foot-fault rate ( P<0.05); compared with the MCAO group, the nefazodone group had significantly decreased mNSS score, increased forelimb grip strength and decreased foot-fault rate ( P<0.05). The percentage of cerebral infarction volume in the nefazodone group was significantly lower than that in the MCAO group ([9.56±1.81]% vs. [21.71±12.26]%, P< 0.05). The MCAO group had statistically higher Iba1 protein expression in ischemic penumbra (7.27±2.88) than the normal control group (1.00±0.64), and the nefazodone group had significantly lower Iba1 protein expression in ischemic penumbra (1.75±0.86) than the MCAO group ( P<0.05). Conclusion:Nefazodone can improve neurological function impairment in MCAO mice by inhibiting the excessive activation of microglia; cytological experiments suggest that its mechanism may be related to the negative regulation of ERK/JNK/NF-κB p65 signaling axis.
4.RCM method-based study on preventive maintenance strategy of heating,ventilation and air-conditioning system in pharmacy intravenous admixture services
Liu-liu ZONG ; Yun-zhi YANG ; Jing ZHAO ; Yun MO ; Dong-hui LAO ; Jian-zhong ZHANG ; Xiao-yu LI ; Qian-zhou LYU
Chinese Medical Equipment Journal 2025;46(10):78-83
Objective To propose a preventive maintenance strategy of the heating,ventilation and air-conditioning(HVAC)system in pharmacy intravenous admixture services(PIVAS)based on the reliability centered maintenance(RCM)method so as to provide references for PIVAS equipment maintenance.Methods Firstly,a HVAC system RCM review team was formed,and the failure modes and impacts of important functional components of the equipment were analyzed to clarify the consequences of the failure of each functional component under the premise of ensuring the safety and integrity of the equipment and with the goal of minimizing the loss of maintenance downtime and the consumption of maintenance resources.Secondly,with a standardized logical decision-making procedure the preventive maintenance strategy was determined and implemented based on the consequences of functional failure.Finally,statistical analyses were carried out on such equipment indicators as performance parameter qualification rate,failure rate and maintenance cost before and after the RCM method-based strategy was executed,in order to evaluate the efficacy of the strategy.Results The RCM method-based preventive maintenance strategy had the performance qualification rate increased from 97.47%to 99.06%(χ2=24.139,P<0.01),the failure rate decreased from 0.24%to 0.03%(χ2=13.519,P<0.01)and the maintenance cost reduced by 11.5%,from RMB 134,200 to 118,700.Conclusion The RCM method-based preventive maintenance strategy provides reliable equipment for PIVAS and lowers the maintenance cost effectively,and references are given for the development of automated and intelligent equipment maintenance strategies for PIVAS.[Chinese Medical Equipment Journal,2025,46(10):78-83]
5.RCM method-based study on preventive maintenance strategy of heating,ventilation and air-conditioning system in pharmacy intravenous admixture services
Liu-liu ZONG ; Yun-zhi YANG ; Jing ZHAO ; Yun MO ; Dong-hui LAO ; Jian-zhong ZHANG ; Xiao-yu LI ; Qian-zhou LYU
Chinese Medical Equipment Journal 2025;46(10):78-83
Objective To propose a preventive maintenance strategy of the heating,ventilation and air-conditioning(HVAC)system in pharmacy intravenous admixture services(PIVAS)based on the reliability centered maintenance(RCM)method so as to provide references for PIVAS equipment maintenance.Methods Firstly,a HVAC system RCM review team was formed,and the failure modes and impacts of important functional components of the equipment were analyzed to clarify the consequences of the failure of each functional component under the premise of ensuring the safety and integrity of the equipment and with the goal of minimizing the loss of maintenance downtime and the consumption of maintenance resources.Secondly,with a standardized logical decision-making procedure the preventive maintenance strategy was determined and implemented based on the consequences of functional failure.Finally,statistical analyses were carried out on such equipment indicators as performance parameter qualification rate,failure rate and maintenance cost before and after the RCM method-based strategy was executed,in order to evaluate the efficacy of the strategy.Results The RCM method-based preventive maintenance strategy had the performance qualification rate increased from 97.47%to 99.06%(χ2=24.139,P<0.01),the failure rate decreased from 0.24%to 0.03%(χ2=13.519,P<0.01)and the maintenance cost reduced by 11.5%,from RMB 134,200 to 118,700.Conclusion The RCM method-based preventive maintenance strategy provides reliable equipment for PIVAS and lowers the maintenance cost effectively,and references are given for the development of automated and intelligent equipment maintenance strategies for PIVAS.[Chinese Medical Equipment Journal,2025,46(10):78-83]
6.Chinese experts' consensus on principles of preoperative hair removal
Yiping MAO ; Jun ZHENG ; Lei LI ; Deyan YANG ; Bing ZHANG ; Lei YANG ; Wang JIA ; Peng KANG ; Hui JIAO ; Yun YANG ; Qi QI ; Shiqing FENG ; Xiao LONG ; Yuewei ZHANG ; Xiaohui WANG ; Lize WANG ; Yuan WEI ; Jichao ZHOU ; Minghui MAO ; Pengju XIN ; Hongyu TAN ; Dahong ZHANG ; Lianxin LIU ; Lei TAO ; Xietong WANG ; Xiaoning YUAN ; Mang CAI ; Li MU ; Fang DU ; Rongzhu CHEN ; Fengmao ZHAO ; Jiuzuo HUANG ; Mingzi ZHANG ; Jie ZHANG ; Baoguo WANG ; Kun WANG ; Fang LUO ; Jinhua ZHANG ; Nong HE ; Ling LYU ; Zhiyong ZONG
Chinese Journal of Nosocomiology 2025;35(10):1441-1449
To formulate an expert consensus on the principles of preoperative hair removal and provide scientific guidance for standardized removal of hair before surgical procedures so as to reduce the incidence of surgical site infections.METHODS Led by the Hospital Management Institute of National Health Commission of the People's Republic of China,this consensus was reached with the joint efforts from the expects of relevant fields such as surgeries,interventional therapies,nursing,and infection prevention and control.The consensus facilitates the classification and evaluation of literatures by following the evidence grade formulated by Oxford Evidence-based Medicine Center and focuses on the association of preoperative hair removal with surgical site infection,it reaches the evidence grade of expert consensus and recommendation intensity by integrating with discussions on meetings and clinical experience of the expects from relevant fields.RESULTS A total of 6 items of consensus were reached by summarizing the latest evidence on the aspects including the indications for preoperative hair removal,tools,range,timing and places.CONCLUSION The consensus,to some extent,make supplements to and complete the exiting regulations and standards.It provides guidance for the medical institutions to carry out the preoperative hair removal.
7.Chinese experts' consensus on principles of preoperative hair removal
Yiping MAO ; Jun ZHENG ; Lei LI ; Deyan YANG ; Bing ZHANG ; Lei YANG ; Wang JIA ; Peng KANG ; Hui JIAO ; Yun YANG ; Qi QI ; Shiqing FENG ; Xiao LONG ; Yuewei ZHANG ; Xiaohui WANG ; Lize WANG ; Yuan WEI ; Jichao ZHOU ; Minghui MAO ; Pengju XIN ; Hongyu TAN ; Dahong ZHANG ; Lianxin LIU ; Lei TAO ; Xietong WANG ; Xiaoning YUAN ; Mang CAI ; Li MU ; Fang DU ; Rongzhu CHEN ; Fengmao ZHAO ; Jiuzuo HUANG ; Mingzi ZHANG ; Jie ZHANG ; Baoguo WANG ; Kun WANG ; Fang LUO ; Jinhua ZHANG ; Nong HE ; Ling LYU ; Zhiyong ZONG
Chinese Journal of Nosocomiology 2025;35(10):1441-1449
To formulate an expert consensus on the principles of preoperative hair removal and provide scientific guidance for standardized removal of hair before surgical procedures so as to reduce the incidence of surgical site infections.METHODS Led by the Hospital Management Institute of National Health Commission of the People's Republic of China,this consensus was reached with the joint efforts from the expects of relevant fields such as surgeries,interventional therapies,nursing,and infection prevention and control.The consensus facilitates the classification and evaluation of literatures by following the evidence grade formulated by Oxford Evidence-based Medicine Center and focuses on the association of preoperative hair removal with surgical site infection,it reaches the evidence grade of expert consensus and recommendation intensity by integrating with discussions on meetings and clinical experience of the expects from relevant fields.RESULTS A total of 6 items of consensus were reached by summarizing the latest evidence on the aspects including the indications for preoperative hair removal,tools,range,timing and places.CONCLUSION The consensus,to some extent,make supplements to and complete the exiting regulations and standards.It provides guidance for the medical institutions to carry out the preoperative hair removal.
8.Bendamustine improves brain edema after cerebral ischemia-reperfusion in mice by inhibiting NETs formation
Yang GENG ; Ningning ZONG ; Lixuan YANG ; Yun XU
Chinese Journal of Neuromedicine 2025;24(10):986-996
Objective:To explore how benzydamine (BA) improves brain edema in mice after cerebral ischemia-reperfusion.Methods:(1) One hundred and twenty 8 week-old male C57BL/6 mice were randomly divided into a sham-operated group, a middle cerebral artery occlusion (MCAO) group, a MCAO+low-dose BA group (L-BA group), and a MCAO+high-dose BA group (H-BA group), with 30 mice in each group. MCAO models in mice of the later 3 groups were established by suture method, while mice in the sham-operated group underwent the same surgical procedure without MCAO. At 6 hours after modeling, mice in the L-BA group and H-BA group were intraperitoneally injected with 5 mg/kg or 10 mg/kg BA, respectively, once daily for 3 days, while mice in the shamoperated group and MCAO group were intraperitoneally injected with same volume of normal saline instead. Dynamics of cerebral perfusion were monitored by laser speckle imaging in MCAO model mice at baseline, during occlusion, and following reperfusion. Three days after modeling, neurological deficits were assessed by modified neurological severity score (mNSS), neurological function was evaluated by forelimb grip strength and rotarod tests; cerebral infarct size was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and neuronal death was assessed by Fluoro-Jade B staining; cerebral edema was quantified by dry-wet weight method, blood-brain barrier (BBB) permeability was assessed by Evans blue dye extravasation, and expressions of tight junction proteins (Claudin-5, zonula occludens-1 [ZO-1]) were detected by immunofluorescent staining; expressions of neutrophil extracellular traps (NETs)-related proteins (citrullinated histone H3 [citH3], myeloperoxidase [MPO] and matrix metalloproteinase 9 [MMP-9]) were determined by Western blotting. (2) Bone marrow neutrophils were extracted from male C57BL/6 mice and randomly divided into a control group, a phorbol 12-myristate 13-acetate (PMA) group, and a PMA+BA group; neutrophils in the PMA group were stimulated with PMA (50 nmol/L), while neutrophils in the PMA+BA group were co-treated with 50 nmol/L PMA and 50 μmol/L BA; and those in the control group were given an equal amount of dimethyl sulfoxide. Sytox Green staining was used to detect the NETs proportion in neutrophils.Results:(1) Baseline cerebral perfusion was robust (1 237.75±98.16 PU), which was markedly reduced during occlusion (297.36±77.63 PU) in the ipsilateral middle cerebral artery territory, and improved following reperfusion (939.21±73.63 PU). Compared with the MCAO group, mice in the L-BA group and H-BA group had lower mNSS score, increased paw grip strength, prolonged rotarod retention time, reduced infarct size, fewer neuronal death, lower brain tissue water content, reduced blood-brain barrier permeability, increased fluorescent intensities of Claudin-5 (0.51±0.11, 0.71±0.04, and 0.83±0.05) and ZO-1 (0.43±0.09, 0.65±0.05, and 0.81±0.03), and decreased protein expressions of citH3 (2.33±0.15, 1.92±0.03, and 1.42±0.04), MPO (2.14±0.08, 1.71±0.06, and 1.37±0.03) and MMP-9 (2.62±0.09, 1.83±0.06, and 1.41±0.05), with significant differences ( P<0.05). All the above changes in the H-BA group were more significant than those in the L-BA group ( P<0.05). (2) Compared with that in the control group (10.00%±8.00%), the proportion of NETs formation per field in both PMA group (85.33%±2.08%) and PMA+BA group (58.46%±5.29%) was significantly increased ( P<0.05); the PMA+BA group showed a significant reduction in NETs formation compared with the PMA group ( P<0.05). Conclusion:BA can alleviate cerebral edema in mice after cerebral ischemia-reperfusion, and its mechanism may be involved in inhibiting NETs formation.
9.Nefazodone improves neurological function impairment in MCAO mice by inhibiting excessive activation of microglia
Lixuan YANG ; Ningning ZONG ; Yang GENG ; Jian CHEN ; Yun XU
Chinese Journal of Neuromedicine 2025;24(10):1013-1024
Objective:To investigate the effect of nefazodone on excessive activation of microglia and its regulatory mechanism, as well as its effect on neurological injury in mice subjected to middle cerebral artery occlusion (MCAO).Methods:(1) BV2 cell line was routinely cultured in vitro, and primary microglia from the cortex of neonatal C57BL/6J mice were cultured. Cell counting kit-8 (CCK-8) assay was employed to assess the effects of nefazodone (0, 10, 20, 30, 50 μmol/L) on viability of BV2 cells and primary microglia to determine the working concentration. BV2 cells and primary microglia were divided into a normal control group, a lipopolysaccharide (LPS) group, and a nefazodone group; cells in the nefazodone group were pretreated with 20 μmol/L nefazodone for 2 h; cells in the LPS group and nefazodone group were stimulated with LPS (0.5 μg/mL for BV2 cells and 0.1 μg/mL for primary microglia) for 24 h; cells in the normal control group received an equivalent volume of solvent. Immunofluorescent staining was used to detect the expressions of ionized calcium-binding adapter molecule 1 (Iba1) and CD68. Reverse transcription quantitative PCR (RT-qPCR) was performed to measure interleukin ( IL) -1β, IL-6, tumor necrosis factor-α ( TNF-α), nitric oxide synthase 2 ( Nos2), C-C motif chemokine ligand 2 ( CCL2), and β-hexosaminidase subunit β ( Hexb) mRNA expressions. ELISA was used to quantify the concentrations of supernatant IL-1β, IL-6, and TNF-α in BV2 cells. Western blotting was applied to detect the protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 cells. Griess reagent assay was used to measure supernatant nitric oxide (NO) level in BV2 cells. Western blotting was also used to assess the protein expressions of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38, nuclear factor kappa B p65 and p-p65 in BV2 cells. (2) Thirty male C57BL/6J mice were randomly divided into a normal control group, a MCAO group, and a nefazodone group, with 10 mice in each group. MCAO model in the MCAO group and nefazodone group was established using suture method; the nefazodone group received an intraperitoneal injection of nefazodone (15 mg/kg) 30 min after modeling, while the normal control group received an equivalent volume of solvent. Three days after modeling, neurological deficits were evaluated using modified neurological severity score (mNSS), and behavioral changes were evaluated by paw grip strength test and foot-fault test. Cerebral infarction volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Iba1 protein expression in the ischemic penumbra was detected by Western blotting. Results:(1) CCK-8 assay showed no significant difference in viability of BV2 cells between the normal control group and 10 or 20 μmol/L nefazodone groups ( P>0.05), and viability of BV2 cells in 30 and 50 μmol/L nefazodone groups was statistically lower than that of normal control group ( P<0.05). Immunofluorescent staining revealed that compared with the normal control group, the LPS group had significantly increased fluorescent intensities of CD68 and Iba1; compared with the LPS group, the nefazodone group had significantly decreased fluorescent intensities of CD68 and Iba1 ( P<0.05). RT-qPCR results indicated that compared with those in the normal control group, the Nos2, CCL2, IL-1β, IL-6, and TNF-α mRNA expressions in both BV2 cells and primary microglia of the LPS group were significantly increased; compared with the LPS group, the nefazodone group had significantly decreased CCL2, IL-1β, and IL-6 mRNA expressions in BV2 cells, and significantly decreased Nos2, CCL2, IL-1β, IL-6, and TNF-α mRNA expressions in primary microglia ( P<0.05). ELISA showed that compared with those in the normal control group, the supernatant IL-1β, IL-6, and TNF-α levels significantly increased in the BV2 cells of LPS group; compared with those in the LPS group, supernatant IL-1β, IL-6 and TNF-α levels statistically decreased in the nefazodone group ( P< 0.05). Western blotting demonstrated that compared with those in the normal control group, the iNOS and COX-2 protein expressions significantly increased in BV2 cells of the LPS group ( P<0.05); compared with those in the LPS group, the iNOS and COX-2 protein expressions in BV2 cells of the nefazodone group statistically decreased ( P<0.05). Griess assay showed that compared with the normal control group, BV2 cells in the LPS group had significantly increased supernatant NO concentration ( P <0.05); compared with the LPS group, BV2 cells in the nefazodone group had significantly decreased supernatant NO concentration ( P<0.05). Western blotting revealed that compared with those in the normal control group, the p-ERK/ERK and p-JNK/JNK ratios significantly increased in BV2 cells of the LPS group ( P<0.05); compared with the LPS group, the p-p65/p65, p-ERK/ERK and p-JNK/JNK ratios significantly decreased in BV2 cells of the nefazodone group ( P<0.05). (2) Behavioral tests showed that compared with the normal control group, the MCAO group had significantly decreased forelimb grip strength and increased foot-fault rate ( P<0.05); compared with the MCAO group, the nefazodone group had significantly decreased mNSS score, increased forelimb grip strength and decreased foot-fault rate ( P<0.05). The percentage of cerebral infarction volume in the nefazodone group was significantly lower than that in the MCAO group ([9.56±1.81]% vs. [21.71±12.26]%, P< 0.05). The MCAO group had statistically higher Iba1 protein expression in ischemic penumbra (7.27±2.88) than the normal control group (1.00±0.64), and the nefazodone group had significantly lower Iba1 protein expression in ischemic penumbra (1.75±0.86) than the MCAO group ( P<0.05). Conclusion:Nefazodone can improve neurological function impairment in MCAO mice by inhibiting the excessive activation of microglia; cytological experiments suggest that its mechanism may be related to the negative regulation of ERK/JNK/NF-κB p65 signaling axis.
10.Factors affecting the postoperative prognosis of penile cancer
Yu-Shu WU ; Zhi-Lei ZHANG ; Tao ZHU ; Zong-Yang LI ; Zhi-Fan GUO ; Yun-Jiang ZANG
National Journal of Andrology 2024;30(6):507-513
Objective:To investigate the risk factors affecting the prognosis of penile cancer after surgery.Methods:We retrospectively analyzed the clinical data on 112 cases of penile cancer treated in Weifang People's Hospital from January 2013 to De-cember 2023.Using the Kaplan-Meier survival curve,χ2 test,Fisher's exact test,and univariate and multivariate Cox risk regression analyses,we compared the clinical characteristics among different groups,and determined the independent prognostic risk factors for cancer-specific survival(CSS)of the patients.Results:The 1-,3-and 5-year CSS rates of the penile cancer patients were 78.2%,66.1% and 63.7%,respectively.Kaplan-Meier analysis indicated a significant correlation of a higher neutrophil-to-lymphocyte ratio(NLR)with a lower CSS rate(P<0.001).Multivariate Cox regression analysis showed high NLR(HR=2.6;95% CI:1.031-6.558;P=0.043)to be an independent risk factor for CSS.Conclusion:Preoperative NLR is an independent risk factor for the prognosis of penile cancer.In addition,older age,farmer or worker occupation,lower education,preoperative lymphocyte-to-monocyte ratio(LMR)≤2.81,preoperative fibrinogen(FIB)≥3.41 g/L,advanced tumor stage and tumor differentiation are associated with the poor prognosis the malignancy.

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