Effects of modified Xuanfuhua Decoction on pain behavior and p38MAPK signaling pathway-mediated spinal cord neuroinflammation in rats with sciatic nerve injury / 国际中医中药杂志

Objective:To observe the effects of modified Xuanfuhua Decoction on pain behaviour and spinal cord neuroinflammation mediated by phosphorylated mitogen-activated protein kinase p38 (p38MAPK) signaling pathway in rats with sciatic nerve injury; To analyse the mechanism of its effects.Methods:Totally 108 SD rats were randomly divided into sham-operation group, model group, pregabalin group, decoction low-, medium- and high-dosage groups, with 18 rats in each group. The CCI model was established by ligation of sciatic nerve in other groups except sham-operation group. On the postoperative day, the decoction low-, medium-, high-dosage groups were gavaged with 2.5, 5.0 and 10.0 g/kg of modified Xuanfuhua Decoction concentrate, respectively. The pregabalin group was gavaged with 15 mg/kg of pregabalin. The sham-operation group and the model group were gavaged with equal amounts of saline once/d for 15 days. Pain behavioural assays were performed before, on the 3rd, 7th, 11th and 15th day of administration respectively. The levels of interleukin (IL)-1β, tumour necrosis factor-α (TNF-α), IL-10 were detected by ELISA method. The expressions of Toll-like receptor 4 (TLR4), nuclear factor-κB p65 (NF-κB p65) were detected by immunohistochemistry staining. The phosphorylated p38MAPK (p-p38MAPK) were measured in the spinal cord by Western blot.Results:Compared with the model group, the scores of spontaneous pain in decoction high-dosage group decreased ( P<0.05), the thermal foot shrinkage latency (TWL) was prolonged ( P<0.05), and the mechanical foot shrinkage reflex threshold (MWT) increased ( P<0.05); the levels of IL-1β and TNF-α in spinal cord tissue of decoction low-, medium- and high-dosage groups decreased ( P<0.05), the level of IL-10 increased ( P<0.05), the average gray values of TLR4 and NF-κB p65 in spinal cord decreased ( P<0.05), and the expression of P-P38MAPK protein decreased ( P<0.05). Conclusion:Modified Xuanfuhua Decoction can effectively improve neurogenic pain in CCI rats, and the mechanism may be related to inhibition of p38MAPK-TLR4 signaling pathway activation-mediated spinal cord neuroinflammation.

Mechanism by which Bushen Yiqi Huayu granule improves callus angiogenesis in osteoporotic rats after fracture operation / 中国组织工程研究

BACKGROUND:Bushen Yiqi Huayu granule has the effect of replenishing qi and eliminating blood stasis,tonifying kidney and smoothing collages,and is often used in the treatment of osteoporosis.At present,there are few studies on the effect of Bushen Yiqi Huayu granule on the callus angiogenesis of osteoporotic fractures. OBJECTIVE:To explore the mechanism of Bushen Yiqi Huayu granule by up-regulating extracellular signal-regulated kinase/mitogen-activated protein kinases(ERK/MAPK)signaling pathway to improve callus angiogenesis after fracturing in osteoporotic rats. METHODS:(1)Bone marrow mesenchymal stem cells from osteoporotic SD rats and bone marrow monocytes from C57BL/6 mice were collected.MTT assay was used to detect different doses of Bushen Yiqi Huayu granule on bone mesenchymal stem cell toxicity.Bone mesenchymal stem cells and bone marrow monocytes were cultured in the medium supplemented with 0 and 1.5 mg/mL Bushen Yiqi Huayu granules,respectively,and osteogenic differentiation and osteoclast differentiation were carried out in vitro.(2)144 SD rats were randomly divided into sham operation group,model group,granule group and granule+PD98059 group with 36 rats in each group.The osteoporotic model was established in the model group,granule group and granule+PD98059 group,and only part of the adipose tissue near the ovary was removed in the sham operation group.8 weeks later,all rats received left tibial osteotomy.In the granule+PD98059 group,5 g/kg Bushen Yiqi Huayu granule was given intragastrically,and 0.3 mg/kg PD98059 was injected into the tail vein.The granule group was given 5 g/kg Bushen Yiqi Huayu granule,and the same volume of normal saline was injected into the tail vein.The sham operation group and model group were given an equal volume of normal saline,and the caudal vein was injected with an equal volume of normal saline.Drug administration was conducted once a day for 8 weeks.Fracture healing and callus angiogenesis were observed by X-ray,micro-computed tomography and microvascular perfusion angiography.Bone mineral density and mechanical strength of the callus were measured.The tibia was observed by hematoxylin-eosin staining and Masson staining.Western blot assay and immunohistochemistry were used to examine the expression of ERK/MAPK signaling pathway-related molecules. RESULTS AND CONCLUSION:(1)Compared with the 0 mg/mL group,the alkaline phosphatase activity,mineralization level,p-ERK1/2 and p-p38 MAPK expression levels of osteocytes increased(P<0.05),while the density and volume of osteoclasts decreased in the 1.5 mg/mL group(P<0.05).(2)Compared with the sham operation group,the fracture healing degree of the granule group was similar at 4 and 8 weeks,but there was no significant difference in Lane-Sandhu score,total callus volume,mineralized callus volume,mineralized callus volume/total callus volume,trabecular thickness,vascular number,spacing,thickness,vascular volume/total volume,bone mineral density,ultimate load of callus,stiffness,p-ERK1/2,p-p38 MAPK,p-IκB-α,and p-p65 expression levels(P>0.05).In the model group and granule+PD98059 group,fracture healing was slow,Lane-Sandhu score,total callus volume,mineralized callus volume,mineralized callus volume/total callus volume,trabecular thickness,vascular number,thickness,vascular volume/total volume,bone mineral density,ultimate load of callus,stiffness,p-ERK1/2,p-p38 MAPK,p-IκB-α,and p-p65 expression levels decreased(P<0.05),and vascular spacing increased(P<0.05),compared with the sham operation group.(3)It is indicated that Bushen Yiqi Huayu granule can improve fracture healing,promote callus angiogenesis and alleviate the symptoms of osteoporosis by enhancing the expression of the ERK/MAPK signaling pathway-related molecules in osteoporotic rats.

Effect of electroacupuncture on P2X4R-p38 MAPK-BDNF signaling pathway in trigeminal ganglion of rats with trigeminal neuralgia / 中华麻醉学杂志

Objective:To evaluate the effect of electroacupuncture on P2X4R-p38 mitogen-activated protein kinase (p38 MAPK)-brain-derived neurotrophic factor (BDNF) signaling pathway in trigeminal ganglion of rats with trigeminal neuralgia.Methods:Thirty-six clean-grade healthy adult male Sprague-Dawley rats, weighing 190-230 g, aged 2-3 months, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (S group), trigeminal neuralgia group (TN group), and electroacupuncture group (E group). The model was developed by chronic constriction of the infraorbital nerve in anesthetized animals. The infraorbital nerve was only exposed without ligation in group S. Rats received electroacupuncture stimulation at the Baihui and Xiaguan acupoints on the affected side for 20 min after developing the model, with a frequency of 80 Hz, twice a day, for 14 consecutive days in E group. Facial mechanical pain threshold (FMT) was measured at 1 day before developing the model and 3, 7, 14, 21 and 28 days after developing the model. The rats were sacrificed after the last behavioral testing, and the trigeminal ganglia were taken for examination of histopathological changes of trigeminal ganglion (by HE staining) and for determination of the expression of P2X4R, p38 MAPK, phosphorylated p38 MAPK (p-p38 MAPK) and BDNF (by Western blot) and contents of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay). Results:Compared with group S, the FMT was significantly decreased at each time point after developing the model, the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased ( P<0.05), the pathological changes of the trigeminal ganglion were obvious in group TN. Compared with group TN, the FMT was significantly increased at each time point after developing the model, and the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased ( P<0.05), and the pathological changes of the trigeminal ganglion were significantly attenuated in group E. Conclusions:The mechanism by which electroacupuncture alleviates trigeminal neuralgia may be related to inhibiting the activity of P2X4R-p38MAPK-BDNF signaling pathway and reducing neuroinflammation in rats.

Effect of Guipitang on ERK1/2 and p38 MAPK in Rats with Myocardial Ischemia / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic effect and mechanism of Guipitang on rats with myocardial ischemia. MethodFifty SD rats were divided into five groups： a control group， a model group， low and high-dose Guipitang （7.52， 15.04 g·kg-1） groups， and a trimetazidine group （0.002 g·kg-1）. By intragastric administration of vitamin D3 and feeding rats with high-fat forage and injecting isoproterenol， the rat model of myocardial ischemia was established. After drug treatment of 15 d， an electrocardiogram （ECG） was performed to analyze the degree of myocardial injury. A fully automatic biochemical analyzer was used to detect the changes in the serum levels of total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C）. Hematoxylin-eosin （HE） staining and Masson staining were used to observe myocardial histopathological changes. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect cardiomyocyte apoptosis. Western blot was adopted to detect the protein levels of extracellular signal-regulated kinase 1/2 （ERK1/2）， phospho-ERK1/2 （p-ERK1/2）， p38 mitogen-activated protein kinase （p38 MAPK）， phospho-p38 MAPK （p-p38 MAPK）， B-cell lymphoma-2 （Bcl-2）-associated X （Bax）， Bcl-2， and cleaved cysteine aspartate proteolytic enzyme （cleaved Caspase-3）. ResultCompared with the control group， the ECG S-T segment decreased in the model group. The serum levels of TC， TG， and LDL-C were increased significantly （P<0.05）. The arrangement of myocardial tissue was disordered， and the proportion of cardiomyocyte apoptosis increased. The protein levels of cleaved Caspase-3， Bax， and p-p38 MAPK in the heart were increased， and the Bcl-2 expression was decreased （P<0.05）. Compared with the model group， the S-T segment downward shift was restored in the low and high-dose Guipitang groups and trimetazidine group， and the levels of TC， TG， and LDL-C were decreased. The protein expression of cleaved Caspase-3 and Bax in the heart dropped， and p-p38 MAPK and p-ERK1/2 protein expressions increased significantly （P<0.05）. The degree of myocardial injury was alleviated， and the proportion of cardiomyocyte apoptosis decreased. Bcl-2 protein expression was increased significantly in the low-dose Guipitang group （P<0.05）. ERK1/2 and p38 MAPK proteins had no significant difference among different groups. ConclusionGuipitang could alleviate myocardial injury and inhibit cardiomyocyte apoptosis in rats by activating the expression of ERK1/2 and p38 MAPK.

Role of P2X4R in maintenance of trigeminal neuralgia in rats: relationship with p38 MAPK/BDNF signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of P2X4 receptor (P2X4R) in the maintenance of trigeminal neuralgia and the relationship with p38 mitogen-activated protein kinase (p38 MAPK)/brain-derived neurotrophic factor (BDNF) signaling pathway in rats.Methods:Forty-eight clean-grade healthy adult male Sprague-Dawley rats, weighing 190-230 g, aged 2-3 months, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (S group), trigeminal neuralgia group (TN group), trigeminal neuralgia+ dimethylsulfoxide (DMSO) group (TN+ DMSO group), and trigeminal neuralgia+ P2X4R specific antagonist 5-BDBD group (TN+ 5-BDBD group). The model was developed by chronic constriction of the infraorbital nerve. The infraorbital nerve was only exposed without ligation in group S. At 3, 7, 10 and 14 days after developing the model, 5 μg/μl 5-BDBD 10 μl was intrathecally injected in TN+ 5-BDBD group, and 2% DMSO 10 μl was intrathecally injected in TN+ DMSO group. The facial mechanical pain withdrawal threshold (MWT) was measured at 1 day before developing the model and 1, 3, 7, 10, 14 and 28 days after developing the model (T 0-6). The rats were sacrificed and the trigeminal ganglia were taken for determination of the expression of P2X4R, p38 MAPK, phosphorylated p38 MAPK (p-p38 MAPK) and BDNF (by Western blot) and contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6 (by enzyme-linked immunosorbent assay). Results:Compared with group S, the MWT was significantly decreased at T 1-6, the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in TN group ( P<0.05). Compared with TN group, the MWT was significantly increased at T 3-6, and the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased in TN+ 5-BDBD group ( P<0.05), and no significant change was found in the indexes mentioned above in TN+ DMSO group ( P>0.05). Conclusions:P2X4R is involved in the maintenance of trigeminal neuralgia in rats, which may be related to the activation of p38 MAPK/BDNF signaling pathway and the increase in inflammatory mediator release.

Effect and mechanism of oxycodone hydrochloride on inflammation and neuronal apoptosis in epileptic rats / 中华老年心脑血管病杂志

Objective To explore the effect of oxycodone hydrochloride on inflammation and neuro-nal apoptosis in epileptic rats through c-Jun amino terminal kinase(JNK)/p38 mitogen activated protein kinase(p38 MAPK)signaling pathway.Methods After epileptic rat model was success-fully constructed,72 epileptic rats were randomly divided into model group,low-,medium-and high-dose oxycodone hydrochloride groups(0.125,0.25 and 0.5 mg/kg),anisomycin(JNK activa-tor 5 mg/kg)and combined group(0.5 mg/kg oxycodone hydrochloride+5 mg/kg anisomycin),with 12 rats in each group.Another 12 healthy rats were selected as control group.In 24 h after the end of administration,the frequency and duration of seizures were recorded for all rats.ELISA was used to detect the serum levels of TNF-α and IL-6,HE staining was employed to observe the his-topathological changes in hippocampal CA1 region,and TUNEL staining was applied to detect the apoptosis of CA1 region neurons.The expression of JNK,p-JNK,p38 MAPK,p-p38 MAPK and Caspase-3 in hippocampal CA1 region was detected by Western blotting.Results Compared with the rats from the control group,those of the model group showed higher frequency and longer du-ration of seizures,higher serum TNF-α and IL-6 levels,increased apoptotic rate of hippocampal CA1 neurons,and elevated p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression(P＜0.05).While low-,medium-and high-dose oxycodone hydrochloride treatment re-versed above changes in frequency and duration of seizures,serum TNF-α and IL-6 levels,neuro-nal apoptosis,p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression(P＜0.05).In the anisomycin group,higher frequency and longer duration of seizures,elevated serum TNF-α and IL-6 levels,increased neuronal apoptotic rate in hippocampal CA1 region,and en-hanced p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression(P＜0.05).Lower frequency and shorter duration of seizures,decreased serum TNF-α and IL-6 levels,and re-duced neuronal apoptotic rate in hippocampal CA1 region were observed in the combined group than the anisomycin group(P＜0.05).The combined group obtained statistically lower p-JNK/JNK ratio,p-p38 MAPK/p38 MAPK ratio and Caspase-3 expression in hippocampal CA1 region than the high-dose group,and opposite results than the anisomycin group(0.89±0.12 vs 0.25± 0.05 vs 1.08±0.16,0.81±0.08 vs 0.21±0.04 vs 0.94±0.12,0.79±0.12 vs 0.26±0.04 vs 0.89± 0.14,P＜0.05).Conclusion Oxycodone hydrochloride can reduce inflammatory response,im-prove epileptic symptoms and pathological damages,and protect neurons in epileptic rats,which is related to the inhibition of JNK/p38 MAPK signaling pathway.

Role of autophagy in morphine preconditioning-induced reduction of OGD/R injury in primary cortical neurons of mice and the relationship with JNK / 中华麻醉学杂志

Objective:To evaluate the role of autophagy in morphine preconditioning-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury in primary cortical neurons of mice and the relationship with c-Jun N-terminal kinase (JNK).Methods:Primary cortical neurons extracted from C57BL/6 neonatal mice within 24 h after birth were divided into 9 groups ( n=24 each) using a random number table method: control group (C group), OGD/R group, morphine preconditioning group (M group), autophagy inhibitor 3-methyladenine (3-MA) group (3-MA group), 3-MA+ morphine preconditioning group (3-MA+ M group), autophagy inhibitor chloroquine group (Ch group), chloroquine + morphine preconditioning group (Ch+ M group), JNK inhibitor SP600125 group (SP group) and SP600125 + morphine preconditioning group (SP+ M group). Morphine preconditioning: morphine was added at a final concentration of 3 μmol/L before OGD/R, and the cells were incubated for 2 h in OGD/R group. In 3-MA, Ch and SP groups, 3-MA 5 mmol/L, chloroquine 50 μmol/L and SP600125 25 μmol/L were added, respectively, and the cells were incubated for 150 min. In 3-MA+ M, Ch+ M and SP+ M groups, 3-MA 5 mmol/L, chloroquine 50 μmol/L and SP600125 25 μmol/L were added, respectively, at 30 min before morphine preconditioning. Then the cells were subjected to oxygen-glucose deprivation for 1 h followed by restoration of oxygen-glucose supply for 24 h. CCK-8 assay was used to detect the neuronal viability. The expression of JNK, phosphorylated JNK (p-JNK), microtubule-associated protein 1 light chain 3 (LC3), p62, Beclin1, caspase-3, and cleaved-caspase-3 was determined by Western blot. The autophagosomes and autolysosomes were counted using LC3-double fluorescent adenovirus transfection, and the neuronal apoptosis rate was determined by TUNEL staining. Results:Compared with C group, the neuronal viability was significantly decreased, the expression of Beclin1 was up-regulated, the expression of p62 was down-regulated, and the LC3Ⅱ/LC3Ⅰ ratio, p-JNK/JNK ratio, the number of autophagosomes and autolysosomes, cleaved-caspase-3/caspase-3 ratio and neuronal apoptosis rate were increased in OGD/R group ( P<0.001). Compared with OGD/R group, the neuronal viability, p-JNK/JNK ratio, LC3Ⅱ/LC3Ⅰ ratio and the number of autophagosomes and autolysosomes were significantly increased, the expression of Beclin1 was up-regulated, and the expression of p62 was down-regulated in M group, the LC3Ⅱ/LC3Ⅰ ratio was significantly decreased, and the expression of p62 was down-regulated in 3-MA group, the LC3Ⅱ/LC3Ⅰ ratio was significantly increased, and the expression of p62 was up-regulated in Ch group ( P<0.001), and no significant change was found in the parameters mentioned above in SP600125 group ( P>0.05). Compared with M group, the neuronal viability was significantly decreased, the LC3Ⅱ/LC3Ⅰ ratio was decreased, and the expression of p62 was up-regulated in M+ 3-MA group, the neuronal viability was significantly decreased, the LC3Ⅱ/LC3Ⅰ ratio was increased, and the expression of p62 was up-regulated in M+ Ch group, and the neuronal viability, LC3Ⅱ/LC3Ⅰ ratio and p-JNK/JNK ratio were significantly decreased, the expression of p62 was up-regulated, the number of autophagosomes and autolysosomes was decreased, the expression of Beclin1 was down-regulated, and the cleaved-caspase-3/caspase-3 ratio and neuronal apoptosis rate were increased in M+ SP group ( P<0.001). Conclusions:Morphine preconditioning can attenuate OGD/R injury by activating JNK, enhancing autophagy and inhibiting apoptosis in primary cortical neurons of mice.

Mechanisms and research progress of targeted therapies on regulation of autophagy by natural compounds from traditional Chinese medicine / 药学学报

Autophagy is an important physiological process that can degrade cell components and maintain cell homeostasis, divided into three types including macroautophagy, microautophagy and chaperon-mediated autophagy generally, and macroautophagy is the most common form. Autophagy can affect the progression of a variety of diseases, such as cancer, neurodegenerative diseases, heart-related diseases, and autoimmune diseases, etc. However, autophagy can promote or inhibit diseases in different circumstances because of the dual roles of autophagy. Therefore, targeted regulating autophagy may be a potential treatment plan for diseases in specific stages of disease development. Now, with the development of traditional Chinese medicine (TCM) resources and the deepening of researches on the modern utilization of TCM, many active compounds from TCM have been discovered that can target autophagy to exert pharmacological activity. Most of the natural compounds activate or inhibit autophagy by affecting the classical PI3K/AKT/mTOR autophagy pathway. In addition, some compounds can also affect autophagy through MAPKs signaling pathways such as MEK/ERK, JNK and p38MAPK. These active compounds exert various biological activities by regulating autophagy, including anti-tumor, inhibiting neurodegenerative diseases, protecting cardiomyocytes, and relief of inflammatory response. In this review, we summarized the active compounds in TCM that affect autophagy by targeting different signaling pathways and their mechanisms of regulating autophagy, also introduced the effects of active compounds on diseases after affecting autophagy. Finally, this paper summarized and prospected the development of targeted autophagy for the treatment of diseases by TCM compounds, hoping to provide clues for subsequent exploration and research.

Research concerning traditional Chinese medicine into correlation between mitogen-activated protein kinases and osteoporosis / 中华创伤骨科杂志

Osteoporosis (OP) is a systemic bone metabolic disease when bone resorption becomes greater than bone formation, resulting in bone mass loss and poor bone structure. The disability or fatality caused by its complications has become a global public problem. As mitogen-activated protein kinases (MAPK) are important molecules that maintain and regulate cellular energy balance, they are closely related to bone metabolism. In clinical practice, traditional Chinese medicine has demonstrated obvious advantages in prevention and treatment of OP. However, there has not been enough comprehensive or systematic summary of the researches into the regulatory mechanisms of this signaling pathway in the treatment of OP by traditional Chinese medicine. Therefore, this paper expounds on the effects of single traditional Chinese herb and compound traditional Chinese herbs on the regulatory mechanisms of MAPK signaling pathway in bone metabolism so that a theoretical basis can be provided for future basic and clinical researches in the prevention and treatment of OP.

Anti-swelling and Analgesic Mechanism of Jianpi Tongluo Prescription from MAPKs Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the anti-swelling and analgesic effects of Jianpi Tongluo prescription （JPTL） and to explore its mechanism initially. MethodA total of 120 ICR mice were divided into normal group， model group， JPTL low-， medium- and high-dose groups （5， 10， 20 g·kg-1） and positive drug （celecoxib， 0.03 g·kg-1） group， with 10 in each group （po，once a day）. Complete freund's adjuvant （CFA） was used to induce the model of chronic inflammatory pain， and xylene-induced ear swelling test， hot plate test and acetic acid writhing test were performed to observe the anti-swelling and analgesic effects of different doses of JPTL in these four acute and chronic models. Further， enzyme-linked immunosorbent assay （ELISA） was used to detect the expressions of prostaglandin E2 （PGE2）， interleukin-1β （IL-1β）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in serum and inflammatory paw of mice with chronic inflammatory pain， and the expressions of aquaporin 1 （AQP1）， aquaporin 3 （AQP3）， cyclooxygenase 1 （COX1）， cyclooxygenase 2 （COX2） and mitogen-activated protein kinases （MAPKs） in inflammatory paw were detected by Western blot， to explore the preliminary mechanism of JPTL. ResultCompared with the conditions in the normal group， there was a significant increase in the ear swelling of xylene-induced model mice， a shortened paw withdrawal latency in the hot plate test （P<0.01）. Compared with the model group， JPTL remarkably increased the inhibition rate of xylene-induced ear swelling （P<0.05， P<0.01）， prolonged the latency period of writhing caused by acetic acid and reduced the number of writhing （P<0.05， P<0.01）. Compared with normal group, the degree of feet swelling in chronic inflammatory pain mice was significantly increased， the threshold of mechanical pain was decreased and the threshold of cold pain was increased （P<0.05， P<0.01）， the protein contents of AQP1 and AQP3 in inflammatory feet were increased， and the contents of IL-1β， IL-6， TNF-α， PGE2 and COX2 in inflammatory feet were increased in serum and/or inflammatory feet. The protein expression levels of p-p38 MAPK， p-JNK and p-ERK in inflammatory feet were increased （P<0.01）. Compared with the model group， JPTL relieved paw swelling of mice with chronic inflammatory pain， elevated mechanical withdrawal threshold while decreased cold withdrawal threshold， with analgesia lasting for 4 h and the optimal time point for analgesia being 2 h after administration （P<0.05， P<0.01）. Moreover， JPTL down-regulated AQP1， AQP3， COX2， p-p38 MAPK， p-JNK and p-ERK in inflammatory paw of mice with chronic inflammatory pain and reduced IL-1β， IL-6， TNF-α， and PGE2 in serum and/or inflammatory paw， but it had no significant effect on COX1 （P<0.05， P<0.01）. ConclusionJPTL has anti-swelling and analgesic effects， and its mechanism is related to inhibiting the production of cytokines and inflammatory mediators via the down-regulation of MAPKs signaling pathway， which provides an experimental basis for the clinical application of JPTL.

Development of small molecule extracellular signal-regulated kinases (ERKs) inhibitors for cancer therapy

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway is widely activated by a variety of extracellular stimuli, and its dysregulation is associated with the proliferation, invasion, and migration of cancer cells. ERK1/2 is located at the distal end of this pathway and rarely undergoes mutations, making it an attractive target for anticancer drug development. Currently, an increasing number of ERK1/2 inhibitors have been designed and synthesized for antitumor therapy, among which representative compounds have entered clinical trials. When ERK1/2 signal transduction is eliminated, ERK5 may provide a bypass route to rescue proliferation, and weaken the potency of ERK1/2 inhibitors. Therefore, drug research targeting ERK5 or based on the compensatory mechanism of ERK5 for ERK1/2 opens up a new way for oncotherapy. This review provides an overview of the physiological and biological functions of ERKs, focuses on the structure-activity relationships of small molecule inhibitors targeting ERKs, with a view to providing guidance for future drug design and optimization, and discusses the potential therapeutic strategies to overcome drug resistance.

Role of AMPK/p38 MAPK/Nrf2 signaling pathway in myocardial ischemia-reperfusion injury in diabetic rats / 中华麻醉学杂志

Objective:To evaluate the role of adenosine monophosphate-dependent protein kinase/p38 mitogen-activated protein kinase/nuclear factor E2-associated factor 2 (AMPK/p38 MAPK/Nrf2) pathway in myocardial ischemia-reperfusion (I/R) injury in diabetic rats.Methods:Clean-grade healthy Sprague-Dawley male rats, aged 2-3 months, weighing 220-280 g, were fed with a high fat diet, and 1% streptozotocin 50 mg/kg was intraperitoneally injected for 4 consecutive days to develop the model of diabetes mellitus.Thirty diabetic rats were divided into 3 groups ( n=10 each) using the random number table method: sham operation group (sham group), myocardial I/R group (I/R group), and AMPK inhibitor compound C+ myocardial I/R group (C+ I/R group). The model of myocardial I/R injury was developed by ligation of the left anterior descending coronary artery for 30 min followed by 120 min reperfusion.Compound C 0.5 mg/kg was injected via the caudal vein at 30 min before ischemia in C+ I/R group, while the equal volume of normal saline was given instead in Sham group and I/R group.At 120 min of reperfusion, the percentage of myocardial infarct size was calculated, the serum concentrations of creatine kinase isoenzymes (CK-MB) and lactic dehydrogenase (LDH) were determined by enzyme-linked immunosorbent assay, the levels of glutathione (GSH), superoxide dismutase (SOD) and reactive oxygen species (ROS) in myocardial tissues were measured by enzyme-linked immunosorbent assay, and the expression of AMPK, phosphorylated AMPK (p-AMPK), phosphorylated p38 MAPK (p-p38 MAPK), Nrf2 and heme oxygenase-1 (HO-1) in myocardium was determined by Western blot. Results:Compared with Sham group, the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased, the levels of GSH and SOD in myocardial tissues were decreased, ROS level was increased, and the expression of AMPK, p-AMPK, p-p38 MAPK, Nrf2 and HO-1 was up-regulated in I/R group ( P<0.05). Compared with I/R group, the percentage of myocardial infarct size and serum CK-MB and LDH levels were significantly increased, the levels of GSH and SOD in myocardial tissues were decreased, ROS level was increased, and the expression of AMPK, Nrf2 and HO-1 was down-regulated in C+ I/R group ( P<0.05). Conclusions:AMPK/p38 MAPK/Nrf2 signaling pathway is involved in the mechanism of endogenous antioxidant stress during myocardial I/R in diabetic rats.

Mechanism of paeoniflorin inhibiting apoptosis of hippocampal neurons of rats induced by lead acetate / 中华劳动卫生职业病杂志

Objective: To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate. Methods: In September 2020, primary hippocampal neuronal cells were isolated and cultured from fetal rats, and identified using cellular immunofluorescent. MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis. MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate. According to the results, different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells, after 24 h, lead acetate was added to the cells, meanwhile, blank and model groups were set up, the content of reactive oxygen species (ROS) , superoxide dismutase (SOD) , lactate dehydrogenase (LDH) , malondialdehyde (MDA) and Caspase-3 were measured. Extracellular signal regulated kinase (ERK) , phosphorylated ERK (p-ERK) , p38 mitogen -activated protein kinases (p38MAPK) , phosphorylated p38MAPK (p-p38MAPK) , c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) protein expression in hippocampal neuronal cells were determined by Western blotting. Results: The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate, MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25 μmol/L. Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80 μmol/L. Compared with the model group, the activity of hippocampal neuronal cells was significantly increased after treating with 20, 40 or 80 μmol/L paeoniflorin (P<0.05) . Compared with the blank group, the ROS activity, LDH release level, MDA content and caspase-3 content were significantly increased (P<0.01) , and the SOD activity was significantly decreased (P< 0.01) in the hippocampal neuronal cells of the model group. Compared with the model group, the ROS activity, LDH release level, MDA content and caspase-3 content were obviously decreased (P<0.05) , SOD activity was significantly increased (P <0.01) after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin. Relative to the model group, the ratio of p-ERK/ERK were significantly up-regulated (P<0.01) , while the ratios of p-p38MAPK/p38MAPK and p-JNK/JNK were significantly down-regulated after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin (P<0.05) . Conclusion: Paeoniflorin may down-regulate the expression of p-p38MAPK and p-JNK protein, up-regulate the expression of p-ERK protein, and inhibit the apoptosis of hippocampal neurons induced by lead acetate through the MAPK signaling pathway.

Arctigenin attenuates paraquat-induced human lung epithelial A549 cell injury by suppressing ROS/p38 mitogen-activated protein kinases-mediated apoptosis / 世界急诊医学杂志（英文）

@#BACKGROUND: Paraquat (PQ)-induced acute lung injury (ALI) and pulmonary fibrosis are common diseases with high mortality but without effective antidotes in emergency medicine. Our previous study has proved that arctigenin suppressed pulmonary fibrosis induced by PQ. We wondered whether arctigenin could also have a protective effect on PQ-induced ALI. METHODS: A PQ-induced A549 cell injury model was used, and the effect of arctigenin was determined by a cell counting kit-8 (CCK-8) cell viability assay. In addition, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining assays and mitochondrial membrane potential assays were performed to evaluate the level of cell apoptosis. The generation of reactive oxygen species (ROS) was reflected by dihydroethidium (DHE) staining and a 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Moreover, immunoblotting studies were used to assess the expression of mitogen-activated protein kinases (MAPKs) and p38 MAPK. RESULTS: Arctigenin attenuated PQ-induced inhibition of A549 cell viability in a dose-dependent manner. Arctigenin also significantly reduced PQ-induced A549 cell apoptosis, as reflected by the TUNEL assay and mitochondrial membrane potential assay, which may result from suppressed ROS/p38 MAPK signaling because we found that arctigenin dramatically suppressed ROS generation and p38 MAPK phosphorylation. CONCLUSION: Arctigenin could attenuate PQ-induced lung epithelial A549 cell injury in vitro by suppressing ROS/p38 MAPK-mediated cell apoptosis, and arctigenin might be considered a potential candidate drug for PQ-induced ALI.

The MAPK signaling pathway: A new target for the treatment of hepatic echinococcosis / 临床肝胆病杂志

The MAPK signaling pathway can mediate a variety of cytokines to participate in the processes of inflammation, cancer, immune disorder, and neurodegenerative diseases, and it also plays an important role in the development and progression of hepatic echinococcosis. This article reviews the structure and regulation of the MAPK signaling pathway and elaborates on the role of the MAPK signaling pathway in hepatic echinococcosis. It is pointed out that the MAPK signaling pathway can activate both the cyst and the host in hepatic echinococcosis, participate in the development and progression of the disease, and exert an impact on its treatment. Drug therapy targeting the MAPK signaling pathway is expected to become a new strategy for the treatment of hepatic echinococcosis.

Effects of neutrophil extracellular traps on proliferation and apoptosis of human amniotic epithelial cells and its possible mechanisms / 中华围产医学杂志

Objective:To investigate the effects of neutrophil extracellular traps (NETs) on the proliferation and apoptosis of human amniotic epithelial cells.Methods:NETs were induced in vitro from the neutrophil cells obtained from the peripheral blood of normal pregnant women before elective cesarean section at full-term. Human amniotic epithelial cell lines (WISH cells) were cultured in vitro, and were divided into four groups:(1) control group: without any stimulus; (2) NETs group: WISH cells were stimulated with NETs (500 ng/ml); (3) NETs+SB203580 (p38 kinase inhibitor) group: WISH cells were pretreated with SB203580 (5 μmol/L) for 30 min and then NETs (500 ng/ml) was added; (4) SB203580 group: only SB203580 was added. After stimulating for 48 h, cell proliferation assay, lactate dehydrogenase(LDH) assay, and flow cytometry assay were used to detect the cell proliferation rate, LDH level of cell supernatant, and cell apoptosis rate among different groups. The results were analyzed and compared using one-way analysis of variance and LSD- t test. Results:(1) Cell proliferation: The cell proliferation ratio in the NETs group was lower than that in the control group [(9.379±0.775)% vs (36.560±1.208)%, LSD- t=20.78, P<0.001]; and the figure in the NETs+SB203580 group [(27.920±0.926)%] was higher than that in the NETs group (LSD- t=14.18, P<0.001). (2)LDH: There was an increased LDH level in the cell supernatant of the NETs group compared with the control group (1.518±0.038 vs 0.274±0.004, LSD -t=44.25, P<0.05), and the LDH level in the NETs+SB203580 group (0.857±0.009) was decreased than that in the NETs group (LSD -t=23.51, P<0.001). (3) Apoptosis: Compared with the control group, the cell apoptosis level of the NETs group was increased [(14.290±0.141)% vs (10.110±0.044)%, LSD- t=21.76, P<0.001]; but that in the NETs+SB203580 group [(10.500±0.218)%] was lower than in the NETs group (LSD- t=19.70, P<0.001). Conclusion:p38/mitogen-activated protein kinases signaling pathway may be involved in the process of NETs, inhibiting proliferation and promoting apoptosis of human amniotic epithelial cells.

Effects of resveratrol on expression of inflammatory cytokines and related genes in benzo (a) pyrene-induced human sebocytes / 中华皮肤科杂志

Objective:To investigate the effect of resveratrol on the expression of inflammatory cytokines and related genes in human SZ95 sebocytes induced by benzo (a) pyrene.Methods:Human SZ95 sebocytes were cultured in vitro, and divided into 4 groups: control group treated with 1‰ dimethyl sulfoxide for 27 hours, resveratrol group treated with 1 × 10 -5 mol/L resveratrol for 24 hours, benzo (a) pyrene group treated with 1 × 10 -5 mol/L benzo (a) pyrene for 3 hours, resveratrol+benzo (a) pyrene group treated with 1 × 10 -5 mol/L resveratrol for 24 hours followed by 1 × 10 -5 mol/L benzo (a) pyrene for 3 hours. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of interleukin (IL) -1α, IL-6, aryl hydrocarbon receptor (AhR) , cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1) in SZ95 sebocytes in the above groups; Western blot analysis was conducted to determine the phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK, expressed as the ratio of phosphorylated to total p38 MAPK) and AhR protein expression; enzyme-linked immunosorbent assay (ELISA) was conducted to detect levels of IL-1α and IL-6 in the cell culture supernatant in each group. One-way analysis of variance was used for comparison of means among multiple groups, and least significant difference- t test was used for multiple comparisons. Results:The mRNA and protein expression of IL-1α in SZ95 sebocytes significantly differed among the control group, resveratrol group, benzo (a) pyrene group and resveratrol+benzo (a) pyrene group (mRNA: 2.045 ± 0.272, 2.058 ± 0.154, 3.124 ± 0.094, 2.185 ± 0.337, protein: 9.132 ± 1.181, 9.429 ± 0.771, 20.361 ± 0.907, 9.917 ± 0.897, F=14.662, 101.705, P < 0.01, < 0.001, respectively) , and were significantly lower in the resveratrol+benzo (a) pyrene group than in the benzo (a) pyrene group (both P < 0.01) . In addition, the phosphorylation level of p38 was significantly higher in the benzo (a) pyrene group than in the control group, resveratrol group and resveratrol+benzo (a) pyrene group ( F=303.129, P < 0.000 1) . The mRNA expression of AhR, CYP1A1 and CYP1B1 was significantly lower in the resveratrol+benzo (a) pyrene group than in the benzo (a) pyrene group ( t=10.64, 33.599, 18.327, respectively, all P < 0.001) . The benzo (a) pyrene group showed significantly decreased protein expression of AhR compared with the resveratrol+benzo (a) pyrene group ( P < 0.001) . Conclusion:Resveratrol can inhibit the environmental pollutant benzo (a) pyrene-induced expression of inflammatory factor IL-1α in SZ95 sebocytes, which is likely mediated by the AhR and p38MAPK pathways.

Effect of erigeron breviscapus on relieving acute ocular hypertension and protecting retinal ganglion cells in glaucoma rats based on mitogen activated protein kinase signaling pathway / 中华眼底病杂志

Objective:To investigate the effect of erigeron breviscapus (EBHM) on ocular hypertension and the protective effect of retinal ganglion cells (RGCs) in rats by regulating mitogen activated protein kinase (MAPK) signaling pathway.Methods:Sixty male Sprague-Dawley rats were divided into control group, model group, low-dose EBHM group (group A), medium-dose EBHM group (group B), and high-dose EBHM group (group C) by random number table method. There were 12 rats in the group, the left eye was used as the experimental eye. The rats of model group, group A, group B, and group C were infused with normal saline through the anterior chamber to construct an acute ocular hypertension model; the control group was given general anesthesia only. Then, 2-30 days after modeling, rats in the control group and model group were given 3 ml of normal saline once a day; rats in group A, group B, and group C were given 0.30, 0.45, and 0.60 g/100 g EBHM by intragastric administration, respectively, 1 time/d. The rat intraocular pressure was measured before modeling and 1, 14, and 30 days after modeling, and the proportion of high intraocular pressure model was measured. Thirty days after modeling, hematoxylin-eosin (HE) staining was used to observe the pathological changes of retinal tissue; immunofluorescence staining was used to detect the changes in the number of RGCs; real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect p38 in the retinas of rats in each group. The relative expression of MAPK and Caspase-3 mRNA; western blot was used to detect p38MAPK and phosphorylation in the retina of rats in each group relative expression of phosphorylate-p38MAPK (p-p38MAPK) and Caspase-3 protein. One-way analysis of variance was used for multi-sample comparison, and SNK-q test was used for comparison between two samples. Results:One day after modeling, none of the rats in the control group developed acute ocular hypertension, and the other groups were successfully modeled. Compared with the model group, the rates of acute ocular hypertension at 14 days after modeling in groups B and C were lower ( χ2=98.701, P<0.05), and the rates of acute ocular hypertension at 30 days after modeling in groups A, B, and C were 0. There was no statistically significant difference in the rates of acute ocular hypertension between 14 and 30 days after modeling in the A, B, and C groups ( P>0.05). The results of HE staining showed that the structure of the retina in the control group was complete, and the layers were clearly visible; the RGCs count was not abnormal, and the morphology was plump and round. The retina of rats in the model group became thinner; the number of RGCs was greatly reduced, the morphology was vacuolated, and the arrangement was sparse. The retina of rats in groups A, B, and C became thicker, and the number of RGCs increased, and the retina structure in group C was better restored. The results of immunofluorescence staining showed that the RGCs counts of rats in groups A, B, and C were higher than those in the model group, and the difference was statistically significant ( F=297.514, P<0.05); pairwise comparison between groups, group A was lower than that of group B and C Group ( q=2.842, 5.263), group B was lower than group C ( q=2.457), the difference was statistically significant ( P<0.05). The results of RT-qPCR and Western blot showed that compared with the model group, the relative expression of Caspase-3 mRNA ( F=267.912) and protein ( F=692.279) and the relative expression of p-p38MAPK protein in the retina of rats in groups A, B and C. The expression level ( F=150.061) all decreased, and the difference was statistically significant ( P<0.05); pairwise comparisons between groups showed that Caspase-3 mRNA ( q=6.977, 15.642) and protein ( q=6.997, 15.642) relative expression levels and p-p38MAPK protein ( q=12.443, 24.358) relative expression levels are lower than groups A and B, group B was lower than group A ( q=11.678, 12.471, 10.204), the difference was statistical academic significance ( P<0.05). Conclusions:EBHM can significantly reduce intraocular pressure in rats with acute ocular hypertension, increase RGCs counts, and reduce retinal damage. Its regulatory mechanism may be related to the MAPK pathway.

Role of IL-1β/JNK pathway in sevoflurane-induced necroptosis of rat hippocampal neurons in vitro / 中华麻醉学杂志

Objective:To evaluate the role of interleukin 1β (IL-1β)/c-Jun N-terminal kinase (JNK) pathway in sevoflurane-induced necroptosis of rat hippocampal neurons in vitro. Methods:Primarily cultured hippocampal neurons of Sprague-Dawley rat fetuses were inoculated into 96-well plates (cell density: 1×10 4 cells/ml, 200 μl/hole) and 6-well plates (cell density: 1×10 6 cells/ml, 2 ml/hole). The cells were divided into 3 groups ( n=20 each) using a random number table method after being cultured for 7 days: control group (group C), sevoflurane group (group S) and IL-1 receptor antagonist group (group I). Group C received routine culture, IL-1 receptor antagonist IL-1ra 1 μg/μl was added, and the cells were incubated for 30 min in group I, and in addition the cells were placed in the incubator containing 2% sevoflurane and cultured for 5 h at 37 ℃ in S and I groups.The cells were collected for microscopic examination of the morphology of neurons (with an inverted microscope) and for determination of the cell necroptosis rate (by flow cytometry), cell viability (by MTT method), and expression of IL-1β, interleukin-1 receptor type I (IL-1RI), interleukin-1 receptor accessory protein (IL-1RAcP), phosphorylated JNK (p-JNK) ), receptor-interacting protein 1 (RIP1), RIP3 and phosphorylated mixed lineage kinase-like (p-MLKL) (by Western blot ). Results:Compared with group C, the cell viability was significantly decreased, the necroptosis rate was increased, and the expression of IL-1β, IL-1RI, IL-1RAcP, p-JNK, RIP1, RIP3 and p-MLKL was up-regulated in group S ( P<0.05). Compared with group S, the cell viability was significantly increased, the necroptosis rate was decreased, and the expression of IL-1β, IL-1RI, IL-1RAcP, p-JNK, RIP1, RIP3 and p-MLKL was down-regulated in group I ( P<0.05). There was no obvious abnormality in the morphology of neurons in group C. The cell body of neurons was shrunk, the processes were broken, and the network between processes was sparse in group S. The cell body was round, and the morphology was close to normal in group I. Conclusion:The mechanism by which sevoflurane induces necroptosis of rat hippocampal neurons in vitro is related to activation of IL-1β/JNK pathway.

Relationship between mechanism underlying methylprednisolone-induced alleviation of ventilator-induced lung injury and p38MAPK/NLRP3 pathway in lung tissues of rats / 中华麻醉学杂志

Objective:To evaluate the relationship between the mechanism underlying methylprednisolone-induced alleviation of ventilator-induced lung injury (VILI) and p38 mitogen-activated protein kinase (p38 MAPK)/nucleotide binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) pathway in lung tissues of rats.Methods:Sixty clean-grade male Sprague-Dawley rats, weighing 270-320 g, aged 4-5 months, were divided into 3 groups ( n=20 each) using a random number table method: control group (group C), mechanical ventilation group (group V), and methylprednisolone group (group M). Group C breathed air spontaneously for 4 h without mechanical ventilation.Group V was mechanically ventilated (RR 40 times/min, V T 40 ml/kg, I∶E 1∶1, PEEP 0, FiO 2 21%) for 4 h. Group M received intravenous methylprednisolone 10 mg/kg at 20 min before mechanical ventilation.At 4 h of mechanical ventilation, broncho-alveolar lavage fluid (BALF) was collected to measure the concentrations of interleukin-1beta (IL-1β), IL-18, and tumor necrosis factor-alpha (TNF-α) and wet/dry lung weight ratio (W/D ratio), and lung tissues were obtained for microscopic examination of the histopathological changes and for detection of the expression of p38MAPK, phosphorylated p38MAPK (p-p38MAPK), NLRP3, apoptosis-related speck-like protein containing a CARD (ASC), and cysteinyl aspartate-specific protease-1 (caspase-1) (using Western blot). Results:Compared with group C, the W/D ratio of lung tissues and concentrations IL-1β, IL-18 and TNF-α in BALF were significantly increased, and the expression of p-p38MAPK, NLRP3, ASC and caspase-1 was up-regulated in group V ( P<0.05), and no significant change was found in group M ( P>0.05). Compared with group V, the W/D ratio of lung tissues and concentrations of IL-1β, IL-18 and TNF-α in BALF were significantly decreased, and the expression of p-p38MAPK, NLRP3, ASC and caspase-1 was down-regulated in group M ( P<0.05). Conclusion:The mechanism by which methylprednisolone alleviates VILI may be related to inhibition of p38MAPK/NLRP3 pathway activity and reduction of inflammatory responses in lung tissues of rats.