Yiqi Huoxue Tongluo Prescription Inhibits Activation of Astrocytes to Relieve Diabetic Neuropathic Pain in Rats via ERK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Yiqi Huoxue Tongluo prescription （YHTP） in the treatment of diabetic neuropathic pain （DNP）. MethodNinety SPF-grade SD male rats were randomized into blank， model， low- （2.25 g·kg-1）， medium- （4.5 g·kg-1）， and high-dose （9 g·kg-1） YHTP， and mecobalamin （0.175 mg·kg-1） groups. Except those in the blank group， the rats in the remaining 5 groups were fed with a high-fat and high-glucose diet and subjected to intraperitoneal injection of low-dose （35 mg·kg-1） streptozotocin （STZ） to establish the model of DNP. The sciatic nerve conduction velocity in DNP rats was measured by the neurophysiological method， and the levels of interleukin-6 （IL-6）， IL-1β， and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of glial fibrillary acidic protein （GFAP） and extracellular signal-regulated kinase （ERK） in the spinal cord. Western blot was employed to measure the protein levels of GFAP and phosphorylated ERK （p-ERK）， and immunofluorescence staining to measure the fluorescence intensity of GFAP and p-ERK in the spinal cord. In the cell experiments， 100 mmol·L-1 high glucose was used to induce the activation of astrocytes （CTX-TNA2） for the modeling of nerve cell injury. The cells were randomized into the normal， model， drug-containing serum （10% YQHT）， inhibitor ［10 mol·L-1 corynoxeine （COR）］， drug-containing serum + inhibitor （10% YHTP + 10 mol·L-1 COR） groups. The levels of pro-inflammatory factors （TNF-α and IL-1β） and the anti-inflammatory factor IL-10 in CTX-TNA2 cells were determined by ELISA， and the protein levels of GFAP and p-ERK in CTX-TNA2 cells by Western blot. ResultThe animal experiments showed that compared with the blank group， the model group presented reduced mechanical withdrawal threshold （MWT）， thermal work limit （TWL）， and nerve conduction velocity， elevated levels of fasting blood glucose， IL-1β， TNF-α， and IL-6， and up-regulated protein levels of GFAP and p-ERK， and mRNA levels of ERK1， ERK2， GFAP （P<0.01）. Compared with model group， YHTP increased the MWT， TWL， and sciatic nerve conduction velocity （P<0.01）， lowered the levels of IL-1β， TNF-α， and IL-6 （P<0.01）， and down-regulated the protein levels of GFAP and p-ERK， and mRNA levels of ERK1， ERK2， GFAP in the spinal cord （P<0.05， P<0.01）. The cell experiments showed that compared with the blank group， the model group had decreased survival rate， elevated levels of pro-inflammatory factors， and up-regulated protein levels of ERK and GFAP （P<0.01）. Compared with the model group， the YHTP-containing serum lowered the levels of IL-1β and TNF-α （P<0.05， P<0.01）， elevated the level of IL-10 （P<0.01）， and down-regulated the protein levels of ERK and GFAP （P<0.01）. ConclusionYHTP may inhibit the activation of astrocytes by inhibiting the ERK signaling pathway to reduce inflammation and thus relieve DNP.

Protective role of vasonatrin peptide in hepatic ischemia-reperfusion injury in rats through activation of extracellular signal-regulated kinase signaling pathway / 中华肝胆外科杂志

Objective:To investigate the protective role of extracellular signal-regulated kinase (ERK) signaling pathway in the process that vasonatrin peptide (VNP) reduces hepatic ischemia-reperfusion injury in rats.Methods:Twenty SD rats, weighting 200-250 g, were randomly divided into four groups and each group has five rats. The four groups were sham operation group (S group), ischemia-reperfusion group (I/R group), VNP group (V group) and PD98059+ VNP group (P+ V group). In the rat model of hepatic warm ischemia and reperfusion, the hepatic artery and portal vein of the left lobe and middle lobe of the liver were clamped with arterial clamp for 45 min followed by reperfusion for 120 min. In the V group, VNP (50 μg/kg) was injected 10 minutes before ischemia. In the P+ V group, PD98059 (2 mg/kg) was injected 20 min before VNP injection followed by VNP administration and I/R treatment. The serum levels of alanine amino transaminase (ALT), aspartate amino transferase (AST), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and the superoxide dismutase (SOD) in liver tissue homogenate and malondialdehyde (MDA) were measured. The histopathology of liver tissue was observed. The contents of p-ERK1/2 were detected by Western blot.Results:Compared with S group, in I/R group and P+ V group the serum levels of ALT [(489.65±11.22), (333.05±24.77) vs. (33.78±4.88) U/L], AST [(651.43±14.99), (503.18±21.48) vs. (154.84±12.32) U/L], TNF-α [(12.83±1.09), (9.64±0.57) vs. (2.11±0.11) ng/L], IL-1β [(7.19±0.62), (5.12±0.22) vs. (1.10±0.49) ng/L], MDA [(8.00±0.88), (5.60±1.01) vs. (2.76±1.29) μmol/mg] increased, while SOD [(54.89±10.60), (68.85±8.33) vs. (126.10±15.63) nmol/mg]decreased (all P<0.05). The histopathology of liver tissue revealed that liver structure damaged more seriously in I/R group and P+ V group. Western blot analysis showed that p-ERK1/2 decreased significantly in I/R group and P+ V group. Compared with I/R group, ALT, AST, MDA, TNF-α and IL-1β decreased significantly and SOD increased significantly in V group (all P<0.05). The histopathology of liver tissue revealed that liver structure was damaged slightly, and p-ERK1/2 increased significantly in V group compared with I/R group ( P<0.05). Conclusion:VNP can significantly reduce hepatic ischemia-reperfusion injury through activation of p-ERK1/2 signaling pathway and inhibition of hepatocyte inflammatory response.

Chinese Medicines Regulate p38 MAPK， ERK1/2， and JNK in Treatment of Osteoporosis： A Review / 中国实验方剂学杂志

With the aging of population， osteoporosis has become one of the main diseases endangering the health of the elderly in China. Therefore， the research on osteoporosis has become a hot spot. Since Chinese medicines demonstrate significant therapeutic effects on osteoporosis， this issue is attracting increasing attention from researchers， especially in the deciphering of the molecular mechanism. This paper introduces the mechanism of the prevention and treatment of osteoporosis by Chinese medicines via the mitogen-activated protein kinase （MAPK） signaling pathway， aiming to provide a theoretical basis for deciphering the mechanism of Chinese medicines in the treatment of osteoporosis and promoting their clinical application. MAPK signaling pathway mainly involves p38 MAPK， extracellular signal-regulated kinase 1/2 （ERK1/2）， c-Jun N-terminal kinase （JNK）， and extracellular signal-regulated kinase 5 （ERK5）. Studies have shown that these proteins play a role in the progression of osteoporosis by regulating cell proliferation， differentiation， and apoptosis. Chinese medicines as a unique therapy with Chinese characteristics has definite efficacy， high safety， and mild side effects. Researchers have proved by experiments that the extracts or compounds of Chinese medicines can significantly mitigate osteoporosis by regulating the proteins involved in the MAPK signaling pathway. Therefore， this article reviews the relevant studies with focus on these proteins.

Effect of Yanghetang-containing Serum on Breast Cancer 4T1 Cells Based on MEK/ERK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Yanghetang （YHT） on breast cancer 4T1 cells and their mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodThe YHT-containing serum was prepared from SD rats. The rats were randomly assigned into a blank group （normal saline） and low-， medium-， and high-dose （5.8， 11.6， 23.2 g·kg-1， respectively） YHT groups. The serum containing 10% YHT in each group was mixed with 90% RMPI 1640 complete medium， and the mixture was used to interfere with the cells. Cell counting kit-8 （CCK-8） method was used to detect the proliferation of the 4T1 cells treated with YHT for 24， 48， 72 h. The apoptosis， migration， and invasion of 4T1 cells were detected by flow cytometry， scratch test， and Transwell assay， respectively. Western blot was employed to determine the expression levels of MEK1/2， phosphorylation （p）-MEK1/2， ERK1/2， p-ERK1/2， and rat sarcoma virus （RAS） protein. ResultCompared with the blank group， the intervention with YHT-containing serum for 24， 48， and 72 h had significant inhibitory effect on 4T1 cell proliferation （P<0.05， P<0.01）. After intervention with YHT-containing serum for 48 h， the apoptosis rate of cells increased （P<0.01）. Compared with the blank group， the intervention with YHT for 24 h and 48 h decreased the healing ability of cells in the scratch test （P<0.01）. The invasive ability of cells treated with the low， medium， and high-dose YHT containing serum showed a decreasing trend （P<0.01）. Compared with the blank group， YHT-containing serum did not change the expression of MEK1/2 and ERK1/2 while down-regulating the expression of p-MEK1/2， p-ERK1/2， and RAS protein （P<0.01）. ConclusionYHT can inhibit the proliferation， migration， and invasion and promote the apoptosis of breast cancer 4T1 cells. In may promote the apoptosis by inhibiting the MEK/ERK signaling pathway and down-regulating the expression of p-MEK1/2， p-ERK1/2， and RAS protein.

Study on the anti-pulmonary fibrosis effect of linarin in vivo and in vitro and its mechanism / 中国药房

OBJECTIVE To investigate the anti-pulmonary fibrosis effect of linarin in vivo and in vitro， and investigate its mechanism preliminarily. METHODS C57BL/6J mice were randomly divided into normal group （carboxymethylcellulose sodium）， model group （carboxymethylcellulose sodium）， positive control group （pirfenidone， 200 mg/kg）， linarin low-dose and high-dose groups （12.5， 25 mg/kg）， with 8 mice in each group. Except for normal group， pulmonary fibrosis model was induced in other groups. After modeling， they were given relevant medicine intragastrically， once a day， for consecutive 14 d. The general situation of mice was observed， and their lung indexes were measured； the levels of tumor necrosis factor-α （TNF-α） and transforming growth factor-β1（ TGF-β1） in serum and interleukin-6 （IL-6） in lung tissue were determined. Hematoxylin-eosin （HE） staining and Masson staining were used to observe the histopathological morphology of lung. The pulmonary fibrosis was scored according to Ashcroft score standard. The expressions of α-smooth muscle actin （α-SMA） and （type Ⅰ collagen， Collagen Ⅰ）， phosphorylated extracellular signal-regulated kinase （p-ERK1/2） and TGF-β1 in lung tissues were detected. HFL1 cells were stimulated by TGF- β1 to form pulmonary fibrosis model in vitro， which were divided into normal group， model group and linarin low-， medium- and high-concentration groups （3.7， 7.4， 14.8 mg/L）. After being cultured for 48 h， the protein expressions of α-SMA， Collagen Ⅰ and p-ERK1/2 in HFL1 cells were detected. RESULTS In vivo， compared with normal group， the lung index of model group and the levels of TNF- α， TGF- β1 and IL-6 were significantly increased （P＜0.01）. There were a large number of inflammatory infiltration and cellular fibrosis lesions in the alveoli， and a large number of collagen depositions. The scores of HE staining and Masson staining were significantly increased （P＜0.01）. The protein expressions of α-SMA， Collagen Ⅰ， p-ERK1/2 and TGF-β1 in lung tissue were up-regulated significantly （P＜0.01）. Compared with model group， above indexes of mice were improved significantly in linarin high-dose group （P＜0.05 or P＜0.01）， and most of indexes （except for lung index） were improved significantly in linarin low-dose group （P＜0.05 or P＜0.01）. In vitro， compared with blank group， the density of cells in the model group increased， and obvious proliferation and other changes occurred； protein expressions of α-SMA， Collagen Ⅰ and p-ERK1/2 were significantly up-regulated （P＜0.05 or P＜0.01）. Compared with model group， the cell density of each concentration group was decreased and the morphology gradually returned to normal； the expressions of above proteins in linarin high-concentration group and the protein expression of p-ERK1/2 in linarin medium-concentration group were down-regulated significantly（P＜0.05 or P＜0.01）. CONCLUSIONS Linarin may regulate ERK and inflammatory pathways to reduce the inflammatory response， thereby exerting anti-pulmonary fibrosis effect.

Extracellular Signal-regulated Kinase 1/2 Signaling Regulates Cell Invasion:a Review / 中国医学科学院学报

Extracellular signal-regulated kinase 1/2 (ERK1/2) is a serine/threoninekinase involved in the signal transduction cascade of Ras-Raf-mitogen-activated protein kinase (MEK)-ERK.It participates in the cell growth,proliferation and even invasion by regulating gene transcription and expression.The occurrence of a variety of diseases such as lung cancer,liver cancer,ovarian cancer,cervical cancer,endometriosis,and preeclampsia,as well the metastasis and disease progression,is closely associated with the regulation of cell invasion by ERK1/2 signaling pathway.Therefore,exploring the regulation of ERK1/2 signaling on cell invasion and its role in pathogenesis of diseases may help to develop more effective treatment schemes.This article introduces recent progress in the regulation of ERK1/2 signaling on cell invasion and the role of such regulation in diseases,with a view to give new insights into the clinical treatment of ERK 1/2-related diseases.

Hippocampus is involved in 17β-estradiol exacerbating experimental occlusal inter- ference-induced chronic masseter hyperalgesia in ovariectomized rats / 北京大学学报(医学版)

OBJECTIVE@#To investigate the influence of chronic masseter hyperalgesia induced by 17β-estradiol (E2) and experimental occlusal interference (EOI) on underlying mechanism in hippocampus of ovariectomized (OVX) rats.@*METHODS@#In the study, 32 OVX rats were randomly divided into 4 groups (8 rats/group): The control group was OVX group, and 0 μg/d E2 (vehicle) injection was started 7 d after OVX without EOI; in the experimental group (1) OVX + E2 group, 80 μg/d E2 injection was started 7 d after OVX without EOI; in the experimental group (2) OVX + EOI group, vehicle injection was started 7 d after OVX and EOI was applied 17 d after OVX; in the experimental group (3) OVX + E2 + EOI group, 80 μg/d E2 injection was started 7 d after OVX and EOI was applied 17 d after OVX. Bilateral masseter muscle mechanical withdrawal thresholds were measured before OVX, 7 days after OVX (before E2 injection), 17 days after OVX (10 days after E2 injection and before EOI) and 24 days after OVX (7 days after EOI). Immunofluorescence staining was used to reveal phospho-extracellular signal regulated kinase 1/2 (p-ERK1/2)-positive neurons in CA3 of hippocampus. The protein expression of p-ERK1/2 in hippocampus was detected using Western Blot.@*RESULTS@#Compared with the control group [left side: (135.3±8.5) g, right side: (135.4±10.8) g], bilateral masseter muscle mechanical withdrawal thresholds of OVX+E2 group [left side: (113.3±5.6) g, right side: (112.5 ± 5.6) g] and OVX+EOI group [left side: (93.3±5.4) g, right side: 90.8±5.5) g] were decreased (P < 0.01). Bilateral masseter muscle mechanical withdrawal thresholds were significantly lower in OVX+E2+EOI group [left side: (81.2±6.2) g, right side: 79.8±7.7) g] than in the control, OVX+E2 and OVX+EOI groups (P < 0.05). The proportion of p-ERK1/2 positive neurons in the CA3 region of the hippocampus was increased in the control, OVX+E2, OVX+EOI and OVX+E2+EOI groups in turn, and the difference between the groups was statistically significant (P < 0.05). p-ERK1/2 protein expression was increased in the control, OVX+E2 and OVX+EOI groups in turn, but the difference was not statistically significant (P>0.05). p-ERK1/2 expression was significantly higher in OVX+E2+EOI group than in the other three groups (P < 0.05).@*CONCLUSION@#High concentration of E2 could exacerbated EOI-induced chronic masseter hyperalgesia in ovariectomized rats, and its central mechanism may be related to the upregulation of the phosphorylation of ERK1/2 in hippocampus.

Effect of extracellular signal-regulated kinase inhibitor on calpain in rat cerebral cortex after cardiopulmonary resuscitation / 中华危重病急救医学

Objective:To explore the effect of extracellular signal-regulated kinase (ERK) inhibitor PD98059 on calpain-related proteins in the brain, and to understand the pathophysiological changes of calpain in cerebral ischemia/reperfusion injury (CIRI).Methods:Forty-two rats were divided into sham operation (Sham) group ( n = 6), model group ( n = 12), dimethyl sulfoxide (DMSO) control group ( n = 12), and PD98059 group ( n = 12) by random number table. The rat model of CIRI induced by cardiac arrest-cardiopulmonary resuscitation (CA-CPR) was reproduced by transesophageal electrical stimulation to induce ventricular fibrillation. In the Sham group, only the basic operations such as anesthesia, tracheal intubation, and arteriovenous catheterization were performed without CA-CPR. The rats in the DMSO control group and PD98059 group were injected with DMSO or PD98059 0.30 mg/kg via femoral vein, respectively, 30 minutes after the restoration of spontaneous circulation (ROSC), and rats in the Sham group and model group were given the same amount of normal saline. The duration of CPR, 24-hour survival rate and neurological deficit score (NDS) after ROSC were recorded. Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological changes of the cerebral cortex. The expressions of phosphorylated ERK (p-ERK), ERK, calpastatin, calpain-1, and calpain-2 were detected by Western blotting. The co-expression of p-ERK and calpain-2 was detected by double immunofluorescence. Results:There were no significant differences in the duration of CPR and 24-hour survival rate among all groups. In the model group, the nuclei of the cerebral cortex were obviously deformed and pyknotic, cells vacuoles and tissues were arranged disorderly, Nissl corpuscles were significantly reduced, NDS scores were also significantly reduced, level of ERK phosphorylation was increased, and calpain-2 protein was significantly up-regulated compared with the Sham group. There was no significant difference in the above parameters between the DMSO control group and the model group. After intervention with PD98059, the pathological injury of brain tissue was significantly improved, Nissl corpuscles were significantly increased, the NDS score was significantly higher than that in the model group [75.0 (72.0, 78.0) vs. 70.0 (65.0, 72.0), P < 0.05], the level of ERK phosphorylation and calpain-2 protein expression were significantly lower than those in the model group [p-ERK (p-ERK/ERK): 0.65±0.12 vs. 0.92±0.05, calpain-2 protein (calpain-2/GAPDH): 0.73±0.10 vs. 1.07±0.14, both P < 0.05], while there was no significant difference in the expressions of calpastatin and calpain-1 in the cerebral cortex among all the groups. Double immunofluorescence staining showed that p-ERK and calpain-2 were co-expressed in cytosol and nucleus, and the co-expression rate of p-ERK and calpain-2 in the model group was significantly higher than that in the Sham group [(38.6±4.3)% vs. (9.2±3.5)%, P < 0.05], while it was significantly lowered in the PD98059 group compared with the model group [(18.2±7.0)% vs. (38.6±4.3)%, P < 0.05]. Conclusions:ERK together with calpain-2 participated in CIRI induced by CA-CPR. PD98059 inhibited the expression of calpain-2 and ERK phosphorylation. Therefore, ERK/calpain-2 may be a novel therapeutic target for CIRI.

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.

Repair effect and mechanism of neural cell adhesion molecule derived peptide P2 on nerve function injury in ischemic stroke rats / 中华行为医学与脑科学杂志

Objective:To clarify the neuroprotective effects of neural cell adhesion molecule (NCAM) derived peptide P2 on in vitro cultured neuron and ischemic stroke rat. Methods:Primary cortical neurons were extracted and cultured, and CCK-8 method was used to observe the protective effect of different concentrations of P2 on cortical neurons under oxygen-glucose deprivation (OGD) conditions.The levels of apoptosis-related proteins and extracellular signal regulated kinase 1/2 (Erk1/2) were observed by Western blot. Clean grade male SD rats were selected for animal experiments. The middle cerebral artery occlusion (MCAO) method was used to establish the rat model of cerebral ischemia/reperfusion injury. The rats with successful model were divided into sham operation group, MCAO group and MCAO+ P2 group according to the random number table, with 12 rats in each group. After operation, rats in MCAO+ P2 group were subcutaneously injected with 1 mg/kg P2 once a day until 14 days after operation, and rats in the other two groups were subcutaneously injected with 0.9% sodium chloride solution of the same volume.Beam-walking test was used to evaluate the motor function of rats.Immunofluorescence staining and Western blot were used to detect the in-situ apoptosis of neuronal cells and the expression of Erk1/2 in ischemic penumbra of rat brains, respectively. All statistical analyses were performed using SPSS 22.0.Repeated measurement ANOVA was used to evaluate the beam-walking experimental data, and one-way ANOVA were used to analyze other experimental data among multiple groups.Results:Compared with OGD group, 0.5, 1.0 and 2.0 μmol/L P2 improved the activity of neurons under OGD conditions, of which 1 μmol/L P2 had the best effect ((2.436±0.284), (1.551±0.410), P<0.05). Western blot showed that the protein levels of bax ((76.120±3.232)%, (88.965±5.208)%, P<0.05), cleaved caspase-3 ((76.736±4.306)%, (97.781±8.111)%, P<0.05) and cleaved caspase-9 ((88.833±6.581)%, (104.962±4.788)%, P<0.05) in 1 μmol/L P2 treated group were all lower than those in OGD group, while the protein levels of bcl-2 ((56.146±3.882)%, (43.170±6.945)%, P<0.05) and phosphorylated Erk1/2 ((73.583±8.557)%, (55. 219±4.615)%, P<0.05) in 1 μmol/L P2 treated group were both higher than those in OGD group. Compared with MCAO group, on the 14th day after P2 intervention, the slip ratio of hindlimb of the paralyzed hind limbs of rats was lower ((23.438±11.540)%, (41.733±13.631)%, P<0.05), the apoptosis rate of neurons around the focus was lower ((13.144±6.485)%, (26. 699±6. 402)%, P<0.05), and the level of phosphorylated Erk1/2 protein in the brain tissues around the infarct focus was higher ((74.062±7.458)%, (53.327±7.093)%, P<0.05). Conclusion:Low doses of neural cell adhesion molecule derived peptide P2 exert neuroprotective effects on OGD neurons and ischemic stroke rats. The underlying mechanism may be related to the activation of Erk.

Effect of Shufeng Tongluo Prescription on Eotaxin and CCR3 Protein Expression and ERK Phosphorylation Level of Asthma Mice / 中国实验方剂学杂志

ObjectiveTo preliminarily explore the mechanism of Shufeng Tongluo prescription （SFTLP） in inhibiting airway inflammation in asthma mice by affecting the expression levels of eotaxin in the serum， CC type chemokine receptor 3 （CCR3）， and extracellular signal-regulated kinase （ERK） phosphorylation in lung tissues. MethodSeventy C57BL/6 mice were randomly divided into a blank group， a model group， low-， medium-， and high-dose SFTLP groups （7.75， 15.5， 30 g·kg-1）， a pertussis toxin （PTX） group， a CCR3 inhibitor （SB328437） group， a phosphoinositide 3-kinase （PI3K） inhibitor （LY294002） group， a p38 protein kinase antagonist inhibitor （SB203580） group， and an ERK inhibitor （PD98059） group. The asthma model was induced in mice by intraperitoneal injection of ovalbumin （OVA） and aluminum hydroxide ［Al（OH）3］ combined with OVA atomization （0.2 mL for all）. After modeling， hematoxylin-eosin staining （HE staining） was used to observe the inflammatory infiltration of lung tissues in mice. Enzyme-linked immunosorbent assay （ELISA） was used to detect the serum levels of eotaxin ［CC chemokine ligand （CCL） 11 and CCL24） in each group. Western blot was used to detect the levels of ERK phosphorylation and CCR3 in lung tissues. ResultCompared with the blank group， the model group showed obvious bronchial constriction， lumen stenosis， damaged alveolar structure， massive inflammatory cell infiltration in lung tissues， mucous plug in the bronchus， edema in the submucosal tissues of the trachea， increased folds， increased serum levels of CCL11 and CCL24 （P<0.01）， and increased expression of CCR3 protein in lung tissues （P<0.05）. The ERK levels in lung tissues of the model group and the PTX group increased （P<0.05）. The level of p-ERK in lung tissues of the model group and the low-dose SFTLP group increased （P<0.05）. As revealed by pathological results， compared with the model group， the high-dose SFTLP group showed relieved lung lesions. The high-dose SFTLP group and the SB328437 group showed reduced CCL11 content （P<0.05）. The low- and high-dose SFTLP group， the PTX group， the SB203580 group， the PD98059 group， and the SB328437 group showed decreased CCR3 protein expression in lung tissues （P<0.05）. The high-dose SFTLP group and the PD98059 group showed reduced p-ERK level （P<0.05）. The PD98059 group showed reduced ERK level （P<0.05）. ConclusionSFTLP can inhibit airway inflammation in asthma， and the mechanism may be related to the inhibition of eosinophil activation by down-regulating CCR3 and CCL11 expression and ERK phosphorylation.

Changes in circadian gene cryptochrome 2 expression in mouse models of psoriasis and HaCaT cells and their underlying mechanisms / 中华皮肤科杂志

Objective:To investigate changes in circadian gene cryptochrome 2 (CRY2) expression in mouse models of psoriasis and HaCaT cells, and to explore underlying mechanisms.Methods:Imiquimod-induced mouse model experiment: 12 C57BL/6 female mice were randomly and equally divided into imiquimod group receiving topical imiquimod treatment for 5 consecutive days and control group receiving no treatment; these mice were sacrificed on day 6, skin tissues were resected from the back of mice, and immunofluorescence staining was performed to determine the CRY2 expression in the epidermis. HaCaT cell transfection experiment: HaCaT cells with small interfering RNA (siRNA) -mediated knockdown of CRY2 served as siRNA-CRY2 group, and siRNA-NC group as control group; 5-ethynyl-2′-deoxyuridine (EdU) staining was performed to evaluate the proliferative activity of the HaCaT cells, real-time fluorescence-based quantitative PCR (qPCR) to determine the mRNA expression of chemokines in the HaCaT cells, and Western blot analysis to determine phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2) . Tumor necrosis factor-α (TNF-α) -stimulated animal and cell experiments: 12 C57BL/6 female mice were randomly and equally divided into TNF-α group subcutaneously injected with TNF-α solution in the ear for 6 days, and phosphate buffered saline (PBS) group subcutaneously injected with the same amount of PBS; the mice were sacrificed on day 7, skin tissues were resected from the ear of mice, and immunofluorescence staining was conducted to determine the CRY2 expression in the epidermis; CRY2-knockdown HaCaT cells stimulated with 50 ng/ml TNF-α for 12 hours served as siRNA-CRY2 + TNF-α group, and siRNA-NC + TNF-α group as control group; qPCR was performed to determine the mRNA expression of chemokines in HaCaT cells in the above groups. Statistical analysis was carried out by using two-independent-sample t test. Results:Immunofluorescence staining showed that the CRY2 protein expression was significantly lower in the mouse dorsal epidermis in the imiquimod group (0.94 ± 0.23) than in the control group (2.30 ± 0.25, t = 3.99, P = 0.016) . Compared with the siRNA-NC group, the siRNA-CRY2 group showed significantly increased proportions of EdU-positive cells (48.13% ± 10.97% vs. 38.23% ± 0.81%, t = 5.00, P = 0.007) , mRNA expression levels of chemokines CXCL1 and CXCL8, as well as significantly increased phosphorylated (p) -ERK1/2 protein expression levels (all P < 0.05) , while there were no significant differences in the CCL20 mRNA expression or ERK1/2 protein expression between the two groups (both P > 0.05) . Immunofluorescence staining showed significantly decreased CRY2 protein expression level in the mouse ear epidermis in the TNF-α group (0.37 ± 0.34) compared with the PBS group (2.04 ± 0.17, t = 4.38, P = 0.012) ; the relative mRNA expression levels of chemokines CXCL1, CXCL8, and CCL20 in HaCaT cells were significantly higher in the siRNA-CRY2 + TNF-α group than in the siRNA-NC + TNF-α group (all P < 0.05) . Conclusion:CRY2 was markedly underexpressed in psoriasis, which might promote the proliferation of keratinocytes and expression of chemokines CXCL1, CXCL8 and CCL20, and TNF-α might be an upstream cytokine that could downregulate CRY2 expression.

Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling / 中国实验方剂学杂志

Objective:To investigate the effect of notoginseng total saponins （TNS） on adriamycin （Adr） resistance in HepG2/Adr cells and the expression and activity of the mechanisms as the modulators of multi-drug resistance， so as to explore the possible mechanism of extracellular signal-regulated kinase （ERK） and protein kinase B （Akt） signaling pathways in reversing the resistance of HepG2/Adr cells mechanism. Method:Effect of TNS on HepG2/Adr cell proliferation was detected by thiazole blue （MTT） method. HepG2/Adr cells were treated with different concentrations （100， 50， 25， 0 mg·L^-1） of TNS and （20 μmol·L^-1） Adr respectively， and a blank group was set. The high-content screening platform was used to detect the accumulation of Adr in HepG2/Adr cells after 40 minutes， 3 hours and 6 hours. Western blot was used to detect the expression of P-glycoprotein /multidrug resistance/ATP binding cassette subfamily B member 1（P-gp/MDR1/ABCB1） and other drug resistance-related proteins and the main protein expression of ERK/Akt signaling pathway. The change of MDR1 on cell membranes was observed by laser confocal microscopy. Result:Compared with HepG2 cells， the expression of MDR1 in HepG2/Adr cells was significantly increased （<italic>P</italic><0.01）. Compared with the Adr group， the half-inhibitory concentration （IC₅₀） of TNS （25， 50， 100 mg·L^-1） and Adr （20 μmol·L^-1） co-administration group on HepG2/Adr cells <italic>in vitro</italic> significantly reduced （<italic>P</italic><0.01）， and the highest reversal multiple was 10 times. Compared with the Adr group， the co-administration group could significantly increase the accumulation of Adr in the cells （<italic>P</italic><0.05） in a dose-dependent manner. Compared with the blank group， the co-administration group could significantly reduce MDR1， ABC semitransporter （ABCG2）， multidrug resistance associated protein （MRP1）， ERK， phosphorylated extracellular regulatory protein kinase （p-ERK）， Akt， phosphorylated protein kinase B （p-Akt）， mammals， rapamycin target protein （mTOR） and phosphorylated mammalian rapamycin target protein （p-mTOR） （<italic>P</italic><0.05）， with the same results in the doxorubicin group. Compared with the blank group， there was no significant difference in the distribution and fluorescence intensity of MDR1 on the cell membrane between the Adr group and the notoginseng total saponins （25 mg·L^-1） group. Compared with the blank group and the doxorubicin group， TNS could significantly reduce the distribution of MDR1 on the cell membrane （<italic>P</italic><0.05）. Conclusion:TNS can inhibit the ERK/Akt pathway， reduce the expression of MDR1， and significantly increase the accumulation of doxorubicin in HepG2/Adr cells， which may be one of the mechanisms of notoginseng total saponins in reversing resistance.

Shikonin Affects Uterine Leiomyoma in Rats by Regulating ERK Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the effect of shikonin on uterine leiomyoma in rats and its molecular mechanism. Method:Sixty female SD rats， of SPF grade and weighing 200-250 g， were randomly divided into the control group， model group， low-， medium-， and high-dose （5， 10， 20 mg·kg^-1） shikonin groups， and mifepristone group. A rat model of uterine leiomyoma was established， and the changes in uterine wet weight， uterine coefficient， and smooth muscle thickness were detected after drug administration for four successive weeks. The pathological changes in uterine tissue were observed by hematoxylin-eosin （HE） staining. The contents of estrogen receptor （ER） and progesterone receptor （PR） in serum and uterus were measured by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of ER， PR， phosphorylated extracellular signal-regulated kinase （p-ERK）， and ERK in the uterine tissue were assayed by Western blot. Result:Compared with the control group， the model group exhibited significantly increased uterine wet weight， uterine coefficient， and smooth muscle thickness （<italic>P</italic><0.01）， uterus deformity， focal smooth muscle cell necrosis and hyperplasia， neutrophil infiltration. elevated serum and uterine ER and PR （<italic>P</italic><0.01）， and up-regulated p-ERK protein expression in the uterine tissue （<italic>P</italic><0.01）. Compared with the model group， shikonin at the middle and high doses and mifepristone significantly reduced the uterine wet weight， uterine coefficient， and smooth muscle thickness （<italic>P</italic><0.01）， relieved the pathological changes in uterus，and lowered serum and uterine ER and PR， and down-regulated the p-ERK protein expression in the uterine tissue （<italic>P</italic><0.05）. In addition， the uterine wet weight， smooth muscle thickness， serum ER， and uterine PR and p-ERK protein expression in the low-dose shikonin group were significantly lower than those in the model group （<italic>P</italic><0.05）. Conclusion:Shikonin produces the anti-uterine leiomyoma activity possibly by inhibiting the activation of ERK pathway.

Mechanism of Xiaozheng Huoluo prescription in preventing liver fibrosis in rats via the ERK5 pathway: An analysis based on network pharmacology / 临床肝胆病杂志

ObjectiveTo investigate the mechanism of Xiaozheng Huoluo prescription (XZHLF) in the prevention and treatment of liver fibrosis based on network pharmacology. MethodsTCMSP database, Chemistry Database, ETCM database, chemical source network database, ChemSrc database, and PubChem database were searched and a literature review was performed to collect the chemical components of each traditional Chinese medicine of XZHLF, Swiss ADME database was used to screen out the active components of each traditional Chinese medicine of XZHLF, and Swiss Target Prediction database was used to predict the targets of these active components; GeneCards and OMIN databases were used to collect the disease targets of liver fibrosis, and Venn diagrams were used to obtain the potential targets of XZHLF in the prevention and treatment of liver fibrosis. Cytoscape 3.7.1 software was used to establish a “drug-active component” network for XZHLF and an “active component-potential target” network for XZHLF in the prevention and treatment of liver fibrosis. Metascape database was used to perform GO and KEGG enrichment analysis of potential targets, and bubble charts were plotted for the top 20 pathways with the highest number of enriched genes. The MAPK signaling pathway among the top 20 KEGG pathways was analyzed to plot an “active component-potential target-pathway” network. Healthy Sprague-Dawley rats were randomly divided into blank control group (K group), model group (M group), colchicine-positive control group (Y group), high-dose XZHLF group (G group), middle-dose XZHLF group (Z Group), and low-dose XZHLF group (D group), and a rat model of liver fibrosis was established by CCl4; the drug was administered simultaneously for 8 weeks. Western blot was used to measure the protein expression of ERK5, p-ERK5, MEK5, and MEKK3 in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsA total of 110 active components were screened out from XZHLF and they acted on 923 targets, which were mapped to 6823 disease targets of liver fibrosis to obtain 647 potential targets. XZHLF might act on multiple protein targets such as EGFR, AKT1, IKBKB, MAPK8, and PDGFRB through the pathways including the MAPK signaling pathway and the biological processes including the regulation of MAPK cascade, thereby playing a role in the prevention and treatment of liver fibrosis. The M group had significant increases in the protein levels of ERK5, p-ERK5, MEK5, and MEKK3 in liver tissue compared with the K, Y, G, Z, and D groups (all P ＜0.05), and the K group had significant reductions in the protein levels of ERK5, p-ERK5, MEK5, and MEKK3 in liver tissue compared with the Y, G, Z, and D groups (all P ＜0.05). ConclusionBased on the method of network pharmacology, it is predicted that XZHLF may prevent and treat liver fibrosis via the MAPK signaling pathway, and it is verified through experiments that XZHLF prevents and treats liver fibrosis via the ERK5 pathway in the MAPK signaling pathway family. The high-dose XZHLF group shows the most obvious anti-liver fibrosis effect.

Protective effect of adrenomedullin on hyperoxia-induced lung injury / 中国当代儿科杂志

OBJECTIVES@#To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions.@*METHODS@#HPMECs were randomly divided into an air group and a hyperoxia group (@*RESULTS@#Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB (@*CONCLUSIONS@#ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.

Matrine inhibits proliferation and migration of HepG2 cells by downregulating ERK1/2 signaling pathways

Objective: To research the effect of matrine on the proliferation and migration of HepG2 cells through extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Methods: HepG2 cell was selected and divided into blank control group, experimental group (matrine 1, 2, and 4 mg/mL), and positive control group (PD98059, ERK1/2 inhibitor). MTT measure was used to detect the effective time and concentration which matrine inhibits HepG2 cells. After 24 h, the effect of effective concentration of matrine on the of morphological changing HepG2 cells was observed. The invasion ability was assayed by transwell method, the expression of ERK1/2 and pERK1/2 were detected through Western blot, and reverse transcription polymerase chain reaction was used to test the expression level of ERK1/2 mRNA. Results: With the increase of matrine concentration, the number of adherent HepG2 cells gradually decreased, the morphologic changes gradually became spherical, some cell morphology was incomplete, and even cell fragments appeared. The proliferation and invasion ability of HepG2 cells decreased. The expression of ERK1/2, pERK1/2, and ERK1/2 mRNA downregulated with the increase of matrine concentration (P < 0.05). Conclusion: Matrine inhibits the proliferation and migration of HepG2 cells by downregulating the ERK1/2 signaling pathway.

Total Panax notoginseng saponin inhibits balloon injury-induced neointimal hyperplasia in rat carotid artery models by suppressing pERK/p38 MAPK pathways

Total Panax notoginseng saponin (TPNS) is the main bioactivity compound derived from the roots and rhizomes of Panax notoginseng (Burk.) F.H. Chen. The aim of this study was to investigate the effectiveness of TPNS in treating vascular neointimal hyperplasia in rats and its mechanisms. Male Sprague-Dawley rats were randomly divided into five groups, sham (control), injury, and low, medium, and high dose TPNS (5, 10, and 20 mg/kg). An in vivo 2F Fogarty balloon-induced carotid artery injury model was established in rats. TPNS significantly and dose-dependently reduced balloon injury-induced neointimal area (NIA) (P<0.001, for all doses) and NIA/media area (MA) (P<0.030, for all doses) in the carotid artery of rats, and PCNA expression (P<0.001, all). The mRNA expression of smooth muscle (SM) α-actin was significantly increased in all TPNS groups (P<0.005, for all doses) and the protein expression was significantly increased in the medium (P=0.006) and high dose TPNS (P=0.002) groups compared to the injury group. All the TPNS doses significantly decreased the mRNA expression of c-fos (P<0.001). The medium and high dose TPNS groups significantly suppressed the upregulation of pERK1/2 protein in the NIA (P<0.025) and MA (P<0.004). TPNS dose-dependently inhibited balloon injury-induced activation of pERK/p38MAPK signaling in the carotid artery. TPNS could be a promising agent in inhibiting cell proliferation following vascular injuries.

Combination of Quercetin, Hirudin and Cinnamaldehyde Promotes Schwann Cell Differentiation and Myelination against High Glucose by Inhibiting ERK Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the therapeutic and synergistic effects of QHC (combination of quercetin (Q), hirudin (H) and cinnamaldehyd (C)) on Schwann cell differentiation and myelination against high glucose (HG) induced injury.@*METHODS@#Primary-culture Schwann cells exposed to HG (50 mmol/L) for 72 h and Schwann cell-dorsal root ganglion (DRG) neuron cocultures exposed to HG (50 mmol/L) for 7 days were employed as in vitro model of diabetic neuropathy. The cells were randomly divided into 10 groups: control (CON, 25 mmol/L glucose), HG (50 mmol/L glucose), HG plus 10 μmol/L quercetin (Q), HG plus 0.04 IU/mL hirudin (H), HG plus 100 nmol/L cinnamaldehyd (C), HG plus 10 μmol/L quercetin and 0.04 IU/mL hirudin (QH), HG plus 10 μmol/L quercetin and 50 nmol/L cinnamaldehyd (QC), HG plus 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (HC), HG plus 10 μmol/L quercetin, 0.04 IU/mL hirudin and 50 nmol/L cinnamaldehyd (QHC) or 10 μmol/L U0126. Cell differentiation was evaluated by periaxin immunofluorescence staining. The protein expression levels of myelin protein zero (P0), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), extracellular signal-regulated kinase (ERK), p-ERK, p-c-Jun, c-Jun, notch intracellular domain (NICD) and the mRNA expression levels of P0, MBP, MAG, Krox-20, Notch1 and Jagged1 were detected by Western blotting and real-time quantitative PCR analysis. The secretion of ciliary neurotrophic factor (CNTF) was determined by enzyme-linked immunosorbent assay (ELISA). The number and length of the myelin segments were evaluated by MBP immunofluorescence staining. The expression and the location of p-ERK in cocultures were detected by MAG and p-ERK immunofluorescence double staining.@*RESULTS@#Co-treatment with Q, C, H and their combination promoted Schwann cell differentiation, increased CNTF secretion, up-regulated the protein and mRNA expressions of myelin, and increased the number and length of the myelin segments (P<0.01 or P<0.05). In particular, the combination therapy of Q, H and C was superior to the respective monotherapy (P<0.01). Combination therapy of QHC exhibited higher inhibitory activities for ERK signaling related molecules than each monomer or the combination of the two monomers (P<0.01).@*CONCLUSION@#QHC combination yielded synergy in promoting Schwann cell differentiation and myelination and the protective effect may involve in the inhibition of ERK signaling pathway, providing scientific evidence for better understanding of combination of Q, H and C in clinical applications.

Value of extracellular signal-regulated kinase 1/2 and p-extracellular signal-regulated kinase 1/2 expression in predicting the efficacy of neoadjuvant chemotherapy in cervical squamous cell carcinoma / 中国医师进修杂志

Objective To explore the value of extracellular signal-regulated kinase (ERK)-1/2 and p-ERK-1/2 expression in predicting the efficacy of neoadjuvant chemotherapy in cervical squamous cell carcinoma.Methods Fifty-seven patients with cervical squamous cell carcinoma who received neoadjuvant chemotherapy from May 2016 to May 2018 in Bazhong Central Hospital of Sichuan Province were selected.The incidence of adverse reactions during treatment was analyzed.The expression of ERK 1/2 and p-ERK 1/2 in cervical squamous cell carcinoma tissues before and after treatment was compared,and the expression levels of ERK 1/2 and p-ERK 1/2 and the effectiveness of neoadjuvant chemotherapy were analyzed.Results After treatment,the effective rate was 68.42％ (39/57),and the incidence of adverse reactions was 24.56％ (14/57).Before treatment,the positive rate of ERK1/2 was 87.72％ (50/57),after treatment,the positive rate of ERK 1/2 was 31.58％ (18/57),and there were statistical differences (P＜ 0.01).Before treatment,the positive rate of p-ERK 1/2 was 75.44％ (43/57),after treatment,the positive rate of p-ERK 1/2 was 19.30％ (11/57),and there was statistical difference (P ＜ 0.01).The effective rate after treatment in pre-treatment ERK 1/2 positive group was significantly lower than that in pre-treatment ERK 1/2 negative group:53.12％ (17/32) vs.88.00％ (22/25),and there was statistical difference (P ＜ 0.05).The effective rate after treatment in pre-treatment p-ERK 1/2 positive group was significantly lower than that in pre-treatment p-ERK 1/2 negative group:60.47％ (26/43) vs.13/14,and there was statistical difference (P ＜ 0.05).Conclusions ERK 1/2 and p-ERK 1/2 in cervical squamous cell carcinoma can predict the efficacy of neoadjuvant chemotherapy.