TNFR1/p38αMAPK signaling in Nex + supraspinal neurons regulates estrogen-dependent chronic neuropathic pain.

Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP). Previously, we have shown that estrogen modulates sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP. The estrogen-dependent role of TNFR1-mediated supraspinal neuronal circuitry in CNP remains unknown. In this study, we interrogated the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that mechanical hypersensitivity induced by chronic constriction injury (CCI) decreases over time in males, but not in females. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38MAPK and NF-κB activation in male cortical tissue; however, p38MAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed a similar recovery from acute pain in male mice following CCI when p38αMAPK was knocked out of supraspinal Nex + neurons (NexCreERT2::p38αMAPKf/f), while chronic pain developed in female mice. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lends therapeutic relief to females following CCI comparable to the response evaluated in male mice. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.

TNFR1/p38αMAPK signaling in Nex+ supraspinal neurons regulates sex-specific chronic neuropathic pain.

Upregulation of soluble tumor necrosis factor (sTNF) cytokine signaling through TNF receptor 1 (TNFR1) and subsequent neuronal hyperexcitability are observed in both animal models and human chronic neuropathic pain (CNP) [1-4]. To test the hypothesis that supraspinal circuitry is critical to pain chronification, we studied the intersect between supraspinal TNFR1 mediated neuronal signaling and sex specificity by selectively removing TNFR1 in Nex + neurons in adult mice (NexCreERT2::TNFR1f/f). We determined that following chronic constriction injury (CCI), pain resolves in males; however, female acute pain transitions to chronic. Subsequently, we investigated two downstream pathways, p38MAPK and NF-κB, important in TNFR1 signaling and injury response. We detected p38αMAPK and NF-κB activation in male cortical tissue; however, p38αMAPK phosphorylation was reduced in NexCreERT2::TNFR1f/f males. We observed similar behavioral results following CCI in NexCreERT2::p38αMAPKf/f mice. Previously, we established estrogen's ability to modulate sTNF/TNFR1 signaling in CNP, which may contribute to female prevalence of CNP [5-9]. To explore the intersection between estrogen and inflammation in CNP we used a combination therapy of an estrogen receptor ß (ER ß) inhibitor with a sTNF/TNFR1 or general p38MAPK inhibitor. We determined both combination therapies lend "male-like" therapeutic relief to females following CCI. These data suggest that TNFR1/p38αMAPK signaling in Nex + neurons in CNP is male-specific and lack of therapeutic efficacy following sTNF inhibition in females is due to ER ß interference. These studies highlight sex-specific differences in pathways important to pain chronification and elucidate potential therapeutic strategies that would be effective in both sexes.

Role of p38 MAPK Pathway in Diabetic Peripheral Neuropathy and Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

Diabetic peripheral neuropathy （DPN） is a symptom and/or sign of peripheral nerve dysfunction that occurs in patients with diabetes mellitus when other causes are excluded. DPN， one of the most common complications of diabetes mellitus， can lead to disability， foot ulcers， and amputation at a later stage. Its pathogenesis is closely related to high glucose-induced inflammatory damage， oxidative stress， mitochondrial disorders， and apoptosis in neural tissues. The p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway is a key mechanism mediating the expression of inflammatory factors， oxidative factors， and apoptotic factors of neural tissues in DPN. The inflammatory response， oxidative stress damage， and apoptosis， induced by the activation of p38 MAPK phosphorylation by factors such as high glucose， can cause cell lipid peroxidation， protein modification， and nucleic acid damage， which results in axonal degeneration and demyelination changes. The current treatment of DPN with western medicine has obvious shortcomings such as adverse effects and addictive tendencies. In recent years， the research on traditional Chinese medicine （TCM） in the prevention and treatment of DPN has gradually increased， and the exploration of Chinese medicine intervention in the p38 MAPK pathway transduction to improve DPN has advanced. The present study reviewed the relations of the p38 MAPK pathway with insulin resistance and peripheral neuropathy and summarized the molecular biological mechanisms involved in the pathological process of DPN， such as inflammation regulation， oxidative stress， polyol pathway regulation， and Schwann cell apoptosis in the past 10 years. In addition， the literature on Chinese medicine monomers， Chinese patent medicines， and Chinese medicine compounds in inhibiting inflammatory reactions， oxidative injury， and apoptosis of DPN peripheral nerves based on the p38 MAPK pathway， resisting axonal degeneration and demyelination changes， improving sensory and motor abnormalities， relieving peripheral pain sensitization， and facilitating nerve conduction mechanism to provide references for the development of new drugs for clinical prevention and treatment of DPN.

Chinese Medicine Intervenes in Diabetic Kidney Disease Based on p38 MAPK Signaling Pathway：A Review / 中国实验方剂学杂志

Diabetic kidney disease （DKD）， a major microvascular complication of diabetes mellitus， serves as the most common cause of end-stage renal disease worldwide. The progression of DKD is closely related to oxidative stress， inflammatory response， apoptosis， and fibrosis in renal tissues activated by high glucose. Numerous studies have shown that the transduction of the p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway is involved in the pathological process of DKD in renal tissues， activating various pathological mechanisms， such as oxidation， inflammation， apoptosis， and fibrosis. Therefore， blocking the transduction of the p38 MAPK signaling pathway is beneficial to alleviating DKD. At present， the main treatment principles of western medicine are glucose lowering， lipid lowering， and blood pressure lowering， as well as medications with new drugs renal sodium-glucose co-transporter 2 （SGLT2）， mineralocorticoid receptor， and endothelin receptor， but the progression of DKD still cannot be stopped. The treatment of DKD by traditional Chinese medicine （TCM） has the advantages of simplicity， low cost， and convenience， and the symptoms and root causes can be both treated. In recent years， the basic research on Chinese medicine intervention in DKD has greatly advanced， and p38 MAPK is the key factor of Chinese medicine intervention in DKD. The present study searched and reviewed the literature on the Chinese medicine intervention in the p38 MAPK signaling pathway in DKD treatment in the past decade. The results showed that p38 MAPK interacted with transforming growth factor-β1 （TGF-β1）， cysteinyl aspartate-specific protease-3 （Caspase-3）， nuclear factor-κB （NF-κB）， and other factors to activate fibrosis， inflammation， oxidative stress， and apoptosis. By acting on p38 MAPK and its upstream and downstream factors， Chinese medicine blocked the pathological processes of DKD and inhibited the pathological injury of DKD and the deterioration of renal function. This study is expected to provide new ideas and directions for the prevention and treatment of DKD with Chinese medicine.

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.

[Effects of aerobic exercise and dietary intervention on testicular oxidative stress in obese mice].

OBJECTIVE: Through aerobic exercise and diet intervention on obese mice, the effects of exercise and diet intervention on testicular oxidative stress and p38MAPK-NF-κB pathway were investigated in obese mice. METHODS: Seventeen C57BL/6J mice were randomly divided into a normal diet group (ND), and 37 mice were divided into a high-fat diet group (HFD), the high-fat diet accounted for 40% of fat. After 12 weeks of feeding, 3 obesity-resistant mice were excluded from the HFD group, and the remaining 34 were successfully modeled. The mice in ND group were then divided into normal diet control group (NC, n=8) and normal diet and exercise group (NE, n=9). The mice in HFD group were divided into obese high-fat diet control group (OC, n=8), obese high-fat diet and exercise group (OE, n=9), obese normal diet group (ONC, n=8), and obese normal diet and exercise group (ONE, n=9). Each group continued to feed for 8 weeks, and the NE, OE and ONE groups performed treadmill exercise for 8 weeks at a speed of 20 m/min, 60 min/d, 6 d/week. Blood and testicular tissue samples were collected 36～40 h after the last exercise. Serum testosterone and testicular oxidative stress (MDA, T-SOD, T-AOC) levels were detected by ELISA, and testicular p38MAPK-NF-κB levels were detected by RT-PCR and Western blot. RESULTS: Compared with the NC group, the body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels in the OC group were increased significantly (P＜0.01), while the levels of testicular SOD, testis coefficient and blood testosterone were decreased significantly (P＜0.01); the body fat parameters of the mice in the NE group were decreased significantly (P＜0.05), and the serum level of testosterone was increased significantly (P＜0.01). Compared with the OC group, the body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels were decreased significantly in the OE group (P＜0.05 or 0.01), and the testicular SOD and blood testosterone levels were increased significantly (P＜0.01); Body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels were decreased significantly in ONC group (P＜0.01), while testicular SOD level and testis coefficient were increased significantly (P＜0.05); Body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels of mice in ONE group were decreased significantly (P＜0.01), while testicular SOD, testis coefficient and blood testosterone levels were increased significantly (P＜0.01). CONCLUSION: Obesity induces oxidative stress in the testis of mice, up-regulates the level of p38MAPK-NF-κB, and reduces the level of blood testosterone; exercise, diet and exercise*diet interventions can reduce testicular oxidative stress and down-regulate testicular p38MAPK-NF-κB levels by reducing body fat.

Effect of Combined Therapy of Lung and Intestine on VIP and p38 MAPK Signaling Pathways in Bronchial Asthma Mice / 中国实验方剂学杂志

ObjectiveTo observe the therapeutic effects of the combined therapy of lung and intestine， a common treatment for pulmonary diseases in traditional Chinese medicine （TCM）， on bronchial asthma mice， and further detect the changes of vasoactive intestinal peptide （VIP） and p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway-related proteins which are closely related to the pathogenesis of asthma， in order to elucidate the mechanism of the combined therapy of lung and intestine in the treatment of bronchial asthma. MethodA total of 60 Kunming mice were randomly divided into normal group， model group， dexamethasone group （0.5 mg·kg-1·d-1）， TCM group （2.73 g·kg-1·d-1）， and lung-intestine treatment group （6.825 g·kg-1·d-1）， 12 mice in each group. All mice except the normal group were sensitized by ovalbumin to induce bronchial asthma. After 30 days of intragastric administration， serum and lung tissue samples were obtained. The content of VIP， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） in the serum of mice in each group was detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA levels of TNF-α， IL-6， and p38 MAPK in lung tissues of mice were detected by real-time quantitative polymerase chain reaction （Real-time PCR）， and the protein levels of TNF-α， IL-6， p38 MAPK， and phosphorylated p38 MAPK （p-p38 MAPK） in lung tissues of mice were assayed by Western blot （WB）. ResultCompared with the normal group， the model group showed decreased content of serum VIP （P<0.05）， increased content of TNF-α and IL-6 （P<0.05）， up-regulated mRNA levels of TNF-α， IL-6， and p38 MAPK， and elevated protein levels of TNF-α， IL-6， and p-p38 MAPK/p38 MAPK in lung tissues （P<0.05）. Compared with the model group， the treatment groups exhibited increased content of serum VIP， TNF-α， and IL-6 （P<0.05）， down-regulated mRNA levels of TNF-α， IL-6， and p38 MAPK， and lower protein levels of TNF-α， IL-6， and p-p38 MAPK/p38 MAPK in lung tissues （P<0.05）. As compared with the lung-intestine treatment group， the serum TNF-α and IL-6 levels in the dexamethasone group were increased （P<0.05）， and the mRNA and protein levels of TNF-α and IL-6 in lung tissues were down-regulated （P<0.05）， while the levels of p38 MAPK， VIP mRNA， and p-p38 MAPK/p38 MAPK protein in lung tissues were up-regulated （P<0.05）. The serum VIP， TNF-α， and IL-6 levels in the TCM group were decreased （P<0.05）， and the mRNA levels of TNF-α， IL-6， p38 MAPK and protein levels of TNF-α， IL-6， p-p38 MAPK/p38 MAPK in lung tissues were up-regulated （P<0.05）， while the level of VIP mRNA in lung tissues was down-regulated （P<0.05）. ConclusionThrough increasing endogenous VIP and inhibiting the excessive activation of p38 MAPK signaling pathway， the combined therapy of lung and intestine can reduce the release of inflammatory factors， inhibit pulmonary inflammation response， and treat bronchial asthma.

Effect of Jingangwan on p38 MAPK，JNK，and IL-1 Content in Osteoporosis Model Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Jingangwan on the expression of osteoclast， c-Jun N-terminal kinase（JNK）， p38 mitogen-activated protein kinase（p38 MAPK）， and interleukin-1（IL-1） in the osteoporosis model rats， explore the mechanism of Jingangwan in the treatment of osteoporosis， and determine the optimal dosing concentration of Jingangwan. MethodFifty-six rats of SPF grade were randomized into a blank group，a sham operation group，a model group， model group，high-， medium-， and low-dose Jingangwan groups （0.72， 0.36， 0.18 g·kg-1·d-1， ig），and an estradiol valerate group （0.009 g·kg-1·d-1， ig）， with eight rats in each group. The rats in the model group， the blank group， and the sham operation group received 3 mL of normal saline， respectively. Samples were collected 12 weeks after drug administration. The number of osteoclasts was observed by tartrate-resistant acid phosphatase （TRAP） staining. Serum levels of JNK， p38 MAPK， and IL-1 were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression levels of p38 MAPK and JNK were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. ResultThe TRAP staining results showed that compared with the model group， the estradiol valerate group and the Jingangwan groups could inhibit the formation of osteoclasts to different degrees. As revealed by ELISA results， compared with the model group and the sham operation group， the model group showed increased serum levels of p38 MAPK， JNK， and IL-1 （P<0.01）， while compared with the model group， all the groups with drug intervention showed decreased levels of p38 MAPK， JNK， and IL-1 （P<0.01）. The serum levels of JNK and IL-1 in the high-dose Jingangwan group were lower than those in the estradiol valerate group （P<0.05）. Real-time PCR results showed that compared with the blank group， the model group showed increased relative mRNA expression of p38 MAPK and JNK in the thighbone （P<0.01）， while compared with the model group， all the groups with drug intervention showed decreased relative mRNA expression of p38 MAPK and JNK in the thighbone （P<0.01）. ConclusionJingangwan can inhibit the formation of osteoblasts，reduce the diameter of the bone marrow cavity，improve bone quality，suppress the production of inflammatory factors，affect the metabolism of the MAPK signaling pathway，and blunt p38 MAPK and JNK activities to inhibit the differentiation and proliferation of osteoblasts and regulate bone metabolism， thereby preventing osteoporosis. Therefore，Jingangwan may be of application value in maintaining bone health and treating osteoporosis.

Effect of Pangshi Antai Zhixue Decoction on Th1/Th2 Balance in Decidua and Membrane of Spontaneous Abortion Rats with Heat Syndrome by Regulating p38 MAPK Signal Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-abortional effect of Pangshi Antai Zhixue decoction and its mechanism in helper T lymphocyte 1 （Th1）/Th2 balance in the decidual tissues of spontaneous abortion rats with heat syndrome， based on the p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway. MethodAconiti Lateralis Radix Praeparata， Zingiberis Rhizoma， and Cinnamomi Cortex decoction was used to replicate the rat model of spontaneous abortion with heat syndrome. The spontaneous abortion rats with heat syndrome were randomly divided into model group， aspirin group （5.25 mg·kg-1）， dydrogesterone group （3.02 mg·kg-1）， Pangshi Antai Zhixue decoction high-dose （44 g·kg-1）， medium-dose （22 g·kg-1）， and low-dose （11 g·kg-1） groups， with ten rats in each group. Ten normal rats were divided into a normal group. Rats in each group were given corresponding drugs， Once a day for 12 d. After 24 h of the last administration， blood was collected from the abdominal aorta. The enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of β-human chorionic gonadotropin （β-HCG）， progesterone （P）， estradiol （E2）， γ interferon （IFN-γ）， and interleukin-4 （IL-4） in rat serum. The uterus and meconium tissues of rats were collected to determine the number and rate of miscarriages. Western blot was used to detect GATA3， T-bet， p38 MAPK， and its phosphorylation in the decidual tissue. ResultAs compared with the normal group， the number of live births， the β-HCG， P， E2， and IL-4 in the serum， and the GATA3 protein expression in the decidual tissue in the model group were reduced （P<0.01）， whereas the number and rate of miscarriages， IFN-γ in the serum， and the expression of p-p38 MAPK and T-bet protein levels in the demolded tissues increased （P<0.01）. As compared with the model group， the number of live births， the β-HCG， P， E2， and IL-4 in the serum， and the GATA3 protein expression in the decidual tissue in the Pangshi Antai Zhixue decoction medium-dose group increased （P<0.01）， whereas the number and rate of miscarriages， IFN-γ in the serum， and the expression of p-p38 and T-bet protein levels in the demolded tissues reduced （P<0.01）. As compared with the aspirin group， the P， E2， and IL-4 in the serum of rats in the dydrogesterone group and the Pangshi Antai Zhixue decoction high-dose and medium-dose groups increased （P<0.01）， the number of live births in the Pangshi Antai Zhixue decoction medium-dose group increased （P<0.01）， and the β-HCG and IFN-γ in the serum of rats in the dydrogesterone group decreased （P<0.01）. The number and rate of miscarriages， IFN-γ in the serum， and T-bet and GATA3 levels in the decidual tissues of rats in the Pangshi Antai Zhixue decoction medium-dose group decreased （P<0.05）. Compared with the Pangshi Antai Zhixue decoction medium-dose group， the low-dose group， high-dose group， and dydrogesterone group showed increased number and rate of miscarriages （P<0.05）， and the high-dose group and dydrogesterone group decreased the number of live birth （P<0.01）. The IFN-γ in the serum and p-p38 MAPK and T-bet protein in the decidual tissue in the low-dose group， and the p-p38 MAPK and T-bet protein in the decidual tissue in the high-dose group all increased （P<0.05）. The β-HCG， P， and E2 in the serum of rats in the Pangshi Antai Zhixue decoction low-dose group， dydrogesterone group， and aspirin group decreased （P<0.01）， and the IL-4 in the serum and GATA3 in the decidual tissue of rats in the Pangshi Antai Zhixue decoction low-dose and high-dose group and the dydrogesterone group decreased （P<0.01）. ConclusionPangshi Antai Zhixue decoction realizes the effect of fetal protection by regulating the activation of p38 MAPK signal pathways and Th1/Th2 balance.

Effect of Pangshi Antai Zhixue Decoction on Th1/Th2 Balance in Decidua and Membrane of Spontaneous Abortion Rats with Heat Syndrome by Regulating p38 MAPK Signal Pathway / 中国实验方剂学杂志

ObjectiveTo explore the anti-abortional effect of Pangshi Antai Zhixue decoction and its mechanism in helper T lymphocyte 1 （Th1）/Th2 balance in the decidual tissues of spontaneous abortion rats with heat syndrome， based on the p38 mitogen-activated protein kinase （p38 MAPK） signaling pathway. MethodAconiti Lateralis Radix Praeparata， Zingiberis Rhizoma， and Cinnamomi Cortex decoction was used to replicate the rat model of spontaneous abortion with heat syndrome. The spontaneous abortion rats with heat syndrome were randomly divided into model group， aspirin group （5.25 mg·kg-1）， dydrogesterone group （3.02 mg·kg-1）， Pangshi Antai Zhixue decoction high-dose （44 g·kg-1）， medium-dose （22 g·kg-1）， and low-dose （11 g·kg-1） groups， with ten rats in each group. Ten normal rats were divided into a normal group. Rats in each group were given corresponding drugs， Once a day for 12 d. After 24 h of the last administration， blood was collected from the abdominal aorta. The enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of β-human chorionic gonadotropin （β-HCG）， progesterone （P）， estradiol （E2）， γ interferon （IFN-γ）， and interleukin-4 （IL-4） in rat serum. The uterus and meconium tissues of rats were collected to determine the number and rate of miscarriages. Western blot was used to detect GATA3， T-bet， p38 MAPK， and its phosphorylation in the decidual tissue. ResultAs compared with the normal group， the number of live births， the β-HCG， P， E2， and IL-4 in the serum， and the GATA3 protein expression in the decidual tissue in the model group were reduced （P<0.01）， whereas the number and rate of miscarriages， IFN-γ in the serum， and the expression of p-p38 MAPK and T-bet protein levels in the demolded tissues increased （P<0.01）. As compared with the model group， the number of live births， the β-HCG， P， E2， and IL-4 in the serum， and the GATA3 protein expression in the decidual tissue in the Pangshi Antai Zhixue decoction medium-dose group increased （P<0.01）， whereas the number and rate of miscarriages， IFN-γ in the serum， and the expression of p-p38 and T-bet protein levels in the demolded tissues reduced （P<0.01）. As compared with the aspirin group， the P， E2， and IL-4 in the serum of rats in the dydrogesterone group and the Pangshi Antai Zhixue decoction high-dose and medium-dose groups increased （P<0.01）， the number of live births in the Pangshi Antai Zhixue decoction medium-dose group increased （P<0.01）， and the β-HCG and IFN-γ in the serum of rats in the dydrogesterone group decreased （P<0.01）. The number and rate of miscarriages， IFN-γ in the serum， and T-bet and GATA3 levels in the decidual tissues of rats in the Pangshi Antai Zhixue decoction medium-dose group decreased （P<0.05）. Compared with the Pangshi Antai Zhixue decoction medium-dose group， the low-dose group， high-dose group， and dydrogesterone group showed increased number and rate of miscarriages （P<0.05）， and the high-dose group and dydrogesterone group decreased the number of live birth （P<0.01）. The IFN-γ in the serum and p-p38 MAPK and T-bet protein in the decidual tissue in the low-dose group， and the p-p38 MAPK and T-bet protein in the decidual tissue in the high-dose group all increased （P<0.05）. The β-HCG， P， and E2 in the serum of rats in the Pangshi Antai Zhixue decoction low-dose group， dydrogesterone group， and aspirin group decreased （P<0.01）， and the IL-4 in the serum and GATA3 in the decidual tissue of rats in the Pangshi Antai Zhixue decoction low-dose and high-dose group and the dydrogesterone group decreased （P<0.01）. ConclusionPangshi Antai Zhixue decoction realizes the effect of fetal protection by regulating the activation of p38 MAPK signal pathways and Th1/Th2 balance.

Lipocalin-2 Regulates Hippocampal Microglial Activation in Poststroke Depression.

Background and Purpose: Microglia play important role in poststroke depression (PSD), however, the exact mechanism was still unclear. The purpose of the study was to study the mechanism of microglial activation in PSD. Methods: 24 rats were randomly divided into three groups: the PSD group (n = 10), the poststroke (PS) group (n = 7), and the sham group (n = 7). Primary hippocampal microglia were isolated and cultured, and recombined LCN2 protein was used to stimulate the cultured microglia. The protein expression of Iba1, P38 MAPK and PP38 MAPK was analyzed by western blotting; the LCN2 expression was measured by RT-qPCR, the serum LCN2 level and the NO level were analyzed by ELISA. Results: Open field test scores (horizontal score, vertical score, and self-grooming score) and the serum LCN2 level were significantly decreased in the PSD group compared with the other two groups (P < 0.05). The serum LCN2 level was positively correlated with the horizontal score and negatively correlated with the self-grooming score in the open field test (P < 0.05). The relative protein level of Iba1 and the LCN2 mRNA level were significantly increased in the hippocampal region compared with other brain regions (P < 0.05), while the relative protein level of Iba1 and the LCN2 mRNA level were significantly increased in the PSD group compared with the other two groups (P < 0.05). The length, supernatant NO level, phagocytic ability and migration ability of LCN2-treated microglia were significantly increased compared with those of untreated microglia (P < 0.05). The relative protein levels of P38 MAPK and the PP38 MAPK significantly increased in hippocampal region in the PSD group and LCN2-treated hippocampal microglia (P < 0.05). Conclusion: Hippocampal microglia are activated during PSD; LCN2 may regulate hippocampal microglial activation by the P38 MAPK pathway in the process of PSD.

PP2A protects podocytes against Adriamycin-induced injury and epithelial-to-mesenchymal transition via suppressing JIP4/p38-MAPK pathway.

Protein phosphatase 2A (PP2A) is one of the major protein serine/threonine phosphatases (PPPs) with regulatory effects on several cellular processes, but its role and function in Adriamycin (ADR)-treated podocytes injury needs to be further explored. Mice podocytes were treated with ADR and PP2A inhibitor (okadaic acid, OA). After transfection, cell apoptosis was detected by flow cytometry. Expressions of podocytes injury-, apoptosis- and epithelial-to-mesenchymal transition (EMT)- and JNK-interacting protein 4/p38-Mitogen-Activated Protein Kinase (JIP4/p38-MAPK) pathway-related factors were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. Interaction between PP2A and JIP4/MAPK pathway was confirmed using co-immunoprecipitation (Co-Ip) assay. In podocytes, ADR inhibited PP2A, Nephrin and Wilms' tumor (WT) 1 expressions yet upregulated apoptosis and Desmin expression, and suppressing PP2A expressionenhanced the effects. PP2A overexpression reversed the effects of ADR on PP2A and podocyte injury-related factors expressions and apoptosis of podocytes. JIP4 was the candidate gene interacting with both PP2A and p38-MAPK pathway, and PP2A overexpression alleviated the effects of ADR on p38-MAPK pathway-related factors expressions. Additionally, in ADR-treated podocytes, PP2A suppression enhanced the effects of ADR, yet silencing of JIP4 reversed the effects of PP2A suppression on regulating p38-MAPK pathway-, apoptosis- and EMT-related factors expressions and apoptosis, with upregulations of B-cell lymphoma-2 (Bcl-2) and E-cadherin and down-regulations of Bcl-2 associated protein X (Bax), cleaved (C)-casapse-3, N-cadherin, Vimentin and Snail. PP2A protects ADR-treated podocytes against injury and EMT by suppressing JIP4/p38-MAPK pathway, showing their interaction in podocytes.

Astaxanthin alleviates neuropathic pain by inhibiting the MAPKs and NF-κB pathways.

Neuropathic pain is a complex condition that usually lasts a lifetime and has a major negative impact on life after injury. Improving pain management is an important and unmet need. Astaxanthin (AST) is a natural marine medicine with effective antioxidant and anti-inflammatory properties and neuroprotective effects. However, few mechanisms can explain the role of AST in the treatment of neuropathic pain. In the present study, we examined its potential to eliminate spinal nerve ligation (SNL) damage by inhibiting the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor-κB (NF-κB) p65 and the inflammatory response. The results of behavior tests indicated the promising role of AST in analgesic effect in SNL mice. AST decreased the neuronal and non-neuronal activation, the levels of the inflammatory signaling mediators (p-ERK1/2 p-p38 MAPK and NF-κB p65) and inflammatory cytokine expression (interleukin [IL]-1, IL-17, IL-6, and tumor necrosis factor-α [TNF-α]. These results suggest that AST is a promising candidate to reduce nociceptive hypersensitization after SNL.

Mechanism of Dahuang Zhechongwan in Inhibiting Silicosis in Mice via p38 MAPK/NF-κB/TGF-β1 Pathway / 中国实验方剂学杂志

Objective:To explore the effects and mechanism of Chinese classical prescription Dahuang Zhechongwan on silicosis in mice. Method:Thirty-six male Kunming mice of SPF grade were randomized into the normal control group， model control group， tetrandrine （Tet， 0.039 mg·kg^-1） group， as well as high- （1.560 g·kg^-1）， medium- （0.780 g·kg^-1）， and low-dose （0.390 g·kg^-1） Dahuang Zhechongwan groups， with six mice in each group. Mice in all groups except for the normal control group underwent static inhalation of silica （SiO₂） dust for 40 consecutive days to induce fibrosis. After 28 days of intervention with corresponding drugs， the mice were sacrificed to collect the serum and lung tissues， with the former used for detecting tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-1<italic>β </italic>（IL-1<italic>β</italic>）， IL-6， and hydroxyproline （HYP） levels by enzyme-linked immunosorbent assay （ELISA） and the latter for observing the pathological changes. Meanwhile， the protein and mRNA expression levels of p38 mitogen-activated protein kinase （p38 MAPK）， nuclear transcription factor-<italic>κ</italic>B （NF-<italic>κ</italic>B）， transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁）， <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA）， Smad2， Smad3， and Smad7 in the lung tissues were determined by Western blot and real-time polymerase chain reaction （Real-time PCR）. Result:Compared with the normal group， the contents of TNF-<italic>α</italic>， IL-1<italic>β</italic>， IL-6 and HYP in the model group were significantly increased， the difference was statistically significant（<italic>P</italic><0.05，<italic>P</italic><0.01）； compared with the model group， the high-dose group of Dahuang Zhechongwan could significantly reduce the contents of TNF-<italic>α</italic>， IL-6 and HYP in the serum of mice（<italic>P</italic><0.05， <italic>P</italic><0.01）， indicating that Dahuang Zhechongwan could reduce the lung inflammation of silicosis mice. At the same time， compared with the normal group， the protein and mRNA expression levels of p38 MAPK， NF-<italic>κ</italic>B p65， TGF-<italic>β</italic>₁， <italic>α</italic>-SMA， Smad2 and Smad3 in the model group were significantly increased（<italic>P</italic><0.05，<italic>P</italic><0.01）， while the protein and mRNA expression levels of Smad7 were significantly decreased（<italic>P</italic><0.01）； compared with the model group， the protein and mRNA expression levels of p38 MAPK， NF-<italic>κ</italic>B p65， TGF-<italic>β</italic>₁， <italic>α</italic>-SMA， Smad2 and Smad3 in the high-dose Dahuang Zhechongwan group were significantly increased the protein and mRNA expression levels were significantly decreased（<italic>P</italic><0.05，<italic>P</italic><0.01）， while Smad7 protein and mRNA expression levels were significantly increased（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Dahuang Zhechongwan ameliorates the alveolar inflammation， extracellular matrix （ECM） deposition， and fibrosis in mice with silicosis possibly by regulating the p38 MAPK/NF-<italic>κ</italic>B/TGF-<italic>β</italic>₁ pathway.

Effect of Xintongtai Regulating p38 MAPK/AP-1 on Traditional Chinese Medicine Syndrome Score and Collagen Fibers in VSMCs of Rabbits with Atherosclerosis： An Exploration Based on Theory of Heart Receiving Qi From Spleen / 中国实验方剂学杂志

Objective:To explore the effects of Xintongtai （XTT） on traditional Chinese medicine （TCM） syndrome score and collagen fibers in vascular smooth muscle cells（VSMCs） of rabbits with atherosclerosis in the regulation of p38 mitogen-activated protein kinase （p38 MAPK）/activator protien-1 （AP-1）signaling pathway. Method:A total of 120 rabbits of SPF grade were randomly divided into the sham operation group， combined phlegm and blood stasis model group， rosuvastatin group， and low-， middle-， and high-dose XTT groups. The rabbit model of atherosclerosis due to combined phlegm and blood stasis was established by exposing them to high-fat diet and balloon injury. Following modeling， the corresponding drugs were administered by gavage for eight weeks （2.3， 4.6， 9.2 g·kg^-1 for low-， middle-， and high-dose XTT groups and 0.55 mg·kg^-1for rosuvastatin group）. At the end of medication， the abdominal aorta was isolated and stained with htoxylin-eosin （HE） for observing the vulnerable plaque. Matrix metalloproteinase-9 （MMP-9） and tissue inhibitor of metalloproteinase-1 （TIMP-1） were detected by immunohistochemistry （IHC）. The collagen fiber decomposition in VSMCs was observed after Masson staining. The protein expression levels of p38 MAPK and AP-1 in aorta was assayed by Western blotting. The combined phlegm and blood stasis syndrome was scored based on TCM syndrome scoring scale. Result:Compared with the model group， XTT at each dose and rosuvastatin significantly decreased MMP-9 content， increased TIMP-1， down-regulated p38 MAPK protein expression， and weakened the nuclear translocation of AP-1 （<italic>P</italic><0.01）. Compared with the low-dose XTT group， the middle- and high-dose XTT groups and rosuvastatin group exhibited obviously lowered MMP-9，elevated TIMP-1， down-regulated p38 MAPK protein expression， and diminished AP-1 nuclear translocation （<italic>P</italic><0.05，<italic>P</italic><0.01）. The TCM syndrome scores of the middle- and high-dose XTT groups and rosuvastatin group were significantly improved as compared with that in the model group （<italic>P</italic><0.05，<italic>P</italic><0.01）. The comparison with the low-dose XTT group revealed a remarkable improvement in TCM syndrome score of the middle- and high-dose XTT groups and rosuvastatin group （<italic>P</italic><0.01）. As demonstrated by Masson staining， the smooth muscle fibers in the model group were arranged in disorder， accompanied by enhanced collagen decomposition， thinned fibrous cap， and increased plaque vulnerability. Compared with the model group， the VSMCs in each XTT group and rosuvastatin group were orderly arranged， manifested as decreased collagen fiber decomposition and increased plaque stability. Conclusion:XTT down-regulates the expression of p38 MAPK and MMP-9， increases the level of TIMP-1， reduces the nuclear translocation of AP-1， diminishes the decomposition of collagen fibers in VSMCs， and improves the score of combined phlegm and blood stasis syndrome. XTT alleviates arteriosclerosis due to combined phlegm and blood stasis by regulating p38 MAPK/AP-1 signaling pathway and downstream cytokines and stabilizing vulnerable plaques.

Coriolus versicolor polysaccharides (CVP) regulates neuronal apoptosis in cerebral ischemia-reperfusion injury via the p38MAPK signaling pathway.

BACKGROUND: This study aims to investigate the regulation of herbal polysaccharide, Coriolus versicolor polysaccharides (CVP), on neuronal apoptosis in a rat cerebral ischemia-reperfusion injury (CIRI) model. We also intend to explore the mechanisms and effectiveness of CVP in the treatment of neuronal apoptosis in CIRI rats, including neurological function, cerebral infarction volume, inflammatory factors, and the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway as well as its downstream protein cleaved-Caspase-3. METHODS: A CIRI model was established in rats using the Longa method of middle cerebral artery occlusion. Neurological function scores and cerebral infarction volumes were measured in CIRI rats. Annexin V-fluorescein isothiocyanate (FITC) were used to measure neuronal apoptosis in CIRI rats. The levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in CIRI rats were determined using enzyme-linked immunosorbent assay (ELISA). A western blot assay was used to measure the protein expression levels of p38MAPK, phospho-p38MAPK (p-p38MAPK), Bcl-2, Bax and cleaved-Caspase-3 in brain tissue of CIRI rats. RESULTS: CVP can effectively improve the neurological function of rats after 6, 12, 24, and 48 h of CIRI. It can also improve the behavioral test, reduce the cerebral infarction volume and inhibit the apoptosis of nerve cells in CIRI rats. The protein expression levels of p-p38MAPK and cleaved-Caspase-3 exhibited a decreasing trend following CVP administration. CONCLUSIONS: CVP can significantly reduce the pathological characteristics of CIRI in rats and inhibit the apoptosis of nerve cells around the lesions. The mechanism of its effectiveness is related to inhibiting the activation of the p38MAPK signaling pathway.

Epigallocatechin-3-Gallate and PEDF 335 Peptide, 67LR Activators, Attenuate Vasogenic Edema, and Astroglial Degeneration Following Status Epilepticus.

Non-integrin 67-kDa laminin receptor (67LR) is involved in cell adherence to the basement membrane, and it regulates the interactions between laminin and other receptors. The dysfunction of 67LR leads to serum extravasation via blood-brain barrier (BBB) disruption. Polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) and pigment epithelium-derived factor (PEDF) bind to 67LR and inhibit neovascularization. Therefore, in the present study, we investigated the effects of EGCG and NU335, a PEDF-derive peptide, on BBB integrity and their possible underlying mechanisms against vasogenic edema formation induced by status epilepticus (SE, a prolonged seizure activity). Following SE, both EGCG and NU335 attenuated serum extravasation and astroglial degeneration in the rat piriform cortex (PC). Both EGCG and NU335 reversely regulated phosphatidylinositol 3 kinase (PI3K)/AKT-eNOS (endothelial nitric oxide synthase) mediated BBB permeability and aquaporin 4 (AQP4) expression in endothelial cells and astrocytes through the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, respectively. Furthermore, EGCG and NU335 decreased p47Phox (a nicotinamide adenine dinucleotide phosphate oxidase subunit) expression in astrocytes under physiological and post-SE conditions. Therefore, we suggest that EGCG and PEDF derivatives may activate 67LR and its downstream effectors, and they may be considerable anti-vasogenic edema agents.

Expression Levels of the Immune-Related p38 Mitogen-Activated Protein Kinase Transcript in Response to Environmental Pollutants on Macrophthalmus japonicus Crab.

Environmental pollution in the aquatic environment poses a threat to the immune system of benthic organisms. The Macrophthalmus japonicus crab, which inhabits tidal flat sediments, is a marine invertebrate that provides nutrient and organic matter cycling as a means of purification. Here, we characterized the M. japonicus p38 mitogen-activated protein kinase (MAPK) gene, which plays key roles in the regulation of cellular immune and apoptosis responses. M. japonicusp38 MAPK displayed the characteristics of the conserved MAPK family with Thr-Gly-Tyr (TGY) motif and substrate-binding site Ala-Thr-Arg-Trp (ATRW). The amino acid sequence of the M. japonicus p38 MAPK showed a close phylogenetic relationship to Eriocheir sinensis MAPK14 and Scylla paramamosainp38 MAPK. The phylogenetic tree displayed two origins of p38 MAPK: crustacean and insect. The tissue distribution patterns showed the highest expression in the gills and hepatopancreas of M. japonicus crab. In addition, p38 MAPK expression in M. japonicus gills and hepatopancreas was evaluated after exposure to environmental pollutants such as perfluorooctane sulfonate (PFOS), irgarol, di(2-ethylhexyl) phthalate (DEHP), and bisphenol A (BPA). In the gills, p38 MAPK expression significantly increased after exposure to all concentrations of the chemicals on day 7. However, on day 1, there were increased p38 MAPK responses observed after PFOS and irgarol exposure, whereas decreased p38 MAPK responses were observed after DEHP and BPA exposure. The upregulation of p38 MAPK gene also significantly led to M. japonicus hepatopancreas being undertested in all environmental pollutants. The findings in this study supported that anti-stress responses against exposure to environmental pollutants were reflected in changes in expression levels in M. japonicusp38 MAPK signaling regulation as a cellular defense mechanism.

(-)-Epigallocatechin-3-gallate induces interferon-λ2 expression to anti-influenza A virus in human bronchial epithelial cells (BEAS-2B) through p38 MAPK signaling pathway.

BACKGROUND: (-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea, has been found to inhibit the influenza virus. However, the mechanism of EGCG anti-influenza virus effect needs to be further explored. METHODS: BEAS-2B cells were treated with different concentrations of EGCG or were treated with EGCG for different times. CCK8 assay was used to detect the cell viability, and quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay were employed to measure the interferon (IFN)-λ2 mRNA and protein expression levels. The phospho-p38 mitogen-activated protein kinase (P-p38 MAPK), phospho-extracellular signal-regulated kinase (P-ERK), and phospho-c-Jun N-terminal kinase (P-JNK) expression were tested by western blot. Then, p38 MAPK, ERK, and JNK inhibitor were used to study the effect of p38 MAPK, ERK, and JNK signaling pathways on IFN-λ2 expression. The BEAS-2B cells were treated with EGCG, EGCG and IFN λ2 neutralizing antibody or control antibody for 12 h, and were infected with influenza A virus (IAV) (H1N1) for 1 h. After 12 h, nucleoprotein (NP) mRNA and protein expression levels of H1N1 were assessed by qRT-PCR and western blot. RESULTS: The IFN-λ2 mRNA and protein expression levels in BEAS-2B cells were up-regulated after EGCG (treatment in time- and dose-dependent manners the concentration range from 0 to 50 µg/mL had no cytotoxicity). Meanwhile, the P-p38 MAPK, P-ERK, and P-JNK expression levels were up-regulated. IFN-λ2 mRNA and protein expression was inhibited after p38 MAPK inhibitor pre-treatment, but not by ERK and JNK inhibitors. Furthermore, the expression of H1N1 NP gene and protein decreased after EGCG pre-treatment, while IFN-λ2 neutralizing antibody attenuated the effect of EGCG inhibiting the expression of H1N1 NP gene and protein. CONCLUSIONS: EGCG inhibited IAV H1N1 by inducing the expression of IFN-λ2 in BEAS-2B cells through the p38 MAPK signaling pathway.

MicroRNA-9 overexpression suppresses vulnerable atherosclerotic plaque and enhances vascular remodeling through negative regulation of the p38MAPK pathway via OLR1 in acute coronary syndrome.

Acute coronary syndrome (ACS) is characterized by atherosclerotic plaque rupture with a high incidence of recurrent ischemic events. Several microRNAs are found to be aberrantly expressed in atherosclerotic plaques. This study aims to investigate the effects of microRNA-9 (miR-9) on vulnerable atherosclerotic plaque and vascular remodeling in ACS and underlying mechanisms. Microarray-based gene expression profiling was used to identify differentially expressed genes related to ACS and regulatory miRNAs. Oxidized low-density lipoprotein (lectin-like) receptor 1 (OLR1) was identified to be aberrantly activated in ACS and regulated by miR-9. OLR1 was verified as a target gene of miR-9 by bioinformatics prediction and dual luciferase reporter gene assay. The atherosclerotic models were induced in ApoE-/- mice, in which the agomir or antagomir of miR-9, or small interfering RNA (siRNA) against OLR1 were separately introduced. Serum lipid levels and expression of vascular remodeling and inflammatory response-related factors were determined, respectively. On the basis of the obtained results, in the atherosclerosis mice treated with the agomir of miR-9 and siRNA against OLR1, the p38-mitogen-activated protein kinase (p38MAPK) pathway was inhibited; levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol, tumor necrosis factor-α, interleukin-6, and vascular endothelial growth factor were reduced, but the high-density lipoprotein cholesterol level was increased, along with decreased vulnerable atherosclerotic plaque area and enhanced vascular remodeling. Taken together, these findings suggested an inhibitory role miR-9 acts in the formation of vulnerable atherosclerotic plaques in ACS mice, along with a promoted vascular remodeling, via a negative feedback regulation of OLR1-mediated p38MAPK pathway.