Small Molecules Accelerate Skin Wound Healing: Shikonin Efficacy and Mechanism of Action in Mice / Las Moléculas Pequeñas Aceleran la Curación de Heridas en la Piel: Eficacia de Shikonin y Mecanismo de Acción en Ratones

SUMMARY: The objective of this study was to investigate the therapeutic wound healing potential and molecular mechanisms of shikonin as small molecules in vitro. A mouse burn model was used to explore the potential therapeutic effect of shikonin; we traced proliferating cells in vivo to locate the active area of skin cell proliferation. Through the results of conventional pathological staining, we found that shikonin has a good effect on the treatment of burned skin and promoted the normal distribution of skin keratin at the damaged site. At the same time, shikonin also promoted the proliferation of skin cells at the damaged site; importantly, we found a significant increase in the number of fibroblasts at the damaged site treated with shikonin. Most importantly, shikonin promotes fibroblasts to repair skin wounds by regulating the PI3K/AKT signaling pathway. This study shows that shikonin can effectively promote the proliferation of skin cell, and local injection of fibroblasts in burned skin can play a certain therapeutic role.

El objetivo de este trabajo fue investigar el potencial terapéutico de cicatrización de heridas y los mecanismos moleculares de la shikonina como moléculas pequeñas in vitro. Se utilizó un modelo de quemaduras en ratones para explorar el posible efecto terapéutico de la shikonina; Rastreamos las células en proliferación in vivo para localizar el área activa de proliferación de células de la piel. A través de los resultados de la tinción para patología convencional, encontramos que la shikonina tiene un buen efecto en el tratamiento de la piel quemada y promueve la distribución normal de la queratina de la piel en el sitio dañado. Al mismo tiempo, la shikonina también promovió la proliferación de células de la piel en el sitio dañado. Es importante destacar que encontramos un aumento significativo en la cantidad de fibroblastos en el sitio dañado tratado con shikonina. Lo más importante es que la shikonina promueve la función reparadora de fibroblastos en las heridas de la piel regulando la vía de señalización PI3K/ AKT. Este estudio muestra que la shikonina puede promover eficazmente la proliferación de células de la piel y que la inyección local de fibroblastos en la piel quemada puede desempeñar un cierto papel terapéutico.

Coenzyme Q10 prevents RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways

Coenzyme Q10 (CoQ10) is a potent antioxidant that is implicated in the inhibition of osteoclastogenesis, but the underlying mechanism has not been determined. We explored the underlying molecular mechanisms involved in this process. RAW264.7 cells received receptor activator of NF-κB ligand (RANKL) and CoQ10, after which the differentiation and viability of osteoclasts were assessed. After the cells were treated with CoQ10 and/or H2O2 and RANKL, the levels of reactive oxygen species (ROS) and proteins involved in the PI3K/AKT/mTOR and MAPK pathways and autophagy were tested. Moreover, after the cells were pretreated with or without inhibitors of the two pathways or with the mitophagy agonist, the levels of autophagy-related proteins and osteoclast markers were measured. CoQ10 significantly decreased the number of TRAP-positive cells and the level of ROS but had no significant impact on cell viability. The relative phosphorylation levels of PI3K, AKT, mTOR, ERK, and p38 were significantly reduced, but the levels of FOXO3/LC3/Beclin1 were significantly augmented. Moreover, the levels of FOXO3/LC3/Beclin1 were significantly increased by the inhibitors and mitophagy agonist, while the levels of osteoclast markers showed the opposite results. Our data showed that CoQ10 prevented RANKL-induced osteoclastogenesis by promoting autophagy via inactivation of the PI3K/AKT/mTOR and MAPK pathways in RAW264.7 cells.

Mechanism of Modified Tianwang Buxindan on Skin of Sleep-deprived Mice Through PI3K/Akt/Nrf2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of modified Tianwang Buxindan （MTBD） on the skin of sleep-deprived （SD） mice and investigate its mechanism. MethodSixty 2-month-old female Kunming mice were randomly divided into a blank group， a model group， a vitamin C （VC， 0.08 g·kg-1）， and MTBD low-， medium-， and high-dose groups （6.5， 12.5， 25 g·kg-1）. Except for the blank group， the other groups were subjected to SD mouse model induction （using multiple platform water environment method for 18 hours of sleep deprivation daily from 15：00 to next day 9：00）， continuously for 14 days， and caffeine （CAF， 7.5 mg·kg-1） was injected intraperitoneally from the 2nd week onwards， continuously for 7 days. While modeling， the blank group and the model group were administered with normal saline （0.01 mL·g-1）， and the other groups received corresponding drugs for treatment. On the day of the experiment， general observations were recorded （such as body weight， spirit， fur， and skin）. After sampling， skin tissue pathological changes were observed under an optical microscope using hematoxylin-eosin （HE） and Masson staining methods. Skin thickness and skin moisture content were measured. Biochemical assay kits were used to detect skin hydroxyproline （HYP） content， skin and serum superoxide dismutase （SOD） activity， and malondialdehyde （MDA） content. Enzyme-linked immunosorbent assay （ELISA） was used to detect serum interleukin （IL）-6， tumor necrosis factor （TNF）-α， and IL-1β levels in mice. Western blot was used to detect skin tissue type Ⅰ collagen （ColⅠ）， type Ⅲ collagen （ColⅢ）， phosphatidylinositol 3-kinase （PI3K）， phosphorylated （p）-PI3K， protein kinase B （Akt）， p-Akt， nuclear factor E2-related factor 2 （Nrf2）， heme oxygenase （HO）-1， and nuclear factor （NF）-κB protein expression. ResultCompared with the blank group， the model group showed varying degrees of changes. In general， signs of aging such as reduced body weight （P<0.01）， listlessness， dull fur color， and formation of wrinkles on the skin appeared. Tissue specimen testing revealed skin thinning， flattening of the dermoepidermal junction （DEJ）， and reduced collagen fibers under the optical microscope. Skin thickness and moisture content decreased， skin tissue HYP content significantly decreased （P<0.01）， skin and serum SOD activity significantly decreased （P<0.01）， and MDA content significantly increased （P<0.01）. Serum IL-6， TNF-α， and IL-1β levels significantly increased （P<0.01）. Skin ColⅠ， ColⅢ， p-PI3K/PI3K， p-Akt/Akt， Nrf2， and HO-1 protein expression significantly decreased （P<0.05， P<0.01）， and NF-κB expression increased （P<0.01）. Compared with the model group， the VC group and the MTBD low-dose group showed increased skin moisture content， HYP content， SOD activity， and ColⅠ， ColⅢ， p-PI3K/PI3K protein expression （P<0.05， P<0.01）， and decreased serum MDA content （P<0.05）. In addition， a decrease in serum IL-6 and IL-1β levels was detected in the MTBD low-dose group （P<0.05）， while the above indicators in the MTBD medium- and high-dose groups improved （P<0.05， P<0.01）. ConclusionSleep deprivation accelerates the aging process of the skin in SD model mice. MTBD can improve this phenomenon， exerting anti-inflammatory and antioxidant effects， and its mechanism of action may be related to the activation of the PI3K/Akt/Nrf2 signaling pathway.

Effect of Faeces Bombycis Extract on Cajal Interstitial Cells in Diabetic Gastroparesis Rats Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic mechanism of Faeces Bombycis on diabetic gastroparesis （DGP） rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein （PI3K/Akt/mTOR） signaling pathway. MethodDGP rat model was prepared by random selection of 15 out of 105 rats as blank group. The rats successfully constructed were randomly divided into model group， high-，medium- and low- dose groups （3.2， 1.6， 0.8 g·kg-1） and moxapride group （1.5 mg·kg-1）， with 12 rats in each group， and were given gavage for 4 weeks. The gastric emptying rate and random blood glucose were measured. The morphological changes of gastric antrum were observed by hematoxylin-eosin （HE） staining， and the expression of the c-Kit gene was analyzed by immunohistochemistry. The apoptosis of Cajal interstitial cells was observed by in situ end labeling （TUNEL） staining， and the protein expressions of PI3K， phosphorylation（p）-PI3K， Akt， p-Akt， mTOR， and p-mTOR were detected by Western blot. ResultCompared with the blank group， the gastric emptying rate of the model group decreased significantly （P<0.01）， and the glandular structure of the gastric antrum was destroyed. The expression of c-Kit decreased （P<0.01）， and the apoptosis of Cajal interstitial cells （ICC） increased. Compared with the model group， the gastric emptying rate in the high， middle， and low-dose groups of Faeces Bombycis extract and mosapride group increased significantly （P<0.01）. The glandular structure of the gastric antrum became closer， and the apoptosis of ICC decreased. The expression of c-Kit in the high dose group of Faeces Bombycis extract increased significantly. After Western blot testing， compared with the blank group， the protein expression of p-Akt/Akt， p-PI3K/PI3K， and p-mTOR/mTOR in the model group increased. Compared with the model group， the protein expression of p-Akt/Akt in the high dose group of Faeces Bombycis extract decreased （P<0.01）， and the protein expression of p-PI3K/PI3K decreased in the middle and low dose groups of Faeces Bombycis extract and mosapride group decreased （P<0.05， P<0.01）. The protein expression of p-mTOR/mTOR decreased in the low dose group of Faeces Bombycis extract （P<0.05）. In terms of random blood glucose， compared with the blank group， the random blood glucose in the model group increased significantly （P<0.01）， and compared with the model group， the random blood glucose in the high and middle dose groups of Faeces Bombycis extract decreased significantly （P<0.05）. Compared with mosapride group， the protein expression of p-Akt/Akt decreased in the high dose group of Faeces Bombycis extract （P<0.05）， and the protein expression of p-PI3K/PI3K increased in the high， middle， and low dose groups of Faeces Bombycis extract （P<0.05， P<0.01）. ConclusionFaeces Bombycis extract can increase gastric emptying rate， reduce ICC apoptosis， and lower random blood glucose in DGP rats. The high dose group of Faeces Bombycis extract has a significant effect on inhibiting ICC apoptosis， and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

Mechanism of Fufang Congrong Yizhi Capsules in treatment of mild cognitive impairment based on network pharmacology / 中国药理学通报

Aim To predict the mechanism of Fufang Congrong Yizhi Capsules (FCYC) in the treatment of mild cognitive impairment (MCI) by network pharmacology method, and further validate it in combination with cellular experiments. Methods TCMSP, Gene-Cards, OMIM and TTD databases, Chinese Pharmacopoeia and related literature were used to screen the active ingredients of FCYC and the targets of MCI treatment. The TCM-compound-target-disease network and PPI of intersection targets were constructed, and the GO and KEGG analysis were performed by the Ehamb bioinformation platform. GO and KEGG analysis were performed through Yihanbo biological information platform. Cell model of MCI was established by PC-12 injury induced by Aβ

Mechanism of EGFR inhibitor AG1478 combined with oxaliplatin in inhibiting PI3K/AKT pathway and promoting autophagy in HI 975 cells / 中国药理学通报

Aim To explore the effect of oxaliplatin combined with epidermal growth factor receptor tyrosine kinase inhibitor AG1478 on autophagy in non-small cell lung cancer H1975 cells. Methods H1975 cells were cultured in vitro using gradient concentrations of AG1478 (0, 5, 10, 15, 20, 25, 30, 35, 40 jjimol • IT

Research progress on the intervention of traditional Chinese medicine in renal interstitial fibrosis based on PI3K/Akt signaling pathway / 中国药房

Renal interstitial fibrosis（RIF） is the main pathological manifestation of chronic kidney disease. Due to the complexity of the mechanism， there is no specific treatment for RIF in clinical practice. The abnormal activation of the phosphatidylinositol 3-kinase （PI3K）/protein kinase B（Akt） signaling pathway and the activation of downstream target genes are key drivers of RIF induction and progression. Traditional Chinese medicine has the characteristics of precise efficacy and minimal toxic side effects， and the occurrence and development of RIF can be regulated by multiple targets and mutual coordination. This review focuses on the PI3K/Akt signaling pathway and summarizes the potential targets and regulatory mechanisms of traditional Chinese medicine in the treatment of RIF. It is found that various effective ingredients （such as sinomenine， mangiferin， coumarin derivates from Hydrangea paniculata， etc.） and formulas （such as Fushengong decoction， Qi-Bang-Yi-Shen formula， etc.） of traditional Chinese medicine can inhibit fibroblast proliferation， improve inflammation and oxidative stress， maintain mitochondrial stability， and slow down ferroptosis through this pathway， thereby delaying the occurrence and progression of RIF.

Isoliensinine affects the proliferation, apoptosis and autophagy of colon cancer SW480 cells through PI3K/Akt/mTOR signaling pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨异莲心碱（Iso）通过PI3K/Akt/mTOR信号通路对结肠癌SW480细胞增殖、凋亡和自噬的影响。方法：用10、20和40 μmol/L的Iso处理结肠癌SW480细胞，CCK-8法、流式细胞术和WB法分别检测Iso对细胞增殖活力、凋亡和自噬相关蛋白LC3Ⅰ、LC3Ⅱ、p62表达的影响。然后，用20 μmol/L的Iso和25 μmol/L的PI3K激活剂740 Y-P分别处理SW480细胞，将细胞分为对照组、740 Y-P组、Iso组和Iso+740 Y-P组，流式细胞术、WB法检测Iso和740 Y-P对各组细胞凋亡及细胞中LC3Ⅰ、LC3Ⅱ、p62、PI3K、p-PI3K、 mTOR和p-mTOR蛋白表达的影响。结果：10、20和40 μmol/L的Iso处理后，SW480细胞增殖活力均显著下降（均P<0.05），细胞凋亡率均显著升高（均P<0.05），LC3Ⅱ/LC3Ⅰ表达均显著上调（均P<0.05），p26蛋白表达显著下调（P<0.05）。Iso和740 Y-P处理后，与对照组相比，740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达均显著下降（均P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著升高（均P<0.05）；Iso组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（均P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著下降（均P<0.05）；与740 Y-P组相比，Iso+740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达升高（P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著下降（均P<0.05）；与Iso组相比，Iso+740 Y-P组细胞凋亡率、LC3Ⅱ/LC3Ⅰ表达下降（均P<0.05），p26、p-PI3K/PI3K和p-mTOR/mTOR表达均显著升高（均P<0.05）。结论：Iso通过抑制PI3K/Akt/mTOR信号通路抑制结肠癌SW480细胞增殖并诱导细胞凋亡和自噬。

Effects of osthole on renal oxidative stress injury in aged spontaneously hypertensive rats by regulating PI3K/Akt/mTOR signaling pathway / 中国临床药理学与治疗学

AIM: To explore the mechanism of osthole on elderly spontaneously hypertensive rats. METHODS: 20-month-old spontaneously hypertensive rats (SHRs) and healthy Wistar-Kyoto (WKY) rats were purchased. SHRs were treated with osthole (i.g.) for 8 weeks. The systolic blood pressure and diastolic blood pressure of rats were monitored. Hematoxylin-eosin staining (H&E), periodic acid-schiff staining (PAS) and Masson staining were used to observe the pathological changes of rat kidney tissues. The activity of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in rat kidney was detected by ELISA kit. PI3K/Akt/mTOR signaling pathway related proteins were detected by western blot. RESULTS: Osthole reduced the systolic and diastolic blood pressure of SHRs, improved the histopathological changes of SHRs kidney, reduced the activity of MDA in SHRs kidney, and increased the activity of SOD and GSH. Osthole reduced the levels of p-PI3K, p-Akt and p-mTOR. CONCLUSION: Osthole reduces the activity of PI3K/Akt/mTOR signaling pathway and exerts a protective effect on renal oxidative stress injury in aged spontaneously hypertensive rats.

Di'ao Xinxuekang activates IRS-1/PI3K/Akt signal pathway to improve insulin resistance in nonalcoholic fatty hepatitis mice / 中国临床药理学与治疗学

AIM: To study the effect and mechanism of Di'ao Xinxuekang (DXXK) on insulin resistance in nonalcoholic steatohepatitis (NASH) mice. METHODS: C57BL/6J mice were randomly divided into normal group and model group. After 16 weeks of high-fat diet, the model group was randomly divided into model group and Pioglitazone group (6.0 mg · kg

Research progress on PI3K/AKT/mTOR signaling pathway mutations and related targeted therapies in triple-negative breast cancer / 中国肿瘤生物治疗杂志

@#[摘 要] 三阴性乳腺癌（TNBC）是最具侵袭性的乳腺癌亚型，PI3K/AKT/mTOR信号通路失调是TNBC最常见的致癌突变之一，靶向PI3K/AKT/mTOR信号通路是治疗TNBC的重要方向。本文着重介绍了PI3K/AKT/mTOR信号通路的机制，TNBC中出现的PIK3CA、AKT1或mTOR的突变，以及失活张力PTEN、PIK3R1或INPP4B的突变或丢失，也展现了布帕尼西、帕他色替、依维莫司等PI3K/AKT/mTOR信号通路靶向药物在治疗TNBC中单独、联合应用和与化疗或免疫疗法联用的疗效，同时论述了目前正在进行的各类临床试验及其未来的前景。

Effect and mechanism of gracillin-induced autophagy in lung cancer A549 cells / 中国药房

OBJECTIVE To investigate the effect and mechanism of gracillin from Reineckia carnea on autophagy in non- small cell lung cancer A549 cells. METHODS Using A549 cells as subjects， the effects of different concentrations of gracillin （0.25， 0.5， 1， 2， 4 μmol/L） on the proliferation of cells were detected by CCK-8 after being treated for different time （12， 24， 48 h）. Compared with the control group without medication， the effect of gracillin （2 μmol/L） on the formation of autophagosomes in cells was observed by transmission electron microscope after 24 h of exposure. The aggregation of GFP-LC3 on autophagosome membrane was detected by GFP-LC3 plasmid transfection after being treated with gracillin （0.25， 0.5， 1， 2 μmol/L） for 24 h. Quantitative real-time PCR and Western blot assay were used to detect the mRNA and protein expressions of family with sequence similarity 102 member A（FAM102A）， the expressions of autophagy-related proteins [p62， Beclin-1， microtubule-associated protein 1 light chain 3B （LC3B）]， and the expressions of phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway-related proteins in A549 cells after being treated with gracillin （0.25， 0.5， 1 and 2 μmol/L） for 24 h. RESULTS Gracillin significantly inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. The IC50 was 2.55 μmol/L at 24 h. After 24 h of gracillin treatment， autophagosomes with bilayer membrane structure were found in the cell cytoplasm， and GFP-LC3 green fluorescent spots on autophagosome membrane were obvious， representing an increasing trend as drug concentration. Compared with the control group， mRNA and protein expressions of FAM102A （0.5， 1， 2 μmol/L groups）， protein expression of Beclin-1 （1， 2 μmol/L groups） and LC3B-Ⅱ/LC3B-Ⅰ ratio （2 μmol/L group） were significantly increased in different concentrations of gracillin groups， while the protein expression of p62 （1， 2 μmol/L groups）， and the protein phosphorylations of Akt （1， 2 μmol/L groups） and PI3K （2 μmol/L group） were all decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Gracillin can promote excessive autophagy in A549 cells by up-regulating mRNA and protein expressions of FAM102A and inhibiting PI3K/Akt signaling pathway， thus inhibiting cell proliferation.

Earthworm Protein Protects Vascular Endothelial Function in Spontaneously Hypertensive Rats via PI3K/Akt/Nrf2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E2-related factor 2 （PI3K/Akt/Nrf2） pathway in the aorta of spontaneously hypertensive rats （SHR） and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction （VED）. MethodTen 10-week-old Wistar Kyoto （WKY） rats and fifty SHR rats were selected for a week of adaptive feeding. WKY rats were selected as the normal group， and fifty SHR rats were randomized according to body weight into model， valsartan （8×10-3 g·kg-1·d-1）， and high-， medium-， and low-dose （0.2， 0.1， 0.05 g·kg-1·d-1， respectively） earthworm protein groups. The normal and model groups were administrated with equal volume of double distilled water by gavage. During the drug intervention period， the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration. After 8 weeks of drug intervention， enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ （Ang-Ⅱ） and endothelin-1 （ET-1） in the serum of rats in each group. The corresponding kits were used to determine the levels of nitric oxide （NO）， malondialdehyde （MDA）， glutathione peroxidase （GPX）， superoxide dismutase （SOD）， and ferrous ion （Fe2+）. Hematoxylin-eosin （HE） staining was employed to observe the changes in the intima of the aorta. Fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA levels of PI3K， Akt， Nrf2， heme oxygenase-1 （HO-1）， and glutathione peroxidase 4 （GPX4） in the aortic tissue. Western blotting was used to determine the protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the thoracic aorta. ResultCompared with the normal group， the model group had decreased body mass， increased irritability， severe endothelial damage， elevated blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， lowered NO level （P<0.01）， and down-regulated mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 in the aortic tissue （P<0.01）. Compared with the model group， drug intervention caused no significant change in the body mass， calmed the rats， alleviated the endothelial damage， lowered blood pressure and serum levels of Ang-Ⅱ， ET1， MDA， and Fe2+ （P<0.01）， elevated the NO level （P<0.05）， and up-regulated the mRNA and protein levels of p-PI3K （Tyr467/199）， PI3K， p-Akt （Ser473）， Akt， Nrf2， HO-1， and GPX4 （P<0.05）. ConclusionThe earthworm protein can exert antihypertensive effects by ameliorating VED in SHR. Specifically， it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.

Effect of Qiwei Baizhusan on Cognitive Dysfunction in Rats with Diabetic Encephalopathy Based on PI3K/Akt/GSK-3β Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the therapeutic effect of Qiwei Baizhusan（QWBZS） on diabetic encephalopathy（DE） rat model， and to explore the possible mechanism of QWBZS in the treatment of DE based on phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt）/glycogen synthase kinase-3β（GSK-3β） signaling pathway. MethodForty-eight SPF male Wistar rats were randomly divided into blank group（8 rats） and high-fat diet group（40 rats）. After 12 weeks of feeding， rats in the high-fat diet group were intraperitoneally injected with 35 mg·kg-1 of 1% streptozotocin（STZ） for 2 consecutive days to construct a DE model， and rats in the blank group were injected with the same amount of sodium citrate buffer. After successful modeling， according to blood glucose and body weight， model rats were randomly divided into model group， low， medium and high dose groups of QWBZS（3.15， 6.3， 12.6 g·kg-1）， combined western medicine group（metformin+rosiglitazone， 0.21 g·kg-1）， with 6 rats in each group. The administration group was given the corresponding dose of drug by gavage， and the blank group and the model group were given an equal volume of 0.9% sodium chloride solution by gavage， 1 time/day for 6 weeks. Morris water maze was used to detect the spatial memory ability of DE rats. Fasting insulin （FINS） level was detected by enzyme-linked immunosorbent assay（ELISA） and insulin resistance index（HOMA-IR） was calculated. Hematoxylin-eosin（HE） staining was used to observe the morphological changes of hippocampus in rats， ELISA was used to detect the indexes of oxidative stress in hippocampal tissues， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect mRNA expression levels of PI3K， Akt， nuclear transcription factor-κB（NF-κB）， tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β） in hippocampus， and Western blot was used to detect the protein expression of PI3K， Akt， phosphorylated（p）-Akt， GSK-3β and p-GSK-3β in hippocampus of rats. ResultCompared with the blank group， FINS and HOMA-IR values of the model group were significantly increased（P<0.01）， the path of finding the original position of the platform was significantly increased， and the escape latency was significantly prolonged（P<0.01）， the morphology of neuronal cells in hippocampal tissues was disrupted， the levels of reactive oxygen species（ROS） and malondialdehyde（MDA） in hippocampus of rats were increased， and the activity of superoxide dismutase（SOD） was decreased（P<0.05， P<0.01）， mRNA expression levels of PI3K and Akt were decreased（P<0.01）， mRNA expression levels of NF-κB， TNF-α and IL-1β were increased（P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly decreased， and the protein expression of GSK-3β was significantly increased（P<0.01）. Compared with the model group， the FINS and HOMA-IR values of the medium dose group of QWBZS and the combined western medicine group were significantly decreased（P<0.01）， the path of finding the original position of the platform and the escape latency were significantly shortened（P<0.01）， the hippocampal tissue structure of rats was gradually recovered， and the morphological damage of nerve cells was significantly improved， the contents of ROS and MDA in hippocampus of rats decreased and the level of SOD increased（P<0.01）， the mRNA expression levels of PI3K and Akt were increased（P<0.01）， and the mRNA expression levels of NF-κB， TNF-α and IL-1β were decreased （P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly increased（P<0.01）， and the expression of GSK-3β was significantly decreased（P<0.01）. ConclusionQWBZS can alleviate insulin resistance in DE rats， it may repair hippocampal neuronal damage and improve learning and cognitive ability of DE rats by activating PI3K/Akt/GSK-3β signaling pathway.

Analysis of Antidepressant Quality Markers of Bupleuri Radix Before and After Vinegar-processing Based on UPLC-Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo analyze the antidepressant quality markers（Q-Marker） of Bupleuri Radix（BP） before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP， and principal component analysis（PCA） orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to identify the differential components in BP that changed significantly before and after vinegar-processing， which were regarded as candidate quality markers（Q-Marker）. Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group， model group， fluoxetine group（2.67 mg·kg-1） and total saponin group（0.72 mg·kg-1）， except the blank group， rats in the other groups were subjected to chronic unpredictable mild stress（CUMS）. Three weeks after the start of modeling， rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks， and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling， 21 days and 28 days after administration， body mass weighing， sucrose preference test and open field test were performed on each group . After 28 days of administration， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase（PI3K）， protein kinase B（Akt）， mammalian target of rapamycin（mTOR）， glycogen synthase kinase-3β（GSK-3β）， forkhead box transcription factor O3a（FoxO3a） and β-catenin in hippocampal tissues of rats in each group， while protein expression levels of PI3K， Akt， mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing， and 9 components such as saikosaponin A， saikosaponin B1， saikosaponin B2， saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network， saikosaponin A， saikosaponin B1， saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests， after 28 d of administration， compared with the blank group， the body mass， sucrose preference index and open field total score of rats in model group， fluoxetine group and total saponin group decreased significantly（P<0.01）. Compared with the model group， the body mass， sucrose preference index and open field total score in total saponin group increased significantly（P<0.01）. Compared with the blank group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the model group decreased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a increased significantly（P<0.05）. Compared with the model group， mRNA expression levels of PI3K， Akt， mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly（P<0.05）， while mRNA expression levels of GSK-3β and FoxO3a decreased significantly（P<0.05）. Compared with the blank group， the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly（P<0.01）， while the protein expression levels of PI3K and FoxO3a increased significantly（P<0.01）. Compared with the model group， the expression level of Akt in hippocampus of the total saponin group increased significantly（P<0.01）， the mTOR expression level was increased but not statistically significant， while the protein expression levels of PI3K and FoxO3a decreased significantly（P<0.01）. ConclusionThe chemical constituents of BP changed greatly after vinegar-processing， and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis， pharmacodynamics， network pharmacology and signaling pathway， which provided a reference for further research on quality control， pharmacodynamic substance basis and processing mechanism of BP.

Effect of South African Herb Hoodia gordonii on Liver Glucolipid Metabolism and PI3K/Akt/FoxO1 Signalling Pathway in db/db Mice / 中国实验方剂学杂志

ObjectiveTo observe the effects of the South African herb Hoodia gordonii （HG） on glucolipid metabolism in diabetic db/db mice and explore the possible mechanisms of HG on the liver of db/db mice based on the phosphoinositide-3 kinase （PI3K）/protein kinase B （Akt）/factor forkhead protein O1 （FoxO1） signaling pathway. MethodA total of 30 db/db mice were randomly divided into five groups according to fasting blood glucose： model group， metformin group （0.195 g·kg-1）， and low dose （0.39 g·kg-1）， medium dose （0.78 g·kg-1）， and high dose （1.56 g·kg-1） HG groups， with six m/m mice in each group， and another six m/m mice were set as normal group. The mice in the normal and model groups were given saline of 9 mL·kg-1 by gavage. Body weight， water intake， and fasting blood glucose of the mice in each group were measured weekly. After six weeks of continuous administration， serum insulin （FINS）， low-density lipoprotein cholesterol （LDL）， total cholesterol （TC）， triglyceride （TG）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， urea， and creatinine （CREA） were measured， and liver sections were embedded and stained with hematoxylin-eosin （HE）， periodic acid-Schiff （PAS）， and oil red O. Protein expression of PI3K p85， p-Akt， and p-FoxO1 in liver was detected by immunohistochemistry. The mRNA expression of PI3K， Akt， and FoxO1 in liver tissue was detected by real-time polymerase chain reaction （Real-time PCR）. ResultAfter six weeks of administration intervention， it was found that fasting blood glucose was significantly downregulated in mice in the three HG groups （P<0.05）. The level of islet resistance index was significantly reduced in both the low and medium dose HG groups （P<0.05）. The expression levels of TC， TG， and LDL were reduced in all HG groups （P<0.05， P<0.01）. Pathologically， HG could alleviate hepatocyte steatosis， reduce the volume and content of lipid droplets in liver， and increase the distribution of glycogen granules in liver to some extent in mice. Immunohistochemical assays revealed that PI3K p85 protein expression was significantly increased in the low， medium， and high dose HG groups compared with the model group （P<0.01）. p-Akt protein expression was significantly increased in the medium and high dose HG groups （P<0.05， P<0.01）. p-FoxO1 protein expression was significantly increased in the low， medium， and high dose HG groups （P<0.05， P<0.01）. Compared with the model group， PI3K mRNA was increased in low dose， medium dose， and high dose HG groups （P<0.05）， and Akt mRNA was increased in high dose HG group （P<0.05）. FoxO1 mRNA was decreased in low dose， medium dose， and high dose HG groups （P<0.05）. ConclusionHG can ameliorate the disorder of glucolipid metabolism in db/db mice， which may be related to its activation of the hepatic PI3K/Akt/FoxO1 signaling pathway.

Berbamine Hydrochloride Ameliorates Sorafenib Resistance by Regulating Autophagy and PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of berbamine hydrochloride on sorafenib resistance in hepatocellular carcinoma cells and the underlying mechanisms. MethodThe sorafenib-resistant cell line SMMC-7721/S was selected by the concentration increment method starting at 1.25 μmol·L-1 sorafenib. Both SMMC-7721 and SMMC-7721/S cells were treated with 0， 2.5， 5， 10， 15， 20 μmol·L-1 sorafenib， and the cell counting kit-8 （CCK-8） assay was employed to determine the half maximal inhibitory concentration （IC50） and calculate the resistance index （RI）. Western blot was conducted to compare the expression of proteins involved in autophagy and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway between SMMC-7721 and SMMC-7721/S cells. Furthermore， SMMC-7721/S cells were treated with 5 μmol·L-1 berbamine hydrochloride alone or in combination with 2.5， 5， 10 μmol·L-1 sorafenib， and the cell growth was assessed by the CCK-8 assay. In addition， SMMC-7721 and SMMC-7721/S cells were treated with 5 μmol·L-1 berbamine hydrochloride alone or in combination with 5 μmol·L-1 sorafenib， and the cell proliferation was examined by the colony formation assay. The immunofluorescence assays with Microtubule-associated protein 1 light chain 3 （LC3） and LysoTracker as probes were employed to assess the lysosomal acidification in SMMC-7721 cells treated with 5 μmol·L-1 berbamine hydrochloride or 0.1 μmol·L-1 autophagy inhibitor bafilomycin A1 （Baf）. Further， the expression of proteins involved in autophagy and PI3K/Akt/mTOR signaling pathway was determined by Western blot and compared between groups. ResultSorafenib showed the IC50 of 9.56 mol·L-1 （P<0.01） and 7.99 mol·L-1 for SMMC-7721/S and SMMC-7721 cells， respectively， at 24 h. The resistance index （RI） of SMMC-7721/S for sorafenib was 1.20 （P<0.01）， which indicated mild resistance. Compared with SMMC-7721 cells， SMMC-7721/S cells exhibited up-regulated expression of p-mTOR， p-Akt， and LC3Ⅱ， down-regulated expression of p62 protein （P<0.01）， and unchanged Akt protein level. CCK-8 and colony formation assays demonstrated that the combination of berbamine hydrochloride and sorafenib exhibited a synergistic effect （Q>1.15）， with berbamine hydrochloride partially reversing the resistance of liver cancer cells to sorafenib. The immunofluorescence detection of LC3 revealed that berbamine hydrochloride and Baf significantly increased LC3 in SMMC-7721 cells. The detection with LysoTracker as the probe showed that berbamine hydrochloride inhibited the acidity of lysosomes in SMMC-7721 cells （P<0.01）， indicating the suppression of autophagy. Berbamine hydrochloride further enhanced the downregulation of p-mTOR and p-Akt protein levels and did not change the Akt protein level in SMMC-7721 cells exposed to sorafenib. Berbamine hydrochloride inhibited the increase in p-mTOR expression， down-regulated the p-Akt protein level， and did not change the total Akt protein level in the SMMC-7721/S cells exposed to sorafenib. ConclusionBerbamine hydrochloride can ameliorate the resistance of liver cancer cells to sorafenib by inhibiting cellular autophagy and the PI3K/Akt/mTOR signaling pathway.

PI3K/AKT/mTOR in synovial fluid extends the proinflammatory response of macrophage polarization in knee osteoarthritis / 安徽医科大学学报

Objective @#Given that the PI3K/AKT/mTOR signaling pathway is associated with the progression of knee osteoarthritis (KOA) , this study aims to investigate whether the polarization induction of synovial macrophages mediated by the PI3K/AKT/mTOR signaling axis is the cause of KOA progression . @*Methods @#The synovial fluid of KOA KL-Ⅱ and KL-Ⅲ patients and normal individuals was collected , and the percentage of M1 macrophages (CD80 , CD86) and M2 macrophages (CD163 , CD206) in the synovial fluid (M1 /M2 ratio) was measured to e- valuate the polarization of macrophage cytokines such as IL-1 , IL-6 , IL-10 , and tumor necrosis factor (TNF) -α, transforming growth factor ( TGF)-βExpression in KOA synovial fluid , and detect and analyze of key molecules PI3K/AKT/mTOR signaling axis PI3K , AKT3 , mTORC1 , and inducible nitric oxide synthase ( iONS) in KOA synovial fluid . @*Results @#Compared with the synovial fluid of normal individuals , the percentage of M1 macrophages (CD80 , CD86) in KOA patients increased (P < 0. 01) , and the M1 /M2 ratio increased ( P < 0. 001) ; The ex- pression of IL-1 , IL-6 , and TNF-αin the synovial fluid of the KOA group was also higher than that of the control group (P < 0. 01) , while the expression of IL-10 and TGF-βin the KOA group was significantly reduced ( P < 0. 01) ; The key proteins PI3K , AKT3 , mTORC1 , and downstream inflammatory factor iONS in the PI3K/AKT/ mTOR signaling pathway in the synovial fluid of the KOA group were higher than those in the control group (P < 0. 01) . @*Conclusion @#In KOA synovial fluid , M1 macrophage polarization plays a dominant role , and the inflam- matory response mediated by M1 macrophage polarization may be the cause of synovitis . At the same time , the PI3K/AKT/mTOR signaling pathway may mediate the polarization of M1 macrophages involved in KOA inflammato- ry response .

DCLK1 Promotes Malignancy of A549 Cell Line by Activating FAK/PI3K/AKT/mTOR Pathway / 肿瘤防治研究

Objective To investigate the effects of doublecortin-like kinase 1 (DCLK1) on the malignant biological behaviors, such as proliferation, migration, and invasion, of A549 cell line and their corresponding mechanisms. Methods DCLK1-overexpressing A549 cell lines were established through lentiviral infection, and DCLK1 expression was validated by using RT-PCR and Western blot analysis. Proliferation ability was assessed with CCK-8 and plate cloning assays, and migration and invasion abilities were examined with Transwell assays. The pathway regulated by DCLK1 in lung adenocarcinoma was analyzed on the basis of the TCGA lung adenocarcinoma cohort with pathway enrichment analysis and verified through Western blot analysis. Results DCLK1 overexpression in A549 cells promoted cell proliferation, migration, and invasion. The inhibition of the FAK/PI3K/AKT/mTOR signaling pathway impaired the DCLK1-mediated malignant behavior of A549 cells. Conclusion DCLK1 promotes the malignant behavior of A549 cells through the activation of the FAK/PI3K/AKT/mTOR signaling pathway.

Icariin Regulates Glucocorticoid-induced Autophagy of Bone Microvascular Endothelial Cells Through PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the impact of icariin （ICA） on autophagy in glucocorticoid-induced bone microvascular endothelial cells （BMECs） mediated by the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodBMECs were isolated and cultured from femoral heads obtained during total hip arthroplasty and identified using immunofluorescence staining. The experimental cells were divided into four groups： A control group， a glucocorticoid group （100 mg·L-1 hydrocortisone）， an ICA group （100 mg·L-1 hydrocortisone+6.7×10-3 mg·L-1 ICA）， and a Rapamycin group （100 mg·L-1 hydrocortisone+6.7×10-3 mg·L-1 ICA+1 mg·L-1 rapamycin）. Autophagy in BMECs was induced using 100 mg·L-1 hydrocortisone. LC3 fluorescence staining was used to observe the peak of autophagy at different time points. Western blot analysis was employed to analyze the expression of autophagy-related proteins and PI3K/Akt/mTOR pathway proteins in each group. Electron microscopy was used to observe autophagosomes and autolysosomes in the cells. ResultHydrocortisone at 100 mg·L-1 induced autophagy in BMECs， reaching a peak at around 5 hours， which then declined with further intervention. Compared to the control group， the glucocorticoid group showed cell membrane damage， disordered organelle arrangement， and a large number of autophagosomes and autolysosomes. Compared to the glucocorticoid group， the ICA group had more intact cell membranes， sparser organelle arrangement， and fewer autophagosomes and autolysosomes. Compared to the ICA group， the Rapamycin group showed cell membrane damage， disordered organelle arrangement， and more autophagosomes and autolysosomes. Compared to the control group， the glucocorticoid group had significantly increased expression of light chain 3B （LC3B）， Atg4B， and p62 （P<0.01）. Compared to the glucocorticoid group， the ICA group showed significantly decreased expression of LC3B， Atg4B， p62， and Beclin-1 （P<0.01）. Compared to the ICA group， the Rapamycin group had significantly increased expression of Atg4B and p62 （P<0.01）. Compared to the control group， the glucocorticoid group had significantly decreased expression of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR （P<0.01）. Compared to the glucocorticoid group， the ICA group showed significantly increased expression of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR （P<0.01）. Compared to the ICA group， the Rapamycin group had significantly decreased expression of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR （P<0.01）. Ubiquitination levels were significantly decreased in the glucocorticoid group compared to the control group （P<0.01）. Compared to the glucocorticoid group， ubiquitination levels were significantly increased in the ICA group （P<0.01）， and significantly decreased in the Rapamycin group compared to the ICA group （P<0.01）. ConclusionThe glucocorticoid-induced autophagy in BMECs is time-dependent. ICA inhibits glucocorticoid-induced autophagy in BMECs， and this effect may be related to the regulation of the PI3K/Akt/mTOR pathway.