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ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg-1）， paeoniflorin （50 mg·kg-1） + inhibitor （20 mg·kg-1） group， and high dose （50 mg·kg-1）， medium dose （25 mg·kg-1）， and low dose （12.5 mg·kg-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.
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ObjectiveTo observe the effects of Hirudo， Notoginseng Radix et Rhizoma， and drug pair on renal pathological morphology and protein phosphatase 2A （PP2A）/adenylate activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signal pathway in rats with chronic renal failure （CRF）. MethodThe 55 male SD rats were randomly divided into a normal group （n=11） and a modeling group （n=44）. The normal group was fed conventionally， and the modeling group was given 0.25 g·kg-1·d-1 adenine by gavage for 28 days to replicate the CRF model. After successful modeling， rats were randomly divided into model group， Hirudo group （3 g·kg-1·d-1）， Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， and Hirudo + Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， with 9 rats in each group. The normal group and model group were given a constant volume of normal saline by intragastric administration for 30 days. At the end of the experiment， the levels of serum creatinine （SCr） and urea nitrogen （BUN） in all groups were measured. The renal pathological morphology changes were observed by hematoxylin-eosin （HE） staining， Masson staining， and electron microscopy. The mRNA expressions of PP2A， AMPK， and mTOR were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of PP2A， AMPK， phosphorylation（p）-AMPK， mTOR， and p-mTOR in renal tissue were detected by Western blot. ResultCompared with the normal group， the renal pathological structure changes were obvious， and the levels of SCr and BUN were significantly increased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression were significantly increased， and the p-AMPK/AMPK was significantly decreased in the model group （P<0.05）. Compared with the model group， the renal pathological morphology changes were significantly improved， and the levels of SCr and BUN were significantly decreased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression in the renal tissue were significantly decreased， and the p-AMPK/AMPK was significantly increased （P<0.05） in all groups after drug intervention. In addition， the effect in the Hirudo+Notoginseng Radix et Rhizoma group was better. The mRNA expression levels of AMPK and mTOR in the renal tissue were not significantly different among the normal group， model group， and other groups. ConclusionThe efficacy of Hirudo and Notoginseng Radix et Rhizoma pairs in improving renal fibrosis in rats with CRF is significantly better than that of the single drug， and its improvement on renal fibrosis in rats with CRF may be related to the regulation of PP2A/AMPK/mTOR signaling pathway.
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ObjectiveTo investigate the regulatory effect of Danggui Shaoyaosan on adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/Unc-51-like kinase-1 （ULK1） signaling pathway in the rat model of metabolism-associated fatty liver disease （MAFLD）. MethodSixty SD rats were randomized into control， model， western medicine （polyene phosphatidylcholine capsules，0.144 g·kg-1）， and low-， medium-， and high-dose （2.44， 4.88， 9.76 g·kg-1， respectively） Danggui Shaoyaosan groups. After being fed with a high-fat diet for 8 weeks， the rats in each group were administrated with corresponding drugs for 4 weeks. At the end of drug treatment， serum and liver tissue were collected for subsequent determination of related indicators. ResultCompared with the control group， the model group showed increased contents of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C） and activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum， increased contents of TC， TG， and free fatty acids （FFAs） in the liver （P<0.01）， and decreased content of high-density lipoprotein cholesterol （HDL-C） in the serum （P<0.01）. Furthermore， the model group showed down-regulated protein levels of p-AMPK， microtubule-associated protein 1 light chain 3B （LC3B） Ⅱ， Beclin1， and ULK1 （P<0.01） and up-regulated protein levels of p-mTOR and ubiquitin-binding protein p62 in the liver （P<0.01）. The hepatic steatosis was obvious and the NAFLD activity score （NAS） and oil red O staining area increased in the model group， （P<0.05， P<0.01）. Compared with the model group， Danggui Shaoyaosan reduced the contents of TC and TG and the activities of ALT and AST in the serum， lowered the levels of TC， TG， and FFA in the liver， down-regulated the protein levels of p-mTOR and p62 （P<0.01）， elevated the serum HDL-C level， and up-regulated the protein levels of p-AMPK， LCBⅡ， Beclin1， and ULK1 in the liver （P<0.05， P<0.01）. Moreover， it alleviated hepatic steatosis and decreased the NAS and oil red O staining area （P<0.05， P<0.01）. ConclusionDanggui Shaoyaosan has therapeutic effect on MAFLD rats by regulating AMPK/mTOR/ULK1 signaling pathway to enhance autophagy.
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ObjectiveTo explore the mechanism of Dihuang Yinzi （DHYZ）in improving astrocyte injury in the brain and regulating energy metabolism and autophagy disorder in Alzheimer's disease （AD） model mice. MethodForty male APP/PS1 transgenic mice aged four months were randomly divided into a model group and a model + DHYZ group （2.5 g·kg-1）， with 20 mice in each group. Forty C57BL/6J mice with the same background and same age were randomly divided into a control group and a control + DHYZ group （2.5 g·kg-1）， with 20 mice in each group. The mice in the control group and the model group were administered with an equal volume of sterilized normal saline by gavage， once a day for 150 days. Novel object recognition test and step-down test were performed to evaluate the learning and memory ability of mice. The expression of glial fibrillary acidic protein （GFAP） in astrocytes was detected by immunofluorescence and Western blot. High-performance liquid chromatography （HPLC） was used to detect adenosine triphosphate （ATP）， adenosine diphosphate （ADP）， and adenosine monophosphate （AMP） in brain tissues of mice， and the data obtained were used to calculate energy charge （EC） levels. The phosphorylation levels of liver kinase B1 （LKB1）， adenosine 5′-monophosphate （AMP）-activated protein kinase （AMPK）， UNC-51-like kinase 1 （ULK1）， and mammalian target of rapamycin （mTOR） and the expression levels of autophagy-related proteins Beclin-1， microtuble-associated protein 1 light chain 3 （LC3）-Ⅱ/LC3-Ⅰ， and p62 in mouse brain were measured by Western blot. ResultCompared with the control group， the model group showed decreased novel object recognition index， shortened retention latency， increased error times in the step-down test， up-regulated protein expression of GFAP， decreased content of ATP， ADP， and EC in brain tissues， elevated AMP ， increased levels of p-AMPK， p-LKB1， and p-mTOR， and protein expression of p62 ， and down-regulated p-ULK1 level and protein expression of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ（P<0.01）， while the above experimental indexes were not significantly different in the control + DHYZ group. Compared with the model group， the model + DHYZ group showed increased novel object recognition index（P<0.05）， prolonged retention latency（P<0.01）， decreased error times（P<0.01） in the step-down test， reduced protein expression of GFAP（P<0.05）， increased content of ATP， ADP， and EC in brain tissues （P<0.05， P<0.01）， decreased AMP content（P<0.05）， reduced p-AMPK， p-LKB1， and p-mTOR levels and protein expression of p62， and up-regulated p-ULK1 level and protein expression of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ（P<0.01）. ConclusionBy protecting astrocytes， DHYZ can improve energy metabolism and autophagy disorder in AD mice to improve the learning and memory ability of model mice.
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Mammalian target of rapamycin (mTOR) controls cellular anabolism, and mTOR signaling is hyperactive in most cancer cells. As a result, inhibition of mTOR signaling benefits cancer patients. Rapamycin is a US Food and Drug Administration (FDA)-approved drug, a specific mTOR complex 1 (mTORC1) inhibitor, for the treatment of several different types of cancer. However, rapamycin is reported to inhibit cancer growth rather than induce apoptosis. Pyruvate dehydrogenase complex (PDHc) is the gatekeeper for mitochondrial pyruvate oxidation. PDHc inactivation has been observed in a number of cancer cells, and this alteration protects cancer cells from senescence and nicotinamide adenine dinucleotide (NAD+) exhaustion. In this paper, we describe our finding that rapamycin treatment promotes pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) phosphorylation and leads to PDHc inactivation dependent on mTOR signaling inhibition in cells. This inactivation reduces the sensitivity of cancer cells' response to rapamycin. As a result, rebooting PDHc activity with dichloroacetic acid (DCA), a pyruvate dehydrogenase kinase (PDK) inhibitor, promotes cancer cells' susceptibility to rapamycin treatment in vitro and in vivo.
Subject(s)
Humans , Sirolimus/pharmacology , Dichloroacetic Acid/pharmacology , Pyruvate Dehydrogenase Complex , TOR Serine-Threonine Kinases , Mechanistic Target of Rapamycin Complex 1 , Neoplasms/drug therapyABSTRACT
Abstract This study aimed to investigate the role and signaling pathways of β3-AR in myocardial ischemia/reperfusion (I/R) injury, which is one of the leading causes of death worldwide. 47 male rats were randomly divided into two main groups to evaluate infarct size and molecular parameters. Rats in both groups were randomly divided into 4 groups. Control (sham), I/R (30 min ischemia/120 min reperfusion), BRL37344 (BRL) (A) (5 µg/kg single-dose pre-treatment (preT) before I/R) and BRL (B) (5 µg/kg/day preT for 10 days before I/R). Infarct size was determined with triphenyltetrazolium chloride staining and analyzed with ImageJ program. The levels of AMPK, SIRT1, mTOR, and p70SK6 responsible for cellular energy and autophagy were evaluated by western blot. Infarct size increased in the I/R group (44.84 ± 1.47%) and reduced in the single-dose and 10-day BRL-treated groups (32.22 ± 1.57%, 29.65 ± 0.55%; respectively). AMPK and SIRT1 levels were decreased by I/R but improved in the treatment groups. While mTOR and p70S6K levels increased in the I/R group, they decreased with BRL preT. BRL preT protects the heart against I/R injury. These beneficial effects are mediated in part by activation of AMPK and SIRT1, inhibition of mTOR and p70S6K, and consequently protected autophagy.
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Diabetic kidney disease （DKD） is one of the typical microvascular complications in patients with diabetes and a major cause of end-stage renal disease， with the pathogenesis remains to be elucidated. It may be associated with hemodynamic effects， genetic factors， kidney inflammatory injury， oxidative stress， autophagy dysregulation， metabolic disorders and so on. Because of its complex mechanism， there are no specific prevention and treatment measures in clinical practice. AMP-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway is a classical pathway involved in the regulation of autophagy. This pathway can be activated for treating DKD. Recent studies have demonstrated that the active components in Chinese medicinal herbs play a role in the prevention and treatment of DKD by directly acting on targeted cells and autophagy targets， which has attracted extensive attention. Researchers have extensively studied the occurrence and development of DKD and the mechanism of drug intervention in DKD， and the results prove that AMPK/mTOR pathway plays a role in the development of this disease. The active components in Chinese medicinal herbs regulate the AMPK/mTOR signaling pathway to affect autophagy， alleviate oxidative stress， inflammation， and extracellular matrix aggregation， and promote the generation of autophagosomes， thus mitigating kidney injury. This paper mainly reviews the relationship between AMPK/mTOR signaling pathway， autophagy， and DKD and the mechanism of active components in Chinese medicinal herbs in mediating autophagy via the AMPK/mTOR pathway， aiming to provide a theoretical basis for the clinical prevention and treatment of DKD.
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ObjectiveTo investigate the effect of Shouwuwan on the synaptic plasticity of hippocampal neurons in the rat model of D-galactose-induced aging via the mammalian target of rapamycin （mTOR） signaling pathway. MethodA total of 50 male SPF-grade SD rats were randomized into normal group， model group， vitamin E （0.018 g·kg-1） group， and low- and high-dose （1.08，2.16 g·kg-1， respectively） Shouwuwan groups. Except the normal group， the other four groups were treated with D-galactose （120 mg·kg-1） for the modeling of aging. The rats were simultaneously administrated with corresponding agents by gavage. After six weeks of modeling， Morris water maze test was carried out to examine the behavioral changes. The whole brain and hippocampus samples were collected. The expression of postsynaptic density protein-95 （PSD-95） and synaptophysin （SYN） in the hippocampus was detected by immunohistochemistry. Golgi staining was employed to observe the changes in the morphology and function of neurons. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were respectively employed to determine the mRNA and protein levels of mTOR， phosphorylated （p）-mTOR， p70 ribosome protein S6 kinase （p70S6K）， phosphorylated （p）-p70S6K， eukaryotic translation initiation factor 4E-binding protein 2 （4EBP2）， and phosphorylated （p）-4EBP2 in the hippocampus. ResultCompared with the normal group， the model group showed slow swimming （P<0.01）， extended total swimming distance （P<0.05）， prolonged latency （P<0.01）， and decreased crossing number （P<0.01）. The modeling inhibited the expression of PSD-95 and SYN in the CA1 region of the hippocampus （P<0.01）， with the weakest staining effect and the smallest region， decreased the intersections of hippocampal neuron dendrites with concentric circles at the concentric distance of 100， 140， 180， and 200 μm from the cell body （P<0.01）， and reduced the length and density of dendritic spine （P<0.01）. In addition， the modeling up-regulated the mRNA levels of mTOR and p70S6K and the protein levels of p-mTOR and p-p70S6K （P<0.01） and down-regulated the mRNA level of 4EBP2 and the protein levels of 4EBP2 and p-4EBP2 （P<0.01）. Compared with the model group， low- and high-dose Shouwuwan increased the average swimming speed （P<0.01）， shortened the latency （P<0.01）， increased the crossing number （P<0.01）， promoted the expression of PSD-95 and SYN in the hippocampal CA1 region （P<0.01）， increased the intersections between hippocampal neuronal dendrites and concentric circles at the concentric distance of 100， 140， 180，200 μm from the cell body （P<0.01）， and increased the number， length， and density of dendritic spine （P<0.01）. Furthermore， Shouwuwan down-regulated the protein levels of p-mTOR and p-p70S6K （P<0.01）， up-regulated the protein levels of 4EBP2 and p-4EBP2 （P<0.05，P<0.01）， down-regulated the mRNA levels of mTOR and p70S6K （P<0.01）， and up-regulated the mRNA level of 4EBP2 （P<0.01）. ConclusionShouwuwan can improve the learning and memory ability of rats exposed to D-galactose， promote the expression of proteins associated with synaptic plasticity， improve the morphology of neurons， repair neural function， reduce neuronal apoptosis， and inhibit mTOR signaling pathway to delay brain aging.
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Colorectal cancer （CRC）， as the third most common cancer in the world， develops from the colonic and rectal epithelial cells. With the rapid development of the global economy， the incidence of CRC in developing countries has been increasing year by year. In the past few years， although preventive colonoscopy screening has improved the survival rate of CRC patients， the majority of cases are still detected after symptoms appear. Currently， the clinical treatment of CRC carries high surgical risks and is prone to recurrence， while radiotherapy and chemotherapy have significant side effects and cause a heavy psychological burden on patients. Therefore， there is currently no ideal treatment protocol for CRC. The phosphatidylinositol 3-kinase/protein kinase B （PI3K/Akt） pathway， as a classical oncogenic pathway， provides potential for the diagnosis and treatment of various malignant diseases， and offers a new direction for the treatment of CRC. In recent years， traditional Chinese medicine （TCM） has become a major focus in cancer treatment due to its advantages in "preventing and treating diseases" and its multiple components， targets， and pathways. Its advantages in having fewer side effect complement western medicine in treatment. Multiple studies have shown that Chinese medicinal monomers and compound formulas can inhibit the proliferation， invasion， migration， and angiogenesis of CRC cells， promote apoptosis and autophagy of cancer cells， and slow down the development of CRC by intervening in the PI3K/Akt signaling pathway， thereby achieving the therapeutic effect on CRC. In recent years， the research progress related to this has been updating rapidly. Previous literature failed to timely incorporate the latest research results， which brought many inconveniences to the literature search of many scholars. Therefore， this article summarized the relevant information from PI3K/Akt pathway， the association between the PI3K/Akt pathway and CRC， and the progress of TCM intervention in the treatment of CRC to provide references for the development of CRC in molecular biology and the clinical development of new drugs in the future.
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ObjectiveTo investigate whether Jiedu Huoxue prescription can induce macrophage autophagy and inhibit inflammatory response to stabilize vulnerable plaques of atherosclerosis （AS） by regulating phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodThirty ApoE-/- mice fed with high-fat diet were randomly assigned into model， low-， medium-， and high-dose （5.35， 10.7， and 21.4 g·kg-1·d-1， respectively） Jiedu Huoxue prescription （Chinese medicine）， and rapamycin （2 mg·kg-1·d-1） groups. Six ApoE-/- mice fed with common diet were used as the control group， and 6 C57BL/6J mice fed with common diet as the blank group. The drugs or equal volume of normal saline were administrated by gavage after 7 weeks of modeling， and the treatment lasted for 4 weeks. The serum levels of lipids and inflammatory cytokines ［monocyte chemoattractant protein-1 （MCP-1） and interleukin-6 （IL-6）］ were measured. Hematoxylin-eosin （HE） staining was employed to observe the pathological changes of the vascular wall of the aortic root. Immunohistochemistry was employed to detect the expression of macrophages/monocytes monoclonal antibody （MOMA-2） and α-smooth muscle actin （α-SMA）. Transmission electron microscopy was employed to count the autophagosomes in the aorta， and Western blot to determine the protein levels of Beclin-1， LC3， PI3K， Akt， and mTOR. ResultCompared with the control group， the model group showed elevated serum levels of lipids， MCP-1， and IL-6 （P<0.05）， inhibited expression of MOMA-2 and α-SMA （P<0.05， P<0.01）， up-regulated protein level of Beclin-1 （P<0.05）， and down-regulated protein levels of PI3K， Akt， and mTOR （P<0.05， P<0.01）. The model group presented obvious atherosclerotic plaques on the inner wall of the aorta， infiltration of inflammatory cells in the plaque， thickened and disarranged vascular intima where the plaque was attached， decreased autophagosomes and mitochondria， and destroyed mitochondrial structure. Chinese medicine and rapamycin groups showed lower levels of total cholesterol， triglycerides， low-density lipoprotein cholesterol， MCP-1， and IL-6 （P<0.05）， higher level of high-density lipoprotein cholesterol （P<0.05）， inhibited expression of MOMA-2 and α-SMA （P<0.05， P<0.01）， higher protein levels of Beclin-1 and LC3Ⅱ （P<0.05， P<0.01）， and lower protein levels of PI3K， Akt， and mTOR （P<0.05， P<0.01） than the model group. Moreover， Chinese medicine and rapamycin groups showed only a small number of atherosclerotic plaques on the inner wall of the aorta， reduced infiltration of inflammatory cells and thickness of the blood vessel wall， and increased autophagosomes and autophagic lysosomes. ConclusionJiedu Huoxue prescription can improve lipid metabolism， enhance macrophage autophagy， and reduce AS-induced inflammation to improve the stability of vulnerable plaques in AS mice by inhibiting the PI3K/Akt/mTOR signaling pathway.
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ObjectiveTo study the mechanism of Danggui Sinitang in mitigating gouty arthritis （GA） in rats by regulating autophagy via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodSixty male SD rats were randomly assigned into normal， model， colchicine （0.3 mg·kg-1）， and low-， medium-， and high-dose Danggui Sinitang （6.54， 13.08， and 26.16 g·kg-1） groups （n=10） and administrated with corresponding drugs by gavage. The rats in the normal group and model group were administrated with equal volume of normal saline by gavage for 7 days. One hour after administration on day 5， the GA model was established by injecting sodium urate suspension （50 g·L-1） into the right ankle joint of rats in other groups except the normal group， and the rats in the normal group were injected with sterile normal saline of the same volume. The swelling and pathological changes of the ankle joint were observed. The serum levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and IL-1β were determined. Western blot was employed to determine the protein levels of PI3K， phosphorylated PI3K （p-PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， mTOR， phosphorylated mTOR （p-mTOR）， microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ （LC3Ⅱ/Ⅰ）， autophagy effector Beclin-1， and ubiquitin-binding protein p62 in the synovial tissue. Real-time fluorescent quantitative PCR （Real-time PCR） was employed to determine the mRNA levels of PI3K， Akt， mTOR， LC3， Beclin-1 and p62. ResultCompared with the normal control， the model group showed increased joint swelling index （P<0.01）， elevated serum levels of TNF-α， IL-6， and IL-1β， inflammatory cell infiltration， and fibrous tissue hyperplasia. In addition， the model group showed up-regulated protein levels of PI3K， p-PI3K， Akt， p-Akt， mTOR， p-mTOR， and p62 and mRNA levels of PI3K， Akt， mTOR， and p62 in the synovial tissue， while it showed down-regulated protein levels of LC3Ⅱ/Ⅰ and Beclin-1 and mRNA levels of LC3 and Beclin-1 （P<0.01）. Compared with the model group， medium- and high-dose Danggui Sinitang alleviated the joint swelling （P<0.01）， lowered the serum levels of TNF-α， IL-6， and IL-1β （P<0.05）， and relieved the inflammatory cell infiltration in the synovial tissue of the ankle joint and the fibrous tissue hyperplasia. Moreover， they down-regulated the protein levels of PI3K， p-PI3K， Akt， p-Akt， mTOR， p-mTOR， and p62 and the mRNA levels of PI3K， Akt， mTOR， and p62 in the synovial tissue （P<0.05）， while they up-regulated the protein levels of LC3Ⅱ/Ⅰ and Beclin-1 and the mRNA levels of LC3 and Beclin-1 （P<0.05）. ConclusionDanggui Sinitang， especially at a high dose， can inhibit PI3K/Akt/mTOR signaling pathway to improve autophagy in the synovial tissue， thereby mitigating GA.
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Osteoporosis （OP）， a common systemic skeletal disease in the elderly， is characterised by bone loss and bone microstructural degeneration. Its clinical manifestations include increased bone fragility and bone pain. Furthermore， OP increases the risk of fracture due to the high bone fragility， which leads to lifelong disability or death， imposing a heavy economic and psychological burden on the patients and their families. The pathogenesis of OP is extremely complex and associated with a variety of factors such as proliferation and differentiation of osteoblasts， impairment of osteoclast activity and function， and abnormalities in autophagy activation. Recent studies have found that mammalian target of rapamycin （mTOR） signaing pathway is involved in the regulation of bone homeostasis， which can promote bone formation and improve bone metabolism and bone microstructure by regulating osteoblast proliferation and differentiation and osteoclast function and activating cellular autophagy， thus playing a crucial role in the prevention and treatment of OP. The prevention and treatment of OP with Chinese medicine has a long history， clear efficacy， multiple targets of action， low adverse effects， and wide medicine sources. Therefore， this paper briefly describes the role of mTOR signaling pathway in the development of OP by reviewing the latest research reports and summarizes in detail the latest research results on the treatment of OP with Chinese medicine extracts and prescriptions via the mTOR signaling pathway. This review aims to provide a basis for the in-depth research on the relationship between mTOR signaling pathway and OP and the clinical application of traditional Chinese medicine in the prevention and treatment of OP.
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Non-small cell lung cancer （NSCLC） is a malignant tumor of the respiratory system with a high incidence. The early symptoms are not typical， and most patients are diagnosed at an advanced stage， which seriously threatens the lives and health of people. Surgery， chemotherapy， and targeted therapy are the main means of treatment at present， but the consequent drug resistance and adverse reactions restrict these treatment methods with certain limitations. In recent years， with the development of traditional Chinese medicine （TCM） in tumor resistance， TCM has attracted extensive attention for its obvious therapeutic effect and fewer adverse reactions. Numerous signaling pathways are involved in the formation and development of NSCLC， where phosphatidylinositol 3 kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway is one of the key regulatory pathways. The PI3K/Akt/mTOR signaling pathway affects the proliferation， invasion， and metastasis of NSCLC cells by affecting the cycle， inhibiting the apoptosis， inhibiting the autophagy of tumor cells， and promoting tumor neovascularization. As revealed by the recent literature， Chinese medicine plays an indispensable role in NSCLC cell autophagy， cell cycle， apoptosis， invasion and metastasis， neovascularization， and reversal of drug resistance by regulating the PI3K/Akt/mTOR signaling pathway. Although some Chinese medicinal extracts or compounds have made great breakthroughs in some mechanisms of action in the treatment of NSCLC， these studies only remain at the level of in vitro cell experiments and animal studies in vivo. Researchers are faced with the great challenge of "transforming the research results of Chinese medicines into clinical applications". Based on the current research status in China and abroad， this paper reviewed Chinese medicine in the intervention in NSCLC through the regulation of PI3K/Akt/mTOR signaling pathway in recent years， in order to open up new ideas for NSCLC drug therapy research and also provide a useful reference for further mechanism research.
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Diabetic kidney disease （DKD）， a chronic kidney disease with unique pathological structural and functional alterations in the kidney， is a common complication of diabetes mellitus （DM）. The majority of researchers believe that the occurrence of this disease is associated with glucose metabolism disorders， oxidative stress， inflammation， endoplasmic reticulum stress， autophagy， and disorders of lipid metabolism and exosome release. The mammalian target of rapamycin （mTOR） signaling pathway， which can maintain glomerular podocyte homeostasis and participate in autophagy， renal fibrosis， oxidative stress， lipid metabolism disorders， and inflammatory response in DKD， has been discovered to play a key role in DKD. Therefore， it has emerged as a novel target for the treatment of DKD. Studies have demonstrated that traditional Chinese medicine can prevent the renal damage in DKD by regulating the mTOR signaling pathway to delay the disease progression and improve the prognosis and the quality of life of the patients. This article summarizes the structure and role of the mTOR signaling pathway in DKD and briefs the research progress in the prevention and treatment of DKD via this signaling pathway by the active components， extracts， and compound prescriptions of Chinese medicines， aiming to present new ideas and approaches for the clinical treatment of DKD with traditional Chinese medicine.
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ObjectiveTo explore the mechanism of Huangqisan （HQS） in regulating autophagy to alleviate hepatic steatosis and improve non-alcoholic fatty liver disease （NAFLD） based on adenosine 5'-monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. MethodThe main chemical components and targets of HQS and NAFLD-related targets were collected from database and the intersection targets were used for Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. The protein-protein interaction （PPI） network was constructed， and in vivo experimental verification was conducted. Sixty C57BL/6J male mice were randomly divided into normal control group （NCD）， model group high-fat diet （HFD）， metformin group （MET， 0.25 g·kg-1）， low-dose Huangqisan group （HQS-L， 0.5 g·kg-1）， and the high-dose Huangqisan group （HQS-H， 1 g·kg-1）， with 12 mice in each group after a one-week acclimatization period. NAFLD model was induced by HFD， and intragastric administration was performed at the same time， once a day for 13 weeks. Random blood glucose， serum total cholesterol （TC）， triglyceride （TG）， non-esterified fatty acid （NEFA）， low density lipoprotein-chdesterol （LDL-C） levels， and liver TG content were determined. The liver weight was weighed， and liver index was calculated. Hematoxylin-eosin （HE） staining， oil red O staining， transmission electron microscope （TEM）， real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot were used to verify the effect and reveal the potential mechanism of C57BL/6J mice in vivo. ResultThrough network pharmacology analysis， combined with previous studies， it was predicted that HQS may improve NAFLD by regulating autophagy via the AMPK/mTOR signaling pathway. The result of in vivo experiment showed that， as compared with NCD group， random blood glucose， body weight， serum TC， LDL-C， NEFA， liver weight， liver index， and liver TG content of mice in the HFD groups were significantly increased （P<0.01）. HE staining showed massive lipid droplets （LDs） vacuolated， oil red O staining showed lipid accumulation in liver cells， and no obvious autophagosomes and autolysosome were observed under TEM. The relative mRNA expression of LC3A、LC3B、AMPKα1 and protein expression of AMPK， phosphory phosphorylated（p）-AMPK， and p-AMPK/AMPK were significantly down-regulated （P<0.01）， while the protein expression of microtubule-associated protein 1 light chain 3 （LC3）Ⅱ/Ⅰ and p-mTOR was significantly up-regulated （P<0.01）. As compared with HFD groups， liver weight， serum TG， and NEFA levels in HQS-L and HQS-H groups were significantly deceased （P<0.05， P<0.01）. HE staining and oil red O staining showed the improvement of liver pathological changes after HQS administration. Under TEM， a small amount of autophagosome and autolysosome were observed. Besides， liver index was significantly decreased in the HQS-L group （P<0.01）， and random blood glucose， serum TC level and liver TG content were significantly decreased in the HQS-H group （P<0.05）. The results of Western blot and Real-time PCR showed that the mRNA expression of LC3A and LC3B and the protein expression of LC3Ⅱ/Ⅰ， p-AMPK， and p-AMPK/AMPK were significantly up-regulated （P<0.01）， while the mRNA expressions of p62 and protein expression of p62 and p-mTOR were significantly down-regulated （P<0.05， P<0.01）. ConclusionHQS may promote autophagy and restore autophagy flux via the AMPK/mTOR signaling pathway to alleviate hepatic steatosis improving NAFLD.
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ObjectiveTo observe the effects of Fuzitang （FZT） on the proliferation of MH7A cells， the human rheumatoid arthritis synovial fibroblasts， and the expression of miR-155 and explore its anti-rheumatoid arthritis mechanism. MethodMH7A cells were cultured in vitro and divided into a blank group， high- （25 g·L-1） and low-dose （12.5 g·L-1） FZT groups， and a positive drug group （hydroxychloroquine， 0.006 25 g·L-1）. The cell proliferation was detected by cell counting kit-8（CCK-8） method， and the change in the MH7A cell cycle was detected by flow cytometry. The mRNA expression of miR-155 and its downstream genes， including SH2 domain-containing inositol 5-phosphatase-1（SHIP-1）， protein kinase B 3（Akt3）， and mammalian target of rapamycin（mTOR）， was detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）， and the protein expression of phosphatidylinositol 3-kinase （PI3K）， Akt3， and mTOR was detected by Western blot. ResultFZT in vitro in a concentration of 6.25 g·L-1 above could inhibit the proliferation of MH7A cells in the significant dose- and time-effect manner. Compared with the blank group， the FZT groups showed increased proportions of cells in the G2/M phase （P<0.05）， and the high-dose FZT group showed a decreased proportion of cells in the G0/G1 phase （P<0.05）. The arresting effect of FZT on the cell cycle was in a significant dose-effect manner. Compared with the blank group， the FZT groups showed down-regulated miR-155 and mTOR mRNA expression （P<0.05）， and the high-dose FZT group showed up-regulated SHIP1 mRNA expression and down-regulated Akt3 mRNA expression （P<0.05）. Compared with the blank group， the FZT groups showed reduced protein expression of PI3K， Akt3， and mTOR （P<0.05）. ConclusionFZT can significantly inhibit the proliferation of MH7A cells， and the mechanism is related to the promotion of the expression of SHIP-1 and down-regulation of the gene expression of the PI3K/Akt3/mTOR signaling pathway by down-regulating the expression of miR-155.
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ObjectiveTo explore the effect and mechanism of Zuogui Jiangtang Tongmai prescription （ZGJTTMP） on astrocytes （ASs） injured by advanced glycation end products（AGEs） combined with oxygen-glucose deprivation （OGD）. MethodCell counting kit-8 （CCK-8） was used to determine the optimal concentration of AGEs and the action time of OGD， and the optimal blood concentration of ZGJTTMP was selected for follow-up experiments. ASs were divided into normal group， model group （AGEs + OGD）， ZGJTTMP group， an adenosine 5'-monophosphate-activated protein kinase （AMPK） inhibitor （Compound C） group， AMPK activator （AICAR） group， and combination group （ZGJTTMP + AICAR）. The morphological changes in ASs in each group were observed under an inverted microscope. The cell survival rate in each group was detected by CCK-8. The content of interleukin-1β（IL-1β）， interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） was detected by enzyme-linked immunosorbent assay （ELISA）. The number of autophagosomes in each group was counted under an electron microscope. The expression of microtubule-associated protein light chain 3 （LC3） was observed by immunofluorescence. The protein expression of LC3， p62， p-AMPK， AMPK， p-mammalian target of rapamycin （mTOR）， mTOR， p-UNC-51 like kinase 1 （ULK1）， and ULK1 was detected by Western blot. ResultAccording to the results of cell survival rate， 200 mg·L-1 AGEs and OGD for 6 h were selected as the optimal modeling conditions for the model group， and 5% was selected as the optimal blood concentration of ZGJTTMP. Under the inverted microscope， the cells were severely damaged after modeling， but the cell injury in the ZGJTTMP group and the Compound C group was significantly improved. As revealed by ELISA results， the content of IL-1β， IL-6， and TNF-α in the model group increased （P<0.01）， and the content of inflammatory factors in the ZGJTTMP group and the Compound C group decreased （P<0.01）. Under the electron microscope， the number of autophagosomes in the model group increased significantly. The immunofluorescence results showed that the expression area of LC3 increased in the model group （P<0.01）， and the ZGJTTMP group and the Compound C group showed decreased number of autophagosomes and reduced expression area of LC3 （P<0.01）. As demonstrated by the results of Western blot， compared with the normal group， the model group showed increased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK （P<0.01） and decreased p62， p-mTOR/mTOR， and p-ULK1/ULK1 （P<0.01）. Compared with the model group， the ZGJTTMP group and the Compound C group showed decreased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK （P<0.01） and increased p62， p-mTOR/mTOR， and p-ULK1/ULK1 （P<0.01）. ConclusionZGJTTMP possesses a protective effect on ASs with inflammatory injury by AGEs combined with OGD， which may be achieved by inhibiting the activation of the AMPK/mTOR/ULK1 pathway related to autophagy， thus inhibiting the overexpression of autophagy.
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ObjectiveTo explore the mechanism of cucurbitacin B （CuB） in inhibiting cell proliferation and glycolysis. MethodCell counting kit-8 （CCK-8） was applied to investigate the effect of different concentrations of CuB （0， 40， 80， 120， 160， 200， 400， and 800 nmol·L-1） on the proliferation of HuCCT1 cells. The effect of different concentrations of CuB （50， 100， and 200 nmol·L-1） on the colony formation ability of HuCCT1 cells was detected by plate cloning assay. The effect of different concentrations of CuB （50， 100， 200 nmol·L-1） on the HuCCT1 cell cycle was analyzed by flow cytometry. Visible spectrophotometry was employed to detect the activity of key glycolytic enzymes hexokinase （HK） and pyruvate kinase （PK）） and changes in glucose consumption， lactate production， and adenosine triphosphate （ATP） production in HuCCT1 cells after administration of different concentrations of CuB （50， 100， 200 nmol·L-1）. Western blotting was used to assay the effect of CuB on the expression of cell cycle-related proteins， proliferation-related proteins， key glycolytic proteins， and Akt/mammalian target of rapamycin （mTOR） pathway-related proteins. ResultAs compared with the blank group， CuB at dose of 160-800 nmol·L-1 after 24 h administration and CuB at dose of 80-800 nmol·L-1 after 48 h administration inhibited the proliferation of HuCCT1 cells in a time- and dose-dependent manner （P<0.05， P<0.01）， and the median inhibitory concentration was 200 nmol·L-1 48 h after administration. CuB can restrain the colony formation ability of HuCCT1 cells in a dose-dependent manner （P<0.01）， and block HuCCT1 cell cycle in G2 phase （P<0.05， P<0.01）. CuB （100 and 200 nmol·L-1） can suppress the activities of HK and PK and reduce cell glucose consumption and production of lactate and ATP （P<0.05， P<0.01）. Western blot results showed that CuB （100 and 200 nmol·L-1） can inhibit the protein levels of cycle-related protein Cyclin B1， proliferating cell nuclear antigen （PCNA）， HK1， HK2， PKM1， PKM2， phosphorylated Akt （p-Akt）， phosphorylated mTOR （p-mTOR）， and phosphorylated ribosomal protein S6 （p-RPS6） （P<0.05， P<0.01）. ConclusionCuB can inhibit aerobic glycolysis in HuCCT1 cells via the Akt/mTOR pathway， thereby affecting cell proliferation.
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ObjectiveTo observe the effects of Scutellariae Radix （SR）-Paeoniae Radix Rubra （PRR） combination of different proportions on the expression of the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear transcription factor κB （NF-κB） and phosphatidylinositol kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathways in liver tissues of rats with hepatic fibrosis and explore the mechanism against hepatic fibrosis. MethodSixty male SD rats of SPF grade were randomly divided into a normal group， a model group， a positive control （silymarin） group， and SR-PRR 1∶1， SR-PRR 1∶2， and SR-PRR 1∶4 groups， with 10 rats in each group. The hepatic fibrosis model was induced in rats except for those in the normal group by intraperitoneal injection of 40% tetrachloromethane （CCl4）-olive oil solution at 3 mL·kg-1， 5 mL·kg-1 for the first time， for 8 weeks， twice per week. After 4 weeks， rats were treated correspondingly at 10 mL·kg-1 by intragastric administration， and the body weight of rats in each group was weighed for 8 weeks. After administration， histopathological changes in the liver were observed. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， hyaluronic acid （HA）， laminin （LN）， albumin （ALB）， alkaline phosphatase （AKP）， and superoxide dismutase （SOD） activities， malondialdehyde （MDA）， and hydroxyproline （HYP） content in liver tissues were detected. The mRNA expression levels of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the model group， SR-PRR combination of different proportions could recover the body weight and improve the pathological injury of the liver. As revealed by enzyme linked immunosorbent assay （ELISA） results， compared with the normal group， the model group showed increased ALT， AST， HA， LN， AKP， MDA， and HYP levels to different degrees （P<0.05）. Compared with the model group， the groups with drug intervention showed decreased levels of ALT， AST， HA， LN， AKP， MDA， and HYP， potentiated SOD activity， and increased level of ALB （P<0.05）. As revealed by Real-time PCR results， compared with the normal group， the model group showed increased mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR （P<0.05）. Compared with the model group， the groups with drug intervention showed reduced mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats （P<0.05）. ConclusionSR-PRR combination of different proportions can improve the histopathological injury in liver tissues caused by CCl4， with the optimal effect observed in the SR-PRR 1∶4 group. SR-PRR may inhibit the development of liver fibrosis by inhibiting the expression of TLR4/MyD88/NF-κB and PI3K/Akt/mTOR signaling pathways， thereby alleviating chemical-induced liver injury.
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Objective To establish a detection system of ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for everolimus concentration in whole blood of liver transplant recipients. Methods The proteins of samples were precipitated with methanol and zinc sulfate, and everolimus-D4 was used as the internal standard. Phenomenex Kinetex PFP column was used. The mobile phase A was water (containing 2 mmol/Lammonium formate and 0.1% formic acid), and the mobile phase B was methanol (containing 2 mmol/L ammonium formate and 0.1% formic acid). The gradient elution was performed with the flow rate of 1 mL/min, the column temperature of 50 ℃ and the injection volume of 1 μL. The multi-reaction monitoring mode was used to quantitatively analyze with electrospray positive ionization. The UPLC-MS/MS detection system required only 100 μL of whole blood, and could achieve a sufficient lower limit of quantification without complicated sample preparation. The total running time was within 4.5 min. Linear regression (1/x2) analysis was performed using peak area of everolimus / peak area of everolimus-D4 (y) and concentration of everolimus/concentration of everolimus-D4 (x) to calculate the calibration function and analyze its accuracy and linear relationship. UPLC-MS/MS was used to detect the trough blood concentration of everolimus in blood samples of 5 recipients after liver transplantation. Results The accuracy of quality control was within 15%, and the linear relationship of everolimus was good in the blood concentration range of 1-100 ng /mL(R2 > 0.990). Trough blood concentration of everolimus measured in blood samples of 5 liver transplant recipients ranged from 3.77 to 9.27 ng/mL. Conclusions The detection system of UPLC-MS/MS in this study is suitable for monitoring the concentration of everolimus in whole blood of liver transplant recipients because of its high accuracy, simple sample processing method and short detection time.