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Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.
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Aim To explore the mechanism of hydroxy-a-sanshool in the treatment of diabetic cardiomyopathy ( DCM) based on label-free quantitative proteomics detection technique. Methods DCM model was established by high fat diet and intraperitoneal injection of streptozotocin ( STZ) . They were divided into control group ( CON group ) , diabetic cardiomyopathy group (DCM group) and hydroxy-a-sanshool treatment group ( DCM + SAN group) . The cardiac function of mice was evaluated by echocardiography, the myocardial morphology was observed by pathology staining, the protective mechanism of hydroxy-a-sanshool on diabetic cardiomyopathy was speculated by proteomic technique , and the expression level of cAMP/PKA signaling pathway and key proteins were verified by Western blotting. Results Cardiac ultrasound and pathology staining showed that hydroxy-a-sanshool had protective effect on the heart of DCM mice. Label-free quantitative proteomic analysis was carried out between DCM + SAN group and DCM group, and 160 differential pro-teins were identified by proteomics, in which 127 proteins were up-regulated and 33 proteins were down regulated ; GO secondary functional annotations showed the biological process, molecular function and cellular component; KEGG enrichment analysis showed that cAMP signaling pathway was the most abundant; protein interaction network showed that PKA as the central node interacted with many proteins in the cAMP signaling pathway. Western blot showed that the relative expression of с AMP, PKA protein in DCM group was significantly lower than that in CON group ( P < 0. 05 ) , while the relative expression of cAMP, PKA protein in DCM + SAN group was significantly higher than that in DCM group ( P < 0. 05 ) . Conclusions Hydroxy-a-sanshool has protective effect on heart function of mice with diabetes, which plays a role through cAMP signaling pathway.
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OBJECTIVE To investigate the effects of polydatin （PD） on cell proliferation， migration， invasion and tumor growth of acute myeloid leukemia （AML）. METHODS Human AML cell KG-1 were divided into normal group， PD low-， medium- and high-concentration groups （10， 30， 60 μmol/L PD）， SQ22536 group [cyclic adenosine monophosphate （cAMP） inhibitor， 100 μmol/L]， high concentration of PD+SQ22536 group （60 μmol/L PD+100 μmol/L SQ22536）. The effects of PD on cell activity， apoptotic rate， invasion and migration ability， cAMP level， the expression of epithelial-mesenchymal transition （EMT） related proteins and protein kinase A （PKA） were investigated. Using BALB/c nude mice as subjects， a transplanted tumor model of AML nude mice was induced by subcutaneous inoculation of KG-1 cell suspension and then divided into control group， PD group， SQ22536 group and PD+SQ22536 group （with 6 mice in each group）. The effects of PD on tumor volume and mass were measured. RESULTS Compared with the normal group or control group， the cell viabilities， the number of migrating cells， the number of invasive cells， the relative expressions of vimentin and Snail as well as the tumor volume and mass were decreased significantly in PD groups， while the apoptotic rates， cAMP levels， the relative expressions of E-cadherin and PKA were significantly increased， with a dose-dependent manner （P＜0.05）. SQ22536 had opposite effects on cells and nude mice compared to PD， and could significantly reverse the anti-tumor activity of PD （P＜0.05）. CONCLUSIONS PD may inhibit the proliferation， migration， invasion and EMT process of KG-1 cells， induce apoptosis， and inhibit tumor growth， by activating the cAMP/PKA signaling pathway， thereby exerting anti-AML effects.
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ObjectiveTo decipher the mechanism of Wenxiao powder in alleviating corticosterone-induced depression-like behaviors in mice. MethodMale ICR mice were randomized into normal， model， paroxetine （20 mg·kg-1）， and low- and high-dose （3.27， 6.54 g·kg-1， respectively） Wenxiao powder groups. The mice in normal and model groups received equal volume of saline. Other groups except the normal group were injected with corticosterone subcutaneously 0.5 h after gavage to induce depression. Mice were tested for depression-like behaviors after drug administration. Enzyme-linked immunosorbent assay （ELISA） was performed to measure the corticosterone content in the serum. Nissl staining was performed to observe the damage of hippocampal neurons. Immunofluorescence staining was employed to observe the expression of double cortin （DCX） in the dentate gyrus （DG） of the hippocampus. Western blot was employed to determine the expression of proteins in the brain-derived neurotrophic factor （BDNF）/tyrosine kinase receptor B （TrkB）/extracellular signal-regulated kinase （ERK）/cAMP-response element-binding protein （CREB） pathway in the hippocampus. ResultCompared with the normal group， the model group showed decreased sucrose preference rate， increased immobility time in the tail suspension test （P<0.01）， and reduced residence time in the central area of the open field and the total movement distance （P<0.05， P<0.01）. In addition， the modeling elevated the corticosterone level in the serum （P<0.01）， decreased the volume and intensified the nuclear staining of hippocampal neurons in the DG area， reduced the expression of DCX in the DG area， and down-regulated the protein levels of BDNF， phosphorylated （p）-TrkB， p-ERK， and p-CREB in the hippocampus （P<0.05， P<0.01）. Compared with the model group， low-dose Wenxiao powder improved the mouse behavivors in the sucrose preference， open field， and tail suspension tests （P<0.05， P<0.01）， and high-dose Wenxiao powder improved the behaviors in the sucrose preference and open field tests （P<0.05， P<0.01）. In addition， Wenxiao powder lowered the serum corticosterone level （P<0.01） and recovered the structure and morphology of neurons with obvious nuclei and presence of Nissl bodies in the DG area of the hippocampus. Moreover， Wenxiao powder at both doses promoted the expression of DCX in the DG area， and high-dose Wenxiao powder up-regulated the protein levels of BDNF， p-TrkB， p-ERK， and p-CREB in the hippocampus （P<0.05， P<0.01）. ConclusionWenxiao powder can alleviate corticosterone-induced depression-like behaviors and promote neurogenesis in mice possibly by activating the BDNF/TrkB/ERK/CREB signaling pathway.
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Studies have suggested that the nucleus accumbens (NAc) is implicated in the pathophysiology of major depression; however, the regulatory strategy that targets the NAc to achieve an exclusive and outstanding anti-depression benefit has not been elucidated. Here, we identified a specific reduction of cyclic adenosine monophosphate (cAMP) in the subset of dopamine D1 receptor medium spiny neurons (D1-MSNs) in the NAc that promoted stress susceptibility, while the stimulation of cAMP production in NAc D1-MSNs efficiently rescued depression-like behaviors. Ketamine treatment enhanced cAMP both in D1-MSNs and dopamine D2 receptor medium spiny neurons (D2-MSNs) of depressed mice, however, the rapid antidepressant effect of ketamine solely depended on elevating cAMP in NAc D1-MSNs. We discovered that a higher dose of crocin markedly increased cAMP in the NAc and consistently relieved depression 24 h after oral administration, but not a lower dose. The fast onset property of crocin was verified through multicenter studies. Moreover, crocin specifically targeted at D1-MSN cAMP signaling in the NAc to relieve depression and had no effect on D2-MSN. These findings characterize a new strategy to achieve an exclusive and outstanding anti-depression benefit by elevating cAMP in D1-MSNs in the NAc, and provide a potential rapid antidepressant drug candidate, crocin.
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Abstract Objective To explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. Methods Eighteen healthy, red-eyed guinea pigs (36 ears) weighing 200-350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 μg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. Results Compared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p< 0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p< 0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p< 0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p< 0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p< 0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. Conclusion DDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence Level 5.
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Purpose: To retrospectively study impact of preoperative posterior segment evaluation on surgical intervention in camp patients recruited for cataract surgery in Gujarat India. Methods: Retrospective analysis of six months data collected from hospital electronic medical record (EMR) system of 9820 admitted patients recruited from screening camp for cataract surgery from 1/10/2019 to 31/3/2020 in Tertiary Eye Hospital in Gujarat, India, has been done. Comprehensive clinical evaluation, of both anterior and posterior segment which included detailed history; best corrected visual acuity (BCVA); intraocular pressure measurement with non?contact tonometer (NCT) and when required with Goldman applanation tonometer; slit lamp examination; and fundus examination with + 90 diopter lens as well as indirect ophthalmoscope as and when indicated. In case there was no view of retina, a B?scan ultrasound was done to rule out any posterior segment pathology. Immediate surgical intervention done was assessed and results analyzed in percentage. Results: Cataract surgery was advised for 8390 patients (85.43%). Surgical intervention for management of glaucoma was done for 68 patients (0.692%). Retina intervention was done for 86 patients. Posterior segment evaluation changed immediate surgical plane of management for 154 (1.57%) patients. Conclusion: Comprehensive clinical evaluation is economical and should be mandatory especially in community services as comorbid conditions like glaucoma, diabetic retinopathy, retinal vein occlusion, and other varied posterior segment diseases contribute significantly to visual disability in elderly age group. It is difficult to follow these patients later if manageable comorbidity is not informed about and if indicated dealt simultaneously for visual rehabilitation of patient.
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OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.
Subject(s)
Animals , Female , Mice , Cell Differentiation , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Mesenchymal Stem Cells , Mice, Inbred C57BL , MicroRNAs/metabolism , Osteocalcin/metabolism , Osteogenesis/genetics , RNA, Messenger/geneticsABSTRACT
OBJECTIVE To investigate the improvement effects of Arisaema Cum Bile on Parkinson’s disease （PD） model mice and its potential mechanism. METHODS Sixty male C57BL/6J mice were randomly divided into normal group， model group， Arisaema Cum Bile low-dose group [0.39 g/（kg·d）]， Arisaema Cum Bile high-dose group [1.56 g/（kg·d）] and positive control drug Levodopa tablet group [80 mg/（kg·d）]， with 12 mice in each group. Except that normal group was given constant volume of normal saline， other groups were given 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine [MPTP，35 mg/（kg·d）] intraperitoneally for 5 consecutive days to induce subacute PD model； after modeling， they were given relevant medicine continuously for 7 d； rod climbing test and line suspension test were performed 1 d before modeling， on the 5th day of modeling and after the last medication. The number of tyrosine hydroxylase （TH）-positive neurons in the substantia nigra of mice were measured by immunofluorescence； the levels of interleukin 1β （IL-1β） and tumor necrosis factor α（ TNF-α） in serum and the levels of IL- E-mail：qhwang668@sina.com 1β， TNF-α， cyclooxygenase-2 （COX-2） and inducible nitric oxide synthase （iNOS） in the substantia nigra of mice were measured by enzyme-linked immunosorbent assay. The expression levels of cAMP-dependent protein kinase catalytic subunit α （PKA C-α）， glutathione peroxidase 4 （GPX4） and ferritin heavy chain polypeptide 1 （FTH1） proteins in the substantia nigra of mice was measured by Western blot. RESULTS After last medicine， compared with the normal group， mice in the model group had significantly longer pole-climbing time （P＜0.01）， significantly lower line suspension scores （P＜0.01）， significantly fewer TH-positive neurons in the substantia nigra （P＜0.01）， significantly higher serum concentrations of IL-1β and TNF-α and nigrostriatal concentrations of IL-1β， TNF-α， COX-2 and iNOS （P＜0.01）， while lower protein expression levels of GPX4， PKA C-α and FTH1 in the substantia nigra （P＜0.05 or P＜0.01）. Compared with the model group， the above indexes of mice were significantly returned in Arisaema Cum Bile high-dose group （P＜0.05 or P＜ 0.01）. CONCLUSIONS Arisaema Cum Bile can improve motor impairment and reduce apoptosis of nigrostriatal TH neurons in MPTP-induced PD mice， and has neuroprotective effects on model mice； this may be related to its inhibition of neuroinflammation and the inhibition of ferroptosis by up-regulating PKA signaling pathway.
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ObjectiveTo observe the effects of modified Shenqiwan on renal function and fibrosis in diabetic nephropathy mice and explore the underlying mechanism based on the glycogen synthase kinase-3β （GSK-3β）/cyclic adenosine monophosphate （cAMP） response element-binding protein （CREB） signaling pathway. MethodFifty male db/db mice and 10 db/m mice were used in this study. The fifty db/db mice were randomly divided into model group， irbesartan group， and low-， medium-， and high-dose modified Shenqiwan groups. The 10 db/m mice were assigned to the normal group. The mice in the low-， medium-， and high-dose modified Shenqiwan groups were administered with modified Shenqiwan in the dosage form of suspension of Chinese medicinal granules by gavage， those in the irbesartan group were given irbesartan suspension by gavage， and those in the normal and model groups were given distilled water of equal volume by gavage. The intervention lasted for 12 weeks. The blood glucose levels， urine albumin-to-creatinine ratio （UACR）， and the protein expression levels of GSK-3β， CREB， transforming growth factor-β1 （TGF-β1）， E-cadherin， Vimentin， fibronectin （FN）， plasminogen activator inhibitor-1 （PAI-1）， and Collagen type Ⅳ （Coll Ⅳ） in the mouse kidneys were recorded before and after treatment. The extent of renal pathological damage was also observed. ResultCompared with the normal group， the model group showed significant increases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， decreased protein expression level of CREB （P<0.05）， and severe renal pathological damage. Compared with the model group， the low-， medium-， and high-dose modified Shenqiwan groups and the irbesartan group showed varying degrees of decreases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， increased expression level of CREB protein （P<0.05）， and improved renal pathological damage. ConclusionModified Shenqiwan can effectively reduce blood glucose levels， improve renal function， and alleviate fibrosis， and the mechanism of action is related to the inhibition of the GSK-3β/CREB signaling pathway.
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OBJECTIVE To study the effects of Yishen daluo decoction on inflammatory factors and cyclic adenosine monophosphate（cAMP）/protein kinase A （PKA）/cAMP response element binding protein （CREB） signal pathway in experimental autoimmune encephalomyelitis （EAE） model mice by inhibiting the expressions of β-arrestin1， and to explore the mechanism of Yishen daluo decoction in the treatment of EAE. METHODS Sixty mice were randomly divided into normal group， model group， TCM group （Yishen daluo decoction 20 g/kg）， positive control group （prednisone acetate 3.9 mg/kg）， β-arrestin1 siRNA adeno- associated virus （AAV-β） group， AAV-β+TCM group， with 10 mice in each group. Except for normal group， EAE model was made in other groups. AAV-β group and AAV-β+TCM group were injected with AAV-β via tail vein to interfere with the expression of β -arrestin1 protein. Starting from the 8th day of modeling， they were given corresponding drug solution/normal saline intragastrically， once a day， for consecutive 14 days. The neurological function score of mice was detected； the pathological and morphological changes were observed in the brain and spinal cord tissues of mice； the serum levels of inflammatory factors [interleukin-2 （IL-2）， IL-23， interferon-γ （IFN-γ）] in mice were determined； the expressions of β-arrestin1， cAMP， PKA and CREB in brain and spinal cord were detected. RESULTS Compared with normal group， neurological function scores， serum levels of inflammatory factors， and protein expressions of β-arrestin1 in brain and spinal cord were significantly increased （P＜0.05 or P＜ 0.01）； protein expressions of PKA， CREB and cAMP in brain and spinal cord were decreased significantly（P＜0.05 or P＜0.01）. The deep staining of cellular shrinkage and aggregation of inflammatory cells were observed in most neurons of the brain and spinal cord， with varying degrees of demyelinating. Compared with model group， the neurological function scores， pathological changes in brain and spinal cord tissues， and most indicators （except for CREB and cAMP proteins in the brain tissue of AAV-β group） were significantly reversed （P＜0.05 or P＜0.01）.Compared with AAV- β group， the neurological function scores， the levels of IFN-γ in serum and β-arrestin1 in spinal cord were significantly decreased （P＜0.05 or P＜0.01）， PKA and cAMP in brain and spinal cord tissues were significantly increased in AAV- β +TCM group （P＜0.05 or P＜0.01）. CONCLUSIONS Yishen daluo decoction can inhibit the expression of β-arrestin1 in the central nervous system thus activating the cAMP/PKA/CREB signaling pathway， relieving nervous system inflammation， and ultimately alleviates the symptoms of EAE.
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ObjectiveTo investigate the mechanism of Buyang Huanwutang in treating diabetic peripheral neuropathy （DPN） via mitochondrial transport. MethodDiabetes in SD rats was induced by a high-carbohydrate/high-fat diet and intraperitoneal injection of streptozotocin （STZ）. The 45 diabetic rats were randomly assigned into a DPN group， an alpha-lipoic acid （60 mg·kg-1·d-1） group， and a Buyang Huanwutang （15 g·kg-1·d-1） group， with 15 rats in each group. Fifteen normal SD rats were fed with the standard diet and set as the control group. The rats were administrated with corresponding drugs by gavage for 12 weeks. The paw withdraw threshold （PWT） and motor nerve conduction velocity （MNCV） were measured at the end of medication， and the sciatic nerve and the bilateral dorsal root ganglia of L4-5 were collected. The injury model of NSC34 cells was established by treating with 50 mmol·L-1 glucose and 250 μmol·L-1 sodium palmitate. The NSC34 cells were then randomly assigned into a blank （10% blank serum） group， a DPN （10% blank serum） group， an apha-lipoic acid （10% apha-lipoic acid-containing serum） group， a Buyang Huanwutang （10% Buyang Huanwutang-containing serum） group， and a Buyang Huanwutang + Compound C （CC） （10% Buyang Huanwutang-containing serum + 10 μmol·L-1 CC） group. The cell intervention lasted for 24 h. The immunofluorescence method， immunohistochemistry， and Western blot were employed to determine the expression levels of phosphorylated adenosine monophosphate-activated protein kinase （p-AMPK）， phosphorylated cAMP-response element binding protein （p-CREB）， kinesin family member 5A （KIF5A）， and dynein cytoplasmic 1 intermediate chain 2 （DYNC1I2）. ResultCompared with the control group， the DPN group of rats showed increased fasting blood glucose （P<0.01）， decreased MNCV and PWT （P<0.01）， down-regulated expression of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.01）， and up-regulated expression of DYNC1I2 （P<0.01）. Compared with the DPN group， drug intervention groups showed increased MNCV and PWT （P<0.01）， up-regulated expression of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.05， P<0.01）， and down-regulated expression of DYNC1I2 （P<0.05， P<0.01）. The Buyang Huanwutang group had higher levels of MNCV and KIF5A （P<0.05） and lower level of DYNC1I2 （P<0.01） than the apha-lipoic acid group. Compared with the blank group， the DPN group of NSC34 cells showed decreased levels of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.01） and increased level of DYNC1I2 （P<0.01）. The apha-lipoic acid group and Buyang Huanwutang group had higher levels of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.05， P<0.01） and lower level of DYNC1I2 （P<0.01） in NSC34 cells than the DPN group. Buyang Huanwutang group had higher KIF5A level （P<0.05） in NSC34 cells than the apha-lipoic acid group. Moreover， the Buyang Huanwutang + CC group had lower levels of KIF5A， DYNC1I2， p-AMPK/AMPK， and p-CREB/CREB （P<0.01） in NSC34 cells than the Buyang Huanwutang group. ConclusionBuyang Huanwutang may regulate mitochondrial anterograde transport via the AMPK/CREB pathway to prevent and treat DPN.
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Vascular dementia (VaD) is the second commonest type of dementia which lacks of efficient treatments currently. Neuroinflammation as a prominent pathological feature of VaD, is highly involved in the development of VaD. In order to verify the therapeutic potential of PDE1 inhibitors against VaD, the anti-neuroinflammation, memory and cognitive improvement were evaluated in vitro and in vivo by a potent and selective PDE1 inhibitor 4a. Also, the mechanism of 4a in ameliorating neuroinflammation and VaD was systematically explored. Furthermore, to optimize the drug-like properties of 4a, especially for metabolic stability, 15 derivatives were designed and synthesized. As a result, candidate 5f, with a potent IC50 value of 4.5 nmol/L against PDE1C, high selectivity over PDEs, and remarkable metabolic stability, efficiently ameliorated neuron degeneration, cognition and memory impairment in VaD mice model by suppressing NF-κB transcription regulation and activating cAMP/CREB axis. These results further identified PDE1 inhibition could serve as a new therapeutic strategy for treatment of VaD.
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OBJECTIVE@#To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism.@*METHODS@#MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells.@*RESULTS@#The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05).@*CONCLUSION@#Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.
Subject(s)
Animals , Mice , Cell Differentiation , Core Binding Factor Alpha 1 Subunit , Glucose/pharmacology , Osteoblasts/drug effects , RNA, Messenger , Signal Transduction , Teriparatide , Cell LineABSTRACT
This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.
Subject(s)
Mice , Animals , Adipose Tissue, Brown , Molecular Docking Simulation , PPAR alpha/metabolism , Adipose Tissue, White , RNA, Messenger/metabolismABSTRACT
ObjectiveTo investigate the effect and mechanism of Aconiti Lateralis Radix Praeparata-Cinnamomi Cortex in regulating the intestinal function in the rat model of slow transit constipation （STC） due to yang deficiency via the vasoactive intestinal peptide （VIP）/cathelicidin antimicrobial peptide （cAMP）/protein kinase A （PKA）/aquaporin （AQP） pathway. MethodSD rats were randomized into 6 groups （n=6）， including a control group， a model group， high-， medium-， and low-dose Aconiti Lateralis Radix Praeparata-Cinnamomi Cortex groups， and a prucalopride group. Other groups except the control group were treated with loperamide hydrochloride combined with ice water by gavage for the modeling of STC due to yang deficiency. The number of fecal pellets， time to the first black stool defecation， fecal water content， intestinal propulsion rate， and score of fecal properties were recorded in each group. At the end of the treatment， the colon was stained with hematoxylin-eosin （HE） to reveal the histopathological changes and Alcian blue/periodic acid-Schiff （AB-PAS） to reveal the secretion of colonic mucus. The enzyme-linked immunosorbent assay （ELISA） was employed to measure the level of VIP in the serum. The mRNA level of AQP in the colon was measured by polymerase chain reaction （Real-time PCR）. Immunohistochemical staining was performed to observe the expression of AQPs in the colon and kidney tissues. Western blot was performed to determine the protein levels of cAMP， PKA， and VIP in the colon tissue. ResultCompared with the control group, the model group had longer time to the first black stool defecation, reduced fecal pellets and water content, reduced Bristol Stool Form Scale score and intestinal propulsion rate, and constipation aggravated（P<0.01）. Moreover, increased the intestinal lesions, reduced the mucus secretion, reduce the serum VIP level, up-regulated the expression levels of AQP1 in the colon and kidney tissues, inhibited the expression of AQP3 and AQP9（P<0.01）., and down-regulated the protein levels of cAMP, PKA, and VIP in the colon tissue. Compared with the model group， the high-dose Aconiti Lateralis Radix Praeparata-Cinnamomi Cortex group had shortened time to the first black stool defecation， increased fecal pellets and water content， increased Bristol Stool Form Scale score and intestinal propulsion rate， and alleviated constipation symptoms. Moreover， high-dose Aconiti Lateralis Radix Praeparata-Cinnamomi Cortex reduced the intestinal lesions， increased the mucus secretion， elevated the serum VIP level（P<0.01）.， down-regulated the expression levels of AQP1 in the colon and kidney tissues， promoted the expression of AQP3 and AQP9（P<0.05，P<0.01）， and up-regulated the protein levels of cAMP， PKA， and VIP in the colon tissue. The medium- and low-dose groups had weaker effect than the high-dose group（P<0.01）. ConclusionHigh-dose Aconiti Lateralis Radix Praeparata-Cinnamomi Cortex can improve the intestinal motility and balance the intestinal water and fluid metabolism by up-regulating the VIP/cAMP/PKA/AQP pathway， thereby mitigating the constipation symptoms in the rat model of slow transit constipation due to yang deficiency.
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Objective:Exploring the role of thyrotropin receptor(TSHR) in lipotoxicity-induced thyroid function damage.Methods:Rat thyroid follicular epithelial cells(RTC) were stimulated with different doses of palmitic acid(PA), and the lipid content of the cells was observed through Oil Red O staining. The expression levels of TSH receptor(TSHR), Ttf1, and SSBP1 mRNA and protein in each group were detected using RT-PCR and Western blot. The TSHR protein level in the cell culture supernatant was measured using ELISA. Membrane TSHR was assessed through immunofluorescence and compared with the control group. We used PA to stimulate the TSHR over-expression(TSHR OE) and normal RTC, as PA+ TSHR OE group and PA group respectively, then testing Tg mRNA and protein, cAMP and Tg in cell supernatants levels, then comparing with the control.Results:RTC were stained into peau d′orange in PA groups. Compared with the control group, we found TTf1, SSBP1 and TSHR mRNA as well as protein levels in PA groups were decreased(all P<0.05), TSHR of the cell membrane and supernatants were reduced(all P<0.05), characterizing dose-dependent changes partly. Moreover, we found in PA group Tg mRNA level was downregulated( P<0.05), Tg protein levels were reduced in the supernatants and cells( P<0.05), cAMP level was decreased in cells( P<0.05); in TSHR OE group, Tg mRNA level was upregulated( P<0.05), Tg protein levels in cells and supernatants were increased(all P<0.05), cAMP level was similar. Compared with the PA group, we found in PA+ TSHR OE group Tg mRNA level was upregulated( P<0.05), Tg protein levels were increased in the supernatants and cells(all P<0.05), cAMP level was elevated in cells( P<0.05). Conclusion:PA induces lipid deposition in RTC, decreased synthesis and secretion of Tg. This effect is likely achieved through the downregulation of the TSHR/cAMP signaling pathway.
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ObjectiveTo investigate the protective effect of Guiqi Baizhu prescription combined with oxaliplatin on the intestinal barrier of tumor-bearing mice with gastric cancer by regulating downstream aquaporin 3 （AQP3） and aquaporin 4 （AQP4） through the vasoactive intestinal peptide （VIP）/cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA） signaling pathway. MethodThe gastric cancer cell lines MFC with a density of 1×107/mL were prepared into cell suspension. The tumor-bearing mouse model of gastric cancer was established by inoculating 0.2 mL cell suspension under the right axilla of mice. After successful modeling， mice were randomly divided into 5 groups， namely， model group， oxaliplatin group （10 mg·kg-1）， and high， medium， and low-dose oxaliplatin + Guiqi Baizhu prescription groups （17.68， 8.84， 4.42 g·kg-1）， with 10 mice in each group， and the remaining 10 mice were set as a blank group. Mice in each group were treated with Chinese medicine， oxaliplatin， or normal saline by gavage or intraperitoneal injection for 14 d. The next day after the last dose， blood was taken from the eyeball to separate serum and take colonic samples. Hematoxylin-eosin （HE） staining was used to observe the changes in tissue morphology. The content of D-lactate acid （D-LA） and diamine oxidase （DAO） in the serum was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expressions of VIP， cAMP， PKA， AQP3， and AQP4 were detected by Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the blank group， the model group showed edema in the colonic submucosa， disordered arrangement of intestinal glands in the mucosal layer， loss of goblet cells， infiltration of inflammatory cells， and villus shedding. However， there were different degrees of improvement in each administration group. As compared with the blank group， the serum levels of DAO and D-LA in the model group were significantly increased （P<0.01）. As compared with the model group， the levels of DAO and D-LA in the high-dose oxaliplatin + Guiqi Baizhu prescription group and the level of D-LA in the medium-dose oxaliplatin + Guiqi Baizhu prescription group were decreased （P<0.05， P<0.01）. As compared with the oxaliplatin group， the levels of D-LA in the high and medium-dose oxaliplatin + Guiqi Baizhu prescription groups were decreased （P<0.05）， and the levels of DAO and D-LA in other administration groups were decreased as well， but the difference had no statistical significance. As compared with the blank group， the mRNA and protein expression levels of VIP， cAMP， PKA， AQP3， and AQP4 in the model group were significantly decreased （P<0.05， P<0.01）. As compared with the model group， the mRNA and protein expression levels of VIP， cAMP， PKA， AQP3， and AQP4 in each administration group were increased， and those in the high-dose oxaliplatin + Guiqi Baizhu prescription group were significantly increased （P<0.05， P<0.01）， while the protein expression level of cAMP in the medium-dose oxaliplatin + Guiqi Baizhu prescription group were increased （P<0.05）. As compared with the oxaliplatin group， the protein expression levels of cAMP in the high-dose oxaliplatin + Guiqi Baizhu prescription group were increased （P<0.05）， and the mRNA and protein expressions of these indexes in the other groups were also increased but the differences were not statistically significant. ConclusionGuiqi Baizhu prescription combined with oxaliplatin can regulate AQP3 and AQP4 through the VIP/cAMP/PKA signaling pathway to protect the intestinal barrier of tumor-bearing mice with gastric cancer.
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Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal chronic interstitial lung disease characterized by a progressive decline in lung function, and current treatment options are limited. cAMP is one of the most important second messengers and plays a key role in relaxing airway smooth muscle cells and reducing inflammation. Phosphodiesterase (PDE) is a superfamily of enzymes, and PDE4 enzymes dominate 11 PDE superfamily enzymes, available in four isoforms-PDE4A, PDE4B, PDE4C and PDE4D, which selectively decompose cAMP, while PDE4 inhibitors increase cAMP levels by preventing cAMP from breaking down, thereby exerting anti-inflammatory, anti-remodeling effects and providing an attractive drug target for the treatment of IPF. This review summarizes knowledge about the association of pulmonary fibrosis with PKE4, as well as emerging preclinical studies and clinical trials regarding PDE4 inhibitors.
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Aim To establish a high-throughput screening cell model for GLP-1 receptor agonists. Methods A pEGFP-GLP-1R-3 C recombinant plasmid was constructed and transfected into HEK293T cells. The cells were screened with G418 and flow cytometry. The established stable cell line was named HEK293TGLP-lR-3C-eGFP cell line. The expression level of GLP-1 R-3C-eGFP protein was confirmed by Western blotting and laser confocal microscopy. Then cyclic adenosine monophosphate (cAMP) response element reporter gene was transfected into the HEK293T-GLP-lR-3C-eGFP cells. The luminescence values were detected by One-Step Luciferase Reporter Gene Assay Kit after stimulation with different concentrations of GLP-1 peptide. The luminescence values reflected the cellular cAMP level, which was verified using the cAMP kit (E L I S A). Results HEK293T-GLP-lR-3C-eGFP cell line was successfully constructed. The relative light unit change trend after stimulation with different concentrations of GLP-1 was similar to that of the cellular cAMP level change trend. The value of Z' in this experiment was 0.52. Conclusions A recombinant HEK293T cell line is established, which can be used for high-throughput screening of GLP-1 receptor agonists.