Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway / 中国中药杂志

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.

Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway / 南方医科大学学报

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.

Improvement effects of sinapic acid on Aβ42-induced injury of PC 12 cells and the mechanism / 中国药房

OBJECTIVE To s tudy the improvement effects of sinapic acid on Aβ42-induced injury of PC 12 cells and the mechanism. METHODS PC12 cells were divided into five groups ：control group ，model group ，sinapic acid group ，phosphoinositide- 3-kinase（PI3K）inhibitor group and extracellular signal-regulated kinase （ERK）inhibitor group. Each inhibitor group was added with LY 294002 and U 0126（10 μmol/L）for 1 h；except for control group ，other four groups were treated with 2 μmol/L Aβ42 for 24 h to replicate the Alzheimer ’s disease cell model ；except for control group and model group ，other three groups were added with 100 μmol/L sinapic acid respectively. After 24 hours of continuous culture ，survival rate of PC 12 cells was detected and the morphology of PC 12 cells was observed. The content of Aβ42，mRNA expression of cAMP response element binding protein （CREB），protein expression of cyclic adenosine monophosphate （cAMP），protein kinase A （PKA），CREB signaling pathway and phosphorylated CREB （p-CREB）were detected. RESULTS After treated with sinapic acid ，the survival rate of PC 12 cells，mRNA expression of CREB and protein expressions of cAMP ，PKA and p-CREB were increased significantly （P＜0.05），while the content of Aβ42 was decreased significantly （P＜0.05）；cell morphology was significantly improved and synapses increased. After intervened with PI 3K and ERK inhibitors ，the survival rate of PC 12 cells，above mRNA and protein expressions were reversed significantly （P＜0.05 or P＜0.01）；cell morphology was irregular ，the fragments increased ，and the synaptic connections decreased. CONCLUSIONS Sinapic acid can improve the survival rate of PC 12 cells injured by A β 42，improve cell （No.2021-KYYWF-0349） morphology and decrease the content of Aβ42，the mechanism of which may be associated with promoting the gene transcription of CREB ， and activating cAMP/PKA/CREB signaling pathway.

Comparison of the effect between electroacupuncture and NSAIDs on pain memory based on cAMP/PKA/CREB pathway in anterior cingulate gyrus / 中国针灸

OBJECTIVE@#To observe the direct intervention effects of electroacupuncture (EA) and non-steroid anti-inflammatory drugs (NSAIDs) on pain memory, and to explore their effects on cAMP/PKA/cAMP pathway in anterior cingulate gyrus (ACC).@*METHODS@#Fifty clean healthy male SD rats were randomly divided into a control group, a model group, an indomethacin group, an EA group and a sham EA group, 10 rats in each group. Except the control group, the pain memory model was established in the remaining four groups by twice injection of carrageenan at foot; 0.1 mL of 2%λ-carrageenan was subcutaneously injected at the left foot of rats; 14 days later, when the pain threshold of rats of each group returned to the basic level, the second injection was performed with the same procedure. The rats in the EA group were treated with EA at bilateral "Zusanli" (ST 36) for 30 min; the rats in the indomethacin group was treated with indomethacin intragastric administration with the dose of 3 mg/kg; the rats in the sham EA group was treated with EA without electricity at the point 0.3 mm forward "Zusanli" (ST 36) with the depth of 2 mm for 30 min; the rats in the control group was not given any invention. All the above interventions were performed 5 h, 1 d, 2 d and 3 d after the second injection of 2% λ-carrageenan. The left-side paw withdrawal thresholds (PWT) were observed before the first injection, 4 h, 3 d, 5 d after the first injection, before the second injection and 4 h, 1 d, 2 d, 3 d after the second injection. Three days after the second injection, the number of positive cells of cAMP, p-PKA, p-CREB and the number of positive cells of protein co-expression in the right ACC brain area were detected by immunofluorescence, and the relative protein expression of p-PKA and p-CREB were detected by Western blot.@*RESULTS@#Compared with the control group, the PWTs in the model group decreased significantly 4 h, 3 d and 5 d after the first injection and 1 d, 2 d and 3 d after the second injection (<0.05); compared with the control group, the positive expression of cAMP, p-PKA and p-CREB in the right ACC brain area in the model group increased significantly (<0.05), and the number of positive cells of the co-expression of cAMP/p-PKA and p-PKA/p-CREB also increased significantly (<0.05). Compared with the model group, indomethacin group and sham EA group, the PWTs in the EA group were increased significantly 1 d, 2 d and 3 d after the second injection (<0.05); compared with the model group, indomethacin group and sham EA group, the positive expression of p-PKA and p-CREB in the right ACC brain area in the EA group decreased significantly (<0.05), and the number of positive cells of co-expression of cAMP/p-PKA and p-PKA/p-CREB was decreased significantly (<0.05). Compared with the model group and sham EA group, the positive expression of cAMP in the right ACC brain area was decreased in the EA group (<0.05).@*CONCLUSION@#EA have a direct intervention effect on pain memory, which have significant advantage over NSAIDs in the treatment of chronic pain. The advantage effect of EA on pain memory may be related to the inhibition of cAMP/PKA/CREB pathway in ACC area.

Antidepressant mechanism of supercritical CO2 extract from Compound Chaigui Precription regulating cAMP signaling pathway / 中草药

Objective: To investigate the antidepressant mechanism of supercritical CO2 extract from Compound Chaigui Precription from the perspective of cAMP signaling pathway. Methods: The rats model were established by chronic unpredictable mild stress (CUMS). The rats were administrated with high, medium and low doses of supercritical CO2 extract from Compound Chaigui Precription and venlafaxine as intervention drugs. The protein expression levels of cAMP, PKA, p-CREB and BDNF in tissues and their corresponding mRNA levels were explored for their antidepressant mechanism. Results: Compared with the blank group, the expression levels of cAMP, PKA, p-CREB and BDNF in the hippocampus of the model group were significantly decreased (P < 0.05, P < 0.01). After the intervention of the Compound Chaigui Precription, compared with the model group, there was a significant difference in the callus of each dose group. At the same time, the mRNA levels of TrkB, PKA, p-CREB and BDNF in the hippocampus of the model group were significantly decreased (P < 0.05, P < 0.01). After the intervention, there was a callback in each dose group compared with the model group, with significant differences. Conclusion: Supercritical CO2 extract from Compound Chaigui Precription can exert antidepressant effect by regulating cAMP-PKA-CREB-BDNF signaling pathway.

Effect of Electroacupuncture of "Weizhong" (BL 40) and "Huantiao" (GB 30) on cAMP-PKA-CREB Signaling of Spinal Cord in Rats with Neuropathic Pain / 针刺研究

OBJECTIVE: We have demonstrated that electroacupuncture (EA) of "Weizhong" (BL 40) and "Huantiao" (GB 30) could evidently relieve mechanical hyperalgesia in spared nerve injury (SNI) rats. The present study was designed to observe the effect of EA on levels of cAMP, and protein kinase A (PKA) and cAMP response element binding protein (CREB) in the lumbar spinal cord of the same pain model rats, so as to explore its mechanisms underlying improvement of neuropathic pain. METHODS: One hundred male SD rats were randomly divided into 5 groups: sham operation, model, EA, AP-5 and L-NAME groups (n＝20 in each group). The sham operation group underwent a simple separation of the sciatic nerve but without ligation and abscission. The neuropathic pain model was established by abscission of the right tibial and common peroneal nerve. EA (2 Hz, 1－3 mA) was applied to right BL 40 and GB 30 for 30 min, once a day for 7 days, starting from day 11 after surgery. For rats of the AP-5 and L-NAME groups, AP-5 (a competitive antagonist for NMDA receptor, 0.7 mg•kg－1•d－1) and L-NAME (a non-selective antagonist for nitric oxide synthase, NOS, 60 mg•kg－1•d－1) were respectively administrated by intraperitoneal injection, once daily for 7 days. The mechanical pain threshold was measured, the cAMP content of spinal cord (L 4－L 6) was determined by radioimmunoassay, and the expression of PKA, p-PKA and CREB proteins of spinal cord (L 4－L 6) was detected by Western blot. RESULTS: The content of cAMP and the expression levels of PKA, p-PKA and CREB were significantly higher in the model group than in the sham operation group (P<0.01), and considerably lower in the EA group than in the model group (P<0.01, P<0.05). Compared to the model group, the expression levels of PKA and p-PKA were significantly decreased in the AP-5 and L-NAME groups (P<0.01, P<0.05). Compared to the EA group, the content of cAMP and the expression levels of CREB were significantly higher (P<0.05), and the expression of p-PKA was significantly lower in the AP-5 and L-NAME groups (P<0.05)．. CONCLUSION: EA intervention-induced down-regulation of cAMP, PKA and CREB levels in the lumbar spinal cord may contribute to its analgesic effect in neuropathic pain rats, suggesting an involvement of reduction of cAMP/PKA/CREB signaling of spinal cord in EA analgesia.

Protective Effect of Mullein Glycoside Polysaccharide Extracting from Cistanche on PC12 Nerve-Cell Damage Model Induced by D-galactose / 中国药学杂志

OBJECTIVE: To explore the protective effect of mullein glycoside polysaccharide of Cistanche deserticola Ma on PC12 nerve-cell model induced by D-galactose. METHODS: The cell survival rate was determined by MTT assays, which provided the basis for selecting mullein glycoside polysaccharide dose and estimated the dose and action time of D-galactose for inducing PC12 nerve cell damage model. After mullein glycoside polysaccharide incubation of PC12 cells, western blotting was used to detect the levels of CREB and p-CREB protein expression. ELISA Kit was used to detect the levels of cyclic adenosine monophosphate(cAMP), cAMP dependent protein kinase(PKA) and brain derived neurotrophic factor(BDNF). The content of MDA, activities of SOD and LDH were measured by their respective kits. RESULTS: (1)After the exposure of the PC12 cells to 16 g·L-1 D-galactose for 40 h, the cell survival rate was (46.67±6.59)%, which has a significant difference compared with the control group(P<0.05), indicating that successful cell aging model was established. (2)Compared with those in model group, mullein glycoside polysaccharide could significantly increase p-CREB expression in dose-dependent manner(r=0.989, P<0.01), content of PKA, cAMP, BDNF and SOD and decrease the levels of MDA and LDH(rMDA=0.875, P<0.05);(rLDH=0.834, P<0.05). However, blockers H-89 could significantly decrease p-CREB expression, PKA, cAMP, SOD and BDNF content(P<0.05), and increase the levels of MDA and LHD(P<0.05). CONCLUSION: The mullein glycoside polysaccharide of Cistanche deserticola Ma has obvious protective effect on PC12 nerve-cell damage model induced by D-galactose and its mechanism relates to the upregulation of cAMP/PKA/ CREB signaling pathways.

In vivo and in vitro studies of protective effect of CDPS onacute aging mouse model induced by D-galactose / 中国药理学通报

Aim To study the protective effect of CDPS on acute aging mouse model induced by D-galactose (D-gal) and its mechanism.Methods (1) The acute aging mouse model was induced by D-gal.After CDPS (25、50、100 mg·kg-1) treatment, the improving effect on learning and memory in mice was examined in vivo.(2) We also established the aging model on PC12 cells in vitro.After CDPS treatment (150、200 mg·L-1), the level of p-CREB in the nucleus was detected by Western blot, and the content of cAMP, PKA and brain derived neurotrophic factor (BDNF) levels were examined by the Elisa kits.Moreover, cAMP, PKA and BDNF were detected in PC12 cells under the condition that H89, the inhibitor of PKA, co-cultured with PC12 cells after CDPS treatment.(3) The UPLC/Q Exactive MS method was developed for determining the concentration of glutamic acid, dopamine and norepinephrine, which secreted in PC12 cells after CDPS treatment.Results (1) In vivo, CDPS significantly improved the memory impairment in aging mice induced by D-gal in the Morris assay.(2) In vitro, CDPS could significantly increase the expression of p-CREB (P<0.05), PKA, cAMP and BDNF (P<0.05).The H-89 abolished the increase of p-CREB (P<0.05), PKA, cAMP and BDNF (P<0.05) in PC12 aging cells induced by D-gal after CDPS treatment.(3) CDPS increased the release of dopamine, norepinephrine, and glutamate secreted in PC12 cells.Conclusion CDPS could significantly improve the learning and memory ability on aging mouse model in vivo, and reversed the damage in PC12 cells induced by D-gal by activating cAMP/PKA/CREB signal cascade, increase the expression of BDNF, and increasing modestly the release of excitatory neurotransmitter.