Paeoniflorin Induces Apoptosis in NSCLC Cells via Activating Hippo Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the apoptosis induced by paeoniflorin （PF） in non-small cell lung cancer （NSCLC） cells and explore its mechanism. MethodCell counting kit-8 （CCK-8） was used to detect the inhibition rates of H1299， H292 and A549 cells with different concentrations of PF （2.5， 5， 10， 20， 25 µmol·L-1）， and to screen suitable concentrations of PF and experimental cells. The inhibitory effect of PF on lung cancer cells was detected by clone formation assay. The effect of PF on cell apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide （PI） double staining. With the right concentration of drugs， levels of apoptosis-associated protein B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved Caspase-3 and Caspase-3 were detected by Western blot. At the same time， the molecular expressions of hypoxia inducible factor -1α （HIF-1α） and Hippo signaling pathway were determined. ResultCompared with the blank group， PF significantly inhibited the growth of H1299， H292 and A549 cells of human lung cancer （P<0.01）. PF significantly induced apoptosis in A549 cells （P<0.01）， decreased the Bcl-2/Bax ratio （P<0.01）， and significantly increased the cleaved Caspase-3 expression （P<0.01）. Compared with those in the blank group， the expression levels of HIF-1α， transcriptional coactivator with PDZ-binding motif （TAZ）， large tumor suppressor 1 （LATS1）， Mps one binding 1 （MOB1） and Yes-associated protein （YAP） in A549 cells of the PF treatment group were significantly decreased （P<0.01）， while the expressions of p-LATS1， p-MOB1 and p-YAP were significantly increased （P<0.01）. At the same time， there was no significant effect on the expression levels of phosphorylated mammalian Ste20-like kinase 1 （p-MST1） and MST1， which did not reach a statistical difference. ConclusionAll data demonstrated that PF showed an anti-tumor effect by improving hypoxic conditions and inhibiting the abnormally activated Hippo signaling pathway， thereby inducing and promoting apoptosis in non-small cell lung cancer.

Improvement effect and mechanism of paeoniflorin on glucose metabolism，inflammation and oxidative stress in rats with gestational diabetes mellitus / 中国药房

OBJECTIVE To explore the effect of paeoniflorin on glucose metabolism， inflammation and oxidative stress in rats with gestational diabetes mellitus （GDM） and its potential mechanism based on nuclear factor-erythroid 2-related factor 2 （Nrf2）/ heme oxygenase-1 （HO-1）/nicotinamide adenine dinucleotide phosphate：quinone oxidoreductase 1 （NQO1） signaling pathway. METHODS The female rats fed with high fat and high sugar diet and the male rats fed with an ordinary diet were caged， the successfully conceived rats were collected， and streptozotocin was injected intraperitoneally once to induce the GDM model. The successfully modeled rats were randomly divided into the model group， metformin hydrochloride group （200 mg/kg metformin by gavage）， paeoniflorin low-， high-dose groups （45， 90 mg/kg paeoniflorin by gavage， respectively）， paeoniflorin+ML385 group （90 mg/kg paeoniflorin by gavage and intraperitoneal injection of 30 mg/kg Nrf2 inhibitor ML385）， with 12 rats in each group； in addition， another 12 conceived rats fed with an ordinary diet were selected as the control group. The rats in each drug group were given the corresponding drug/normal saline， once a day， for 2 consecutive weeks. Glucose metabolism indexes [fasting blood glucose （FBG）， fasting insulin （FINS）， insulin resistance index （HOMA-IR）]， serum inflammatory factors [interleukin-6 （IL-6）， tumor necrosis factor- α （TNF- α）] and renal tissue oxidative stress indexes [superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）] were detected； the pathological changes of renal tissue were observed， and the protein expressions of Nrf2， HO-1 and NQO1 in renal tissue were detected. RESULTS Compared with the control group， the renal tissue lesions of the model group were obvious， including glomerular atrophy， edema degeneration of renal tubular epithelial cells and a large number of inflammatory cell infiltration； the levels of FBG and FINS， HOMA-IR， the levels of IL-6 and TNF-α in serum， and the level of MDA in renal tissue were significantly increased （P＜0.05）， while the levels of SOD and GSH-Px and the protein expressions of Nrf2， HO-1 and NQO1 in renal tissue were significantly decreased （P＜0.05）. Compared with the model group， the renal tissue lesions of rats in paeoniflorin low-dose and high-dose groups were reduced， the above quantitative indexes were significantly improved， and the improvement effect was better in high-dose group （P＜0.05）， while ML385 could significantly reverse the improvement effect of paeoniflorin on the above indexes （P＜0.05）. CONCLUSIONS Paeoniflorin can improve the abnormal glucose metabolism， inflammation and oxidative stress damage of renal tissue in GDM rats， which may be related to the activation of Nrf2/HO-1/NOQ1 signaling pathway.

Protective Mechanism of Paeoniflorin on Mice with Ulcerative Colitis Based on AMPK/mTOR Autophagy Pathway / 中国实验方剂学杂志

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.

Paeoniflorin ameliorates diabetic cognitive dysfunction by inhibiting TLR4/NF-κB signaling in ovariectomized mice / 中华内分泌代谢杂志

Objective:To investigate the effect of paeoniflorin on toll-like receptor 4(TLR4)/nuclear transcription factor(NF-κB) signaling pathway of streptozotocin combined with ovariectomized mice, and to explore whether it can improve the cognitive impairment of ovariectomized diabetic mice.Methods:Ninety female C57BL/6J mice were divided into SHAM group, ovariectomy group, diabetes group(intraperitoneal injection of STZ 50 mg·kg -1·d -1 for 5 consecutive days), dual model group(DM modeling and OVX operation), paeoniflorin low-dose intervention group(OVX+ STZ+ L-PF 50 mg·kg -1·d -1), paeoniflorin high-dose intervention group(OVX+ STZ+ H-PF 100 mg·kg -1·d -1; all groups n=15). After 8 weeks of paeoniflorin intervention, their cognitive function was tested by behavioral experiments(Morris water maze and Y maze). The pathological changes of hippocampal tissue were observed by HE and Nissl staining. The mRNA expressions of TLR4, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) in hippocampal tissues were detected by real-time fluorescence quantitative PCR. The expression of TLR4, NF-κB P65, TNF-α, IL-6, IL-1β, β-amyloid protein(Aβ), tau proteins, and p-tau proteins were detected by Western blot. Results:Compared with SHAM group, the learning and memory ability of ovariectomy group, diabetes group and dual model group decreased, hippocampal cells were damaged, and the expression of related gene mRNA and protein were increased, especially in dual model group; Compared with dual model group, paeoniflorin intervention could delayed the learning and memory impairment, improve cognitive function, reduce the degree of hippocampal injury, and decrease the expression levels of related gene mRNA and protein, The above changes were the most pronounced at paeoniflorin high-dose intervention group.Conclusion:Paeoniflorin improves cognitive dysfunction in ovariectomized diabetic mice by inhibiting TLR4/NF-κB signaling pathway.

Optimization of the extraction process of Shangke Huoxue Granule by central composite design-response surface methodology / 国际中医中药杂志

Objective:To optimize the extraction process of Shangke Huoxue Granule.Methods:Taking the factors of extraction solvent multiple, extraction time and extraction times as investigation factors, and extraction amount of ferulic acid, paeoniflorin and the ratio of extraction as comprehensive evaluation indices, one-factor experimental design and central composite design-response surface methodology were adopted to optimize the extraction process of Shangke Huoxue Granule.Results:The binomial fitting equation was Y=96.16+2.42 A+0.63 B-3.76 AB-1.57 A2-1.87 B2 ( P<0.01). The optimal extraction process parameters were confirmed to be adding 16 times of water, 64 minutes each time, twice. The deviation rates between the measured values of three verification experiments and the predicted value were 2.00%, 3.23% and 0.66%. Conclusion:The established model of central composite design-response surface methodology has high predictability and the optimized extraction process is stable and feasible.

Effects of paeoniflorin on inflammation and fibrosis in db / db mice via regulating NLRP3 inflammasome pathway / 安徽医科大学学报

Objective @#To investigate the effect of paeoniflorin (PF) on liver injury mediated by NLRP3 inflamma- some pathway in db / db mice．@*Methods @#In this study，db / db mice were used as the mice model of type 2 diabetic while db / m mice were used as control group．The mice were randomly divided into six groups : db / m group，db / m + PF group，db / db group，db / db + PF 25 mg / kg group ，db / db + PF 50 mg / kg group，db / db + PF 100 mg / kg group．After 12 weeks of PF gavage，mouse serum samples were collected to detect the levels of alanine aminotrans- ferase(ALT) ，aspartate aminotransferase(AST) ，total cholesterol(TC) ，triglyceride(TG) and free fat acid(FFA) . HE staining，oil red O staining and Sirius red staining were used to observe the degree of pathological injury of liv- er．The expression of F4 /80，α-SMA and Col Ⅲ protein was detected by immunohistochemistry．The expression of interleukin-1 β ( IL-1 β ) ， interleukin-18 ( IL-18 ) ， tumor necrosis factor-α ( TNF-α ) ， NOD-like receptor 3 (NLRP3) ，apotpsis associated spck-like protein ( ASC) and cysteinyl asparate specific proteinase-1 ( Caspase-1 ) was detected by Western blot. @*Results @#Compared with db / m group，the levels of serum ALT，AST，TC，TG and FFA increased in db / db group，and these indexes decreased after PF gavage． Compared with db / m group，his- topathological examination of the liver revealed increased hepatic tissue lipid accumulation，inflammatory cell infil- tration，and collagen deposition in db / db mice，and PF treatment reduced hepatic lipid accumulation，inflammatory cell infiltration，and collagen deposition．Meanwhile，compared with db / m group，the expression of proinflammato- ry cytokines (IL-1 β , IL-18 and TNF-α) ，F4 /80，α-SMA，Col Ⅲ , NLRP3，ASC and Caspase-1 protein in the liver of db / db mice increased，and the expression of these proteins decreased after gavage with PF．@*Conclusion@#PF inhibited the NLRP3 inflammatory pathway and attenuated liver inflammation and fibrosis in db / db mice.

Therapeutic effects of benzoylaconitine and paeoniflorin in rats with collagen-induced arthritis

Abstract To explore the effects and mechanisms of benzoylaconitine and paeoniflorin on collagen-induced arthritis (CIA) rats. Weight, paw swelling, arthritis index and joint pathologic changes were examined in each group after CIA induction. PGE2, IL-1ß, IL-6, IL-10, TNF-α, VEGF, MMP-3, IgG and anti-CII Ab were assessed by ELISA; STAT1 and STAT3 expressions were analyzed immunohistochemically, and the ultrastructure of synovial cells was observed by transmission electron microscopy. Therapeutic effects were determined in CIA rats via injecting benzoylaconitine and paeoniflorin, which could alleviate the degree of swelling and arthritis index (AI) and pathological lesions of the sacroiliac gland; decrease the levels of PGE2, IL-1ß, TNF-α, VEGF and IgG in serum; reduce STAT1 and STAT3 expression in the membrane tissue; and inhibit the secretion and proliferation of synovial cells. These results showed that benzoylaconitine and paeoniflorin could significantly palliate the arthritic symptoms of CIA rats, and better therapeutic effects could be achieved if the two components were used in combination

Content determination of five constituents in Shenfukang Ⅱ capsule by HPLC / 中国药房

OBJECTIVE To develop an HPLC method for the simultaneous dete rmination of morroniside ，loganin，paeoniflorin， salvianolic acid B and icariin in Shenfukang Ⅱ capsule. METHODS The determination was performed on Agilent 5 TC-C18 column with mobile phase consisted of acetonitrile- 0.1％ phosphate acid （gradient elution ）at the flow rate of 1 mL/min. The column temperature was 30 ℃，and detection wavelength was set at 240 nm. The sample size was 10 μL. RESULTS The linear range of morroniside，loganin，paeoniflorin，salvianolic acid B and icariin were 4.80-240.00，4.84-242.00，7.00-350.00，4.72-236.00 and 5.18-259.00 μg/mL（r≥0.999 8），respectively. RSDs of precision ，stability and reproducibility tests were all lower than 3％（n＝6）. Average recoveries were 97.22％-101.36％ with the RSDs of 1.19％-2.43％（n＝6）. The contents of above 5 components in 5 batches of samples were 2.019 3-2.360 0，1.624 2-1.847 1，5.637 7-6.828 0，5.015 9-5.717 0 and 1.208 8-1.754 6 mg/g，respectively. CONCLUSIONS The method is simple ，accurate and reproducible. It can improve the quality control level of Shenfukang Ⅱ capsule.

Pharmacokinetic behavior and brain tissue distribution of paeoniflorin combined with normal and toxic doses of strychnine in rats after percutaneous administration / 中国中药杂志

This study aims to establish a rapid and sensitive UPLC-MS/MS method for simultaneously determining the content of strychnine and paeoniflorin in plasma and brain tissue of rats, and compare the pharmacokinetic behavior and brain tissue distribution of paeoniflorin combined with normal and toxic doses of strychnine in rats after percutaneous administration. Compared with those in the toxic-dose strychnine group, the AUC_(0-t), AUC_(0-∞), and C_(max) of strychnine decreased by 51.51%, 45.68%, and 46.03%, respectively(P<0.01), and the corresponding values of paeoniflorin increased by 91.41%, 102.31%, and 169.32%, respectively(P<0.01), in the compatibility group. Compared with the normal-dose strychnine group, the compatibility group showed insignificantly decreased C_(max), AUC_(0-t), and AUC_(0-∞) of strychnine, increased C_(max) and T_(max) of paeoniflorin(P<0.01), 66.88% increase in AUC_(0-t), and 70.55% increase in AUC_(0-∞) of paeoniflorin. In addition, the brain tissue concentration of strychnine decreased and that of paeoniflorin increased after compatibility. The combination of paeoniflorin with normal dose and toxic dose of strychnine can inhibit the percutaneous absorption of strychnine, and greatly promote the percutaneous penetration of paeoniflorin, whereas the interaction mechanism remains to be explored. The UPLC-MS/MS method established in this study is easy to operate and has good precision. It is suitable for in vivo study of pharmacokinetic behavior and brain tissue distribution of paeoniflorin and strychnine after percutaneous administration in rats, which provides reference for the safe and rational clinical use of strychnine and the combined use of drugs, and lays a solid foundation for the development of external preparations containing Strychni Semen.

Mechanism of paeoniflorin inhibiting apoptosis of hippocampal neurons of rats induced by lead acetate / 中华劳动卫生职业病杂志

Objective: To investigate the effect and underlying mechanism of paeoniflorin on hippocampal neuron apoptosis induced by lead acetate. Methods: In September 2020, primary hippocampal neuronal cells were isolated and cultured from fetal rats, and identified using cellular immunofluorescent. MTT assay was used to measure the cell viability to determine the concentration and time of lead acetate-induced hippocampal neuron apoptosis. MTT was also used to evaluate the effect of paeoniflorin concentration on the apoptosis of hippocampal neurons induced by lead acetate. According to the results, different concentrations of paeoniflorin were selected to intervene hippocampal neuron cells, after 24 h, lead acetate was added to the cells, meanwhile, blank and model groups were set up, the content of reactive oxygen species (ROS) , superoxide dismutase (SOD) , lactate dehydrogenase (LDH) , malondialdehyde (MDA) and Caspase-3 were measured. Extracellular signal regulated kinase (ERK) , phosphorylated ERK (p-ERK) , p38 mitogen -activated protein kinases (p38MAPK) , phosphorylated p38MAPK (p-p38MAPK) , c-Jun N-terminal kinase (JNK) and phosphorylated JNK (p-JNK) protein expression in hippocampal neuronal cells were determined by Western blotting. Results: The isolated and cultured hippocampal neurons were identified by immunofluorescence chemical staining and then treated with lead acetate, MTT results showed that lead acetate had the best toxicity effect when treated for 24 h at a concentration of 25 μmol/L. Paeoniflorin showed no cytotoxic effect on hippocampal neuronal cells when the concentrations below 80 μmol/L. Compared with the model group, the activity of hippocampal neuronal cells was significantly increased after treating with 20, 40 or 80 μmol/L paeoniflorin (P<0.05) . Compared with the blank group, the ROS activity, LDH release level, MDA content and caspase-3 content were significantly increased (P<0.01) , and the SOD activity was significantly decreased (P< 0.01) in the hippocampal neuronal cells of the model group. Compared with the model group, the ROS activity, LDH release level, MDA content and caspase-3 content were obviously decreased (P<0.05) , SOD activity was significantly increased (P <0.01) after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin. Relative to the model group, the ratio of p-ERK/ERK were significantly up-regulated (P<0.01) , while the ratios of p-p38MAPK/p38MAPK and p-JNK/JNK were significantly down-regulated after hippocampal neuronal cells were treated with 40 or 80 μmol/L paeoniflorin (P<0.05) . Conclusion: Paeoniflorin may down-regulate the expression of p-p38MAPK and p-JNK protein, up-regulate the expression of p-ERK protein, and inhibit the apoptosis of hippocampal neurons induced by lead acetate through the MAPK signaling pathway.

Paeoniflorin improves myocardial injury via inhibition of Src/VE-cadherin pathway in septic rats / 中华内科杂志

Objective:To study the protective effect and mechanism of paeoniflorin (pae) on myocardial injury in septic rats.Methods:Sprague-Dawley (SD) rats were randomly divided into 4 groups with 10 rats in each group. Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml 5% dimethyl sulfoxide (DMSO)solution independently in control group and DMSO group. Rats were intraperitoneally injected with 1.4 ml normal saline and 1.4 ml pae independently, then with 0.1 ml lipopolysaccharide (LPS) 1 hour later in sepsis group and pae group. Enzyme linked immunosorbent assay (ELISA) was used to detect serum cardiac troponin I (cTnI) levels and myocardial tissue tumor necrosis factor alpha (TNFα), interleukin(IL)-6, IL-1β, chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), vascular cell adhesion molecule 1 (VCAM-1) levels. Evans blue (EB) method was used to detect the EB content of myocardial tissue. HE staining method was used to observe the pathological changes, real-time quantitative polymerase chain reaction (RT-qPCR) to detect mRNA expression levels of the above molecules, and Western-blot to detect vascular endothelium-cadherin (VE-cadherin), phosphorylated p38 mitogen-activated protein kinase (P-p38MAPK), phosphorylated Src protein (P-Src), Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) levels.Results:Compared with control group, cTnI level and the EB content in sepsis group increased significantly, and the myocardial inflammatory cell infiltration was obvious. The cTnI level and EB content in pae group were significantly reduced, and myocardial inflammatory cell infiltration was reduced [cTnI: (227.7±15.9)pg/ml vs. (312.9±17.9)pg/ml;EB: (13.2±2.3)μg/g vs. (23.8±2.9)μg/g; P<0.05]. Compared with control group, the levels of TNFα, IL-6, IL-1β, CXCL1, CXCL2, and VCAM-1 in sepsis group were increased. Compared with sepsis group, the above-mentioned molecular levels of pae group were significantly decreased [TNFα: (63.39±9.55)pg/ml vs. (126.54±19.17)pg/ml ;IL-6: (64.03±8.82)pg/ml vs. (85.60±9.52)pg/ml;IL-1β: (69.52±9.23)pg/ml vs. (130.45±15.10)pg/ml;CXCL1: (2 600.19±379.54)pg/ml vs. (4 903.89±533.42)pg/ml;CXCL2: (93.71±10.83)pg/ml vs. (127.24±13.92)pg/ml;VCAM-1: (112.22±13.49)pg/ml vs. (149.32±15.65)pg/ml, both P<0.05]. RT-qPCR results showed that the mRNA expressions of TNFα, IL-6, IL-1β, CXCL1, CXCL2 and VCAM-1 in the sepsis group were increased compared with the control group; Compared with sepsis group, the IL-6 mRNA (1.271±0.139 vs. 1.920±0.191, P<0.05), IL-1βmRNA (1.180±0.130 vs. 1.817±0.191, P<0.05), VCAM-1 mRNA (1.088±0.144 vs. 1.460±0.166, P<0.05) expression decreased significantly in the pae group. Compared with control group, the levels of P-p38MAPK and P-Src in sepsis group increased, and the level of VE-cadherin decreased. Compared with sepsis group, the levels of p38MAPK and P-p38MAPK in pae group were significantly decreased, and the level of VE-cadherin was increased (p38MAPK/β-actin: 1.125±0.078 vs. 1.520±0.164; P-p38MAPK protein: 1.639±0.133 vs. 2.112±0.222; both P<0.05). Conclusion:Paeoniflorin could improve the permeability of cardiac microvascular endothelium in sepsis rats and inhibit the secretion and expression of inflammation-related proteins and genes, which might be related to the inhibition of Src/VE-cadherin pathway by paeoniflorin.

The healing effect of paeoniflorin on the wound and regulation of NGF/Akt/GSK3β pathway on rats with diabetic foot / 国际中医中药杂志

Objective:To analyze the healing effect of paeoniflorin on the wound of rats and regulation of NGF/Akt/GSK3β pathway on rats with diabetic foot.Methods:60 rats were randomly divided into normal control group, model group, low, medium and high dose paeoniflorin groups, 12 rats in each group. Diabetic foot model was established by intraperitoneal injection of streptozotocin (STZ) and electrothermal scald. Paeoniflorin groups were injected intraperitoneally with 20 mg/kg, 40 mg/kg and 80 mg/kg paeoniflorin, once a day for 21 days. The wound healing rate of the rats was measured. The fasting blood glucose was measured by blood glucose meter, HbAlc, TC, LDL-C and TG were measured by automatic biochemical analyzer. Serum CRP, IL-6, IL-1β and TNF-α were measured by ELISA. The histopathological changes of the wound were examined by HE staining, the levels of NGF, Akt and GSK3 β mRNA in skin tissue were measured by real-time quantitative polymerase chain reaction (RTq-PCR), the protein and phosphorylation levels of NGF, Akt and GSK3 β were determined by Western blot.Results:Compared with the model group, the healing rate the rats' wounded surface in the low, medium and high dose groups of paeoniflorin was increased ( P<0.05). The levels of fasting blood glucose, HbAlc, TC, LDL-C, TG levels and serum CRP, IL-6, IL-1β, TNF-α was decreased ( P<0.05). The expression of rat skin tissue NGF mRNA (0.83±0.12, 3.17±0.11, 4.54±0.25 vs. 0.31±0.06), Akt mRNA (1.71±0.14, 2.96±0.27, 4.10±0.34 vs. 0.97±0.20) increased ( P<0.05), expression of GSK3β mRNA (4.28±0.35, 2.82±0.14, 1.22±0.33 vs. 7.62±0.43) decreased ( P<0.05), expression of NGF (0.46±0.02, 0.70±0.04, 0.87±0.04 vs. 0.30±0.06), Akt (0.51±0.09, 0.63±0.03, 0.79±0.06 vs.0.41±0.05),p-NGF/NGF (0.47±0.06, 0.61±0.04, 0.83±0.07 vs. 0.25±0.03), p-Akt/Akt(0.54±0.08, 0.83±0.11,0.96±0.07 vs. 0.13±0.05) was increased( P<0.05), the expression of GSK3β (0.67±0.05, 0.54±0.04,0.45±0.03 vs. 0.86±0.05), and the ratio of p-GSK3β/GSK3β (0.78±0.09, 0.64±0.07, 0.42±0.07 vs. 0.97±0.05) was decreased ( P<0.05), and the changes of each index were dependent on the dose of paeoniflorin. Conclusion:Paeoniflorin can regulate the level of blood glucose and blood lipid, inhibit the level of serum inflammatory factors and promote the healing of the wound in diabetic foot rats, and its mechanism may be related to the regulation of NGF/Akt/GSK3β pathway.

Effect of paeoniflorin on motor behavior and dopamine neurons of mice with Parkinson's disease by activating autophagy / 中华神经医学杂志

Objective:To observe the effect of paeoniflorin on motor behavior and dopamine neurons of mice with Parkinson's disease (PD) by activating autophagy.Methods:Forty C57BL/6 mice were randomly divided into normal control group, model group, paeoniflorin group and paeoniflorin+3-methyl adenine (3-MA) group ( n=10). The later 3 groups were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetmhydropyridine (MPTP) at a dosage of 30 mg/(kg·d) for a consecutive 7 d to establish subacute PD models. Meanwhile, mice in the paeoniflorin group and paeoniflorin+3-MA group were intraperitoneally injected with paeoniflorin (30 mg/[kg·d]) or paeoniflorin (30 mg/[kg·d])+3-MA (2 mg/[kg·d]), respectively. Eight d after modeling, pole test and traction test were used to evaluate the locomotor ability of mice in each group. Then, the substantia nigra of the midbrain was taken; TUNEL was used to detect the nerve cell apoptosis; immunohistochemical staining was used to detect the number of tyrosine hydroxylase (TH) positive cells and the expressions of α-synuclein (α-Syn), lysosomal associated membrane protein 2A (LAMP2A) and microtubule-associated protein 1 light chain 3-II (LC3-II); Western blotting was used to detect the LAMP2A and LC3-II protein levels. Results:As compared with model group, paeoniflorin group had significantly shortened rod climbing time, significantly higher scores in traction test, significantly decreased number of apoptotic nerve cells, significantly increased number of TH-positive cells and expressions of LAMP2A and LC3-II, significantly decreased α-Syn expression, and significantly increased protein levels of LAMP2A and LC3-II ( P<0.05). The paeoniflorin+3-MA group had significantly prolonged rod climbing time, significantly lower scores in traction test, significantly increased number of apoptotic nerve cells, significantly decreased number of TH-positive cells and expressions of LAMP2A and LC3-II, significantly increased expression of α-Syn, and significantly decreased protein levels of LAMP2A and LC3-II as compared with the paeoniflorin group ( P<0.05). Conclusion:Paeoniflorin can improve dyskinesia in PD mice by inducing autophagy to remove α-Syn and protect dopaminergic neurons.

Mechanism of paeonol combined with paeoniflorin against myocardial ischemia injury:based on proteomics / 中国中药杂志

The study aims to investigate the effect of the compatibility of paeonol and paeoniflorin(hereinafter referred to as the compatibility) on the expression of myocardial proteins in rats with myocardial ischemia injury and explore the underlying mechanism of the compatibility against myocardial ischemia injury. First, the acute myocardial infarction rat model was established by ligation of the anterior descending branch of the left coronary artery. The model rats were given(ig) paeonol and paeoniflorin. Then protein samples were collected from rat cardiac tissue and quantified by tandem mass tags(TMT) to explore the differential proteins after drug intervention. The experimental results showed that differential proteins mainly involved phagocytosis engulfment, extracellular space, and antigen binding, as well as Kyoto encyclopedia of genes and genomes(KEGG) pathways of complement and coagulation cascades, syste-mic lupus erythematosus, and ribosome. In this study, the target proteins and related signaling pathways identified by differential proteomics may be the biological basis of the compatibility against myocardial ischemia injury in rats.

Chemical Constituents and Pharmacological Effects of Paeoniae Radix Rubra： A Review / 中国实验方剂学杂志

Paeoniae Radix Rubra is a traditional Chinese medicine commonly used in clinical practice， it is mostly wild and widely distributed in different areas of China. In addition， the plant of Paeoniae Radix Rubra also has ornamental value. Modern phytochemical researches showed that the chemical constituents of Paeoniae Radix Rubra were complex. Up to now， more than 300 chemical constituents have been found， mainly including monoterpene glycosides， triterpenoids， flavonoids， tannins， phenolic acids， saccharides， steroids， volatile oils and so on. Among them， the content of monoterpene glycosides was the highest， and the types of volatile oil were the most. Paeoniae Radix Rubra has a wide range of pharmacological effects， exerting different curative effects in multiple systems such as blood， cardiovascular， nervous and digestive system. It can protect myocardial cells and nerve cells， stabilize microcirculation， anti-endotoxin， anti-atherosclerosis， reduce pulmonary hypertension， anti-depression， protect liver， anti-gastric ulcer， anti-tumor， slow down aging， treat Parkinson's syndrome and diabetes and its complications， anti-radiation， anti-inflammatory， anti-virus and so on. Through reviewing the literature on chemical constituents and pharmacological effects of Paeoniae Radix Rubra， it was found that total glycosides and monomers such as paeoniflorin， albiflorin， benzoylpaeoniflorin and gallic acid may be the main active components of Paeoniae Radix Rubra. At present， the research on Paeoniae Radix Rubra mainly focused on monoterpene glycosides， while the research on flavonoids and volatile oil in Paeoniae Radix Rubra was less. It is suggested that research on these two components should be strengthened in the future.

Study on Improvement Effects and Mechanism of Paeoniflorin on Myocardial Injury in Type 2 Diabetic Model Rats / 中国药房

OBJECTIVE：To study the improvement effects of paeon iflorin（PF）on myocardial injury in type 2 diabetes mellitus（T2DM）model rats and its mechanism. METHODS ：The experiment was set up in the normal group ，model group ， positive control group （metformin 90 mg/kg），PF high-dose ，medium-dose and low-dose groups （90，60，30 mg/kg），with 8 rats in each group. Except for normal group ，other groups were given high-glucose and high-fat diet and intraperitoneal injection of streptozotocin （30 mg/kg） to induce T 2DM model. After modeling ， administration groups were given relevant medicine intragastrically，normal group and model group were given constant volume of normal saline intragastrically ，once a day ，for consecutive 4 weeks. The body weight ，fasting blood glucose and oral glucose tolerance were measured ；serum levels of glycosylated serum protein （GSP），total cholesterol （TC），triacylglycerol（TG），glutathione peroxidase （GSH-Px），superoxide dismutase（SOD），malondialdehyde（MDA），creatine kinase isoenzyme-MB （CK-MB） and troponin Ⅰ （cTn Ⅰ） were determined. The pathomorphological changes of myocardium were observed. The apoptosis index of rat cardiomyocytes was （ detected. The protein expression of B-cell lymphoma 2 （Bcl-2），Bcl-2 related X protein （Bax）and caspase- 3 in rat myocardium were detected by immunohistochemistry and Western blot. RE SULTS：Compared with normal group ，the body weight ，serum levels of GSH-Px and SOD ，protein expression of Bcl- 2 in myocardium were decreased significantly in model group（P＜0.01）；while fasting blood glucose ，area under blood glucose curve ，serum levels of biochemical indexes （GSP，TC， TG，MDA，CK-MB，cTnⅠ），cardiomyocyte apoptosis index ，protein expression of Bax and caspase- 3 in myocardium were increased significantly （P＜0.05 or P＜0.01）. The arrangement of myocardium was relatively irregular ，and some muscle fibers were broken. Compared with model group ，except for body weight ，serum levels of SOD and MDA ，the protein expression of Bax in myocardium in PF low-dose group ， above indexes of PF groups were reversed significantly （P＜0.05 or P＜0.01）. CONCLUSIONS：PF can regulate glycolipid metabolism ，enhance antioxidant ability ，inhibit cardiomyocyte apoptosis and improve myocardial injury in T 2DM model rats ；the mechanism may be associated with increasing the protein expression of Bcl- 2 and down-regulating the protein expression of Bax and caspase- 3 in myocardium.

Paeoniflorin regulates gut microbiota and bile acids metabolism in colitis mice / 药学学报

The current study was designed to evaluate the modulatory effects of paeoniflorin on the dysregulated gut microbiota as well as the disturbed fecal bile acids (BAs) in colitis mice. After approved by Xi'an Jiaotong University Ethics Committees (Approval No. XJTU2019-679), the animals were randomly distributed into the control (Con), colitis, low dose paeoniflorin (PF-L, 25 mg·kg-1·d-1), high dose paeoniflorin (PF-H, 50 mg·kg-1·d-1) and 5-aminosalicylic acid (5-ASA, 50 mg·kg-1·d-1) groups. Colitis was induced by administering 3% (w/v) DSS in drinking water for 7 days. Paeoniflorin and 5-ASA were dissolved in water and administered to the appropriate groups by oral gavage over the 7-day period. The mice were monitored daily, and the disease activity index (DAI) comprising of body weight loss, stool consistency and gross blood was measured. The pathological changes of colon were evaluated by HE staining; the levels of inflammatory cytokines in colonic tissue were determined by ELISA; the gut permeability was measured by FITC-dextran. Microbiota analysis based on 16S rDNA and targeted metabolomics for BAs were used to evaluate the composition of gut microbiota and fecal BAs pool. The results showed that administration of paeoniflorin markedly alleviated the inflammatory response and intestinal barrier dysfunction in DSS-induced colitis. Importantly, these ameliorative effects of paeoniflorin were accompanied by the improvements of disturbed composition of gut microbiota and the dysmetabolism of bile acids in feces. Finally, we performed Spearman's correlation analysis between the fecal BAs and gut microbiota genera, and found that Lactobacillus has a strong positive correlation with DCA and LCA which were reported to confer the beneficial effects of maintaining intestinal homeostasis. Taken together, paeoniflorin might improve the intestinal homeostasis in colitis mice via modulating gut microbiota and fecal BAs metabolism.

Protective effect of paeoniflorin on H / 中国天然药物

This study was to investigate the protective effect of paeoniflorin (PF) on hydrogen peroxide-induced injury. Firstly, "SMILES" of PF was searched in Pubchem and further was used for reverse molecular docking in Swiss Target Prediction database to obtain potential targets. Injury-related molecules were obtained from GeenCards database, and the predicted targets of PF for injury treatment were selected by Wayne diagram. For mechanism analysis, the protein-protein interactions were constructed by String, and the KEGG analysis was conducted in Webgestalt. Then, cell viability and cytotoxicity assay were established by CCK8 assay. Also, the experimental cells were allocated to control, model (200 μmol·L

HPLC fingerprint of Moutan Cortex in different growth period / 国际中医中药杂志

Objective:To establish the HPLC fingerprint method for assessing the quality of Moutan Cortex, and to determine the contents of paeonol, paeoniflorin, gallic acid, hydroxyl-paeoniflorin and benzoyl-paeoniflorin of Moutan Cortex in different growth period. Methods:Diamonsil Plus C18 column (250 mm × 4.6 mm, 5 μm) was used with the mobile phase comprising acetonitrile-0.05% formic acid solution and the flow rate of 1.0 ml/min with gradient elution manner. The detected wavelength was 230 nm for paeoniflorin and benzoyl-paeoniflorin, 267 nm for gallic acid, 258 nm for hydroxyl-paeoniflorin and 274 nm for paeonol with temperature column of 25 ℃. Then putting chromatograms into Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica (2012A) to evaluate the similarity of Moutan Cortex in different growth period; then putting peak area data into SPSS software for cluster analysis and the clustering effect was determined. Results:The HPLC fingerprints established with this method has 23 shared peaks and 5 of them were identified, namely, paeonol, paeoniflorin, gallic acid, hydroxyl-paeoniflorin and benzoylpaeoniflorin. The similarity of Moutan Cortex in different years was between 0.850-0.991. This method has good linear relation ( r≥0.999 5), RSDs of precision, stability tests and reproducibility were lower than 1.6% ( n=6). Different growth periods of Moutan Cortex have obvious influence on the concentration of five compounds. Conclusion:This method is useful to evaluate and discriminate Moutan Cortex at different growth periods so as toprovide scientific reference on the harvest,industrialization and evaluation of Moutan Cortex.

Study on specific chromatogram and content determination of multi-index components about material reference of classic prescription of Huaganjian / 中草药

Objective: To establish the UPLC specific chromatogram and HPLC content determination methods of multi-index components about the material reference of classical Huaganjian and build its quality control system. Methods: According to the ancient books and combining with the previously inspected process, 18 batches of Huaganjian material reference from different origins were prepared. The specific chromatogram was established by using UPLC. Similarity was calculated by using TCM Chromatographic Fingerprint Similarity Evaluation Software (2012). Combining with orthogonal partial least squares discriminant analysis, we excavated the main components that affected the quality of Huaganjian material reference from different batches and origins. Three of these index components (paeoniflorin, hesperidin, paeonol) from prescription sovereign drug, minister drug, and assistant drug were selected and used as indicators for content determination of Huaganjian material reference. HPLC content determination methods were established and the content of 18 batches of samples was determined respectively. Results: The similarity of the specific chromatogram was ≥ 0.989. Thirty-three common peaks were calibrated, and eight common peaks were identified by chemical composition (gallic acid, geniposide, paeoniflorin, hesperidin, didymin, paeonol, sinensetin, and 3,5,6,7,8,3',4'- heptamethoxyflavone). Nine index components that affected the stability between batches were found out (Peak 31, 20, 11, 13, 22, 33, 21, 29, 1). Paeoniflorin, hesperidin, and paeonol were selected as content determination indicators. The content range of these components in material reference was 1.28%-1.95% paeoniflorin, 0.91%-1.02% hesperidin, 0.48%-0.57% paeonol. Conclusion: The quality control method of the material reference of classic prescription Huaganjian was established preliminarily through the UPLC specific chromatogram and HPLC content determination of index components. This method was rapid, simple, feasible, reproducible, stable and could provide a theoretical basis for the subsequent development and quality control of Huaganjian preparations.