Hedysari Radix Polysaccharide Regulates Apoptosis of Smooth Muscle Cells in Gastric Antrum of Rat Model of Diabetic Gastroparesis via IGF-1/PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Hedysari Radix polysaccharide on the apoptosis of gastric sinus smooth muscle cells and explore the underlying mechanism via the insulin-like growth factor-1 （IGF-1）/phosphatidylinositol 3-kinase （PI3K）/serine-threonine kinase （Akt） pathway in the rat model of diabetic gastroparesis （DGP）. MethodSixty-two Wistar male rats were randomized into a blank group （n=12） and a modelling group （n=50）. The rat model of DGP was established by small-dose multiple intraperitoneal injections of streptozotocin combined with an irregular high-fat and high-sugar diet for 4 weeks. The modeled rats were randomized into model group， mosapride citrate （1.35 mg·kg-1）， and high-， medium-， and low-dose （200， 100， and 50 mg·kg-1， respectively） Hedysari Radix polysaccharide groups. The rats were administrated with corresponding drugs by gavage， and those in the blank and model groups with equal volumes of pure water by gavage once a day for 8 consecutive weeks. The random blood glucose and body mass were measured every 2 weeks， and gastric emptying rate was calculated. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of smooth muscle in gastric antrum， and terminal deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to detect the apoptosis of smooth muscle cells in the gastric antrum. The expression of IGF-1， phosphorylated （p）-PI3K， and p-Akt in the smooth muscle of gastric sinus tissue was detected by immunohistochemistry. Western blot was employed to determine the protein levels of IGF-1， p-PI3K/PI3K， p-Akt/Akt， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in the smooth muscle of the gastric antrum. ResultCompared with the blank group， the model group showed elevated random blood glucose at all time points （P<0.01）， decreased body mass and gastric emptying rate （P<0.01）， increased apoptotic index of smooth muscle cells in the gastric antrum （P<0.01）， down-regulated protein levels of IGF-1， p-PI3K/PI3K， p-Akt/Akt， and Bcl-2， and up-regulated protein level of Bax （P<0.01）. Compared with the model group， the 8 weeks of drug administration lowered the random blood glucose， increased the body mass and gastric emptying rate （P<0.05， P<0.01）， decreased the apoptotic index of smooth muscle cells in the gastric antrum （P<0.05， P<0.01）， up-regulated the protein levels of IGF-1， p-PI3K/PI3K， p-Akt/Akt， and Bcl-2， and down-regulated the protein level of Bax （P<0.05， P<0.01）. Compared with the mosapride citrate group，the administration of low-dose Hedysari Radix polysaccharide for 6 and 8 weeks lowered the random blood glucose and decreased the body mass （P<0.05， P<0.01），low and medium-dose Hedysari Radix polysaccharide decreased the gastric emptying rate and the apoptotic index of smooth muscle cells in the astragaloside low-dose group decreased （P<0.05）. The protein levels of IGF-1，p-PI3K/PI3K，p-Akt/Akt and Bcl-2（low dose）were down-regulated and the protein level of Bax was up-regulated by low doses of Hedysari Radix polysaccharide （P<0.05， P<0.01）. Compared with high-dose Hedysari Radix polysaccharide， low-dose Hedysari Radix polysaccharide elevated random blood glucose and reduced body mass after 6 and 8 weeks of administration （P<0.05， P<0.01）， and the low and medium doses decreased the gastric emptying rate， increased the apoptotic index of smooth muscle cells in the gastric antrum （P<0.05， P<0.01）， down-regulated the protein levels of IGF-1， p-PI3K/PI3K， p-Akt/Akt， and Bcl-2， and up-regulated the protein level of Bax （P<0.05， P<0.01）. Compared with the medium-dose group，the low-dose group of Hedysari Radix polysaccharide had lower body mass，lower gastric emptying rate in rats，higher apoptotic index of smooth muscle cells in gastric sinus tissue after 6 and 8 weeks of administration （P<0.05， P<0.01）， and lower protein expression of IGF-1，p-PI3K/PI3K，p-Akt/Akt. ConclusionHedysari Radix polysaccharide protects the smooth muscle cells in gastric antrum against apoptotic injury and promotes gastric motility by activating the IGF-1/PI3K/Akt signaling pathway， as manifested by the up-regulated expression of IGF-1， p-PI3K， p-Akt， and Bcl-2 and down-regulated expression of Bax.

Comparison on Ameliorating Effect of Arisaematis Rhizoma and Arisaematis Rhizoma Preparatum on Allergic Asthma in Rats / 中国实验方剂学杂志

ObjectiveTo investigate the therapeutic effects and difference in the effects of Arisaematis Rhizoma （AR） before and after processing （i.e.， Arisaematis Rhizoma Preparatum， ARP） with Zingiberis Rhizoma Recens-Alumen on allergic asthma in rats and to provide a basis for the theory of processing improving the efficacy. MethodA rat model of allergic asthma was established in 70 SD rats by intraperitoneal injection of ovalbumin （OVA）-aluminum hydroxide. The rats were administrated with the aqueous extracts of AR （1.2， 0.3 g∙kg-1） and ARP （1.2， 0.3 g∙kg-1） aqueous extracts by gavage， and montelukast sodium （0.001 g∙kg-1） was used as the positive drug. The T helper cell type 1/type 2 （Th1/Th2） ratio in the serum and bronchoalveolar lavage fluid （BALF） and percentages of inflammatory cells in BALF were determined. Polymerase chain reaction （PCR） was employed to determine the mRNA level of mucin 5AC （MUC5AC） in the lung tissue. The pathological changes in the lung tissue were observed by hematoxylin-eosin （HE） staining and PAS staining. Immunohistochemical assay was employed to measure the expression of c-Jun amino-terminal kinase （JNK）， extracellular signal regulated protein kinase （ERK）， and p38 mitogen-activated protein kinase （p38 MAPK） in rat lung tissue. Western blot was employed to determine the protein levels of ERK， p-ERK， JNK， p-JNK， p38， p-p38 in the lung tissue. The effects of AR and ARP were compared based on overall desirability. ResultCompared with the blank group， the levels of interleukin-12 （IL-12） and γ interferon （IFN-γ） in serum and BALF of rats in the model group were significantly lower （P<0.05， P<0.01）， and the levels of interleukin-4 （IL-4）， interleukin-5 （IL-5） and interleukin-13 （IL-13） were significantly higher （P<0.05， P<0.01）. Compared with the model group， the serum and BALF contents of IL-12 and IFN-γ in rats in the montelukast sodium group， high-dose AR group and high-dose ARP group were significantly higher （P<0.05， P<0.01）， and the contents of IL-4， IL-5 and IL-13 were significantly lower （P<0.05， P<0.01）， and the serum contents of IFN-γ in rats in the low-dose AR group and low-dose ARP group were in BALF was significantly higher （P<0.05） and IL-4 and IL-13 were significantly lower （P<0.05， P<0.01）， the percentages of macrophages， lymphocytes， neutrophils， and eosinophils were reduced in BALF， and the expression of JNK/ERK/p38 MAPK signaling pathway and MUC5AC protein was inhibited in lung tissues. Overall assessment of the normalized analysis revealed that the ARP group was slightly more potent than the AR group after administration of the same dose. ConclusionAR and ARP can effectively treat allergic asthma by inhibiting JNK/ERK/p38 MAPK signaling pathway， and the effect is better after concoction， which can provide data support for its "concoction efficiency".

Modified Tongqiao Huoxuetang Down-regulates PI3K/Akt Pathway to Treat Basilar Artery Dolichoectasia / 中国实验方剂学杂志

ObjectiveTo establish a mouse model of basilar artery dolichoectasia （BAD） and explore the mechanism of modified Tongqiao Huoxuetang （JTQHX） in regulating BAD via phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） pathway. MethodSixty C57/BL6 female mice were randomized into sham operation （injected with 10 U·mL-1 inactivate elastase）， model， atorvastatin calcium tablets （2.6 mg·kg·d-1）， and low- and high-dose （crude drug 3.4， 17 g·kg-1·d-1， respectively） JTQHX groups. The mouse model of BAD was established by injection with 10 U·mL-1 elastase. After 14 days of modeling， the sham operation group and model group were administrated with equal volumes of pure water by gavage， and other groups with corresponding drugs for 2 months. The levels of interleukin-6 （IL-6） and calpain （LpA） in the serum were measured by enzyme-linked immunosorbent assay （ELISA）. Verhoeff 's Van Gieson （EVG） staining was employed to observe the pathological changes of blood vessels. Terminal-deoxynucleotidyl transferase mediated nick end labeling （TUNEL） was employed to examine the apoptosis rate of vascular smooth muscle cells （VSMCs）. Image Pro Plus was used to observe and calculate the curvature index， elongation length， percentage increase in vessel diameter， and curvature angle of the basilar artery vessels in mice. Western blot was employed to determine the expression levels of PI3K and Akt in the vascular tissue. ResultCompared with the sham operation group， the model group showed lowered IL-6 level （P<0.01）， no significant change in LpA level， increased apoptosis of VSMCs （P<0.01）， and increased curvature index， elongation length， percentage increase in vessel diameter， and curvature angle （P<0.01）. Furthermore， the modeling up-regulated the protein levels of PI3K and Akt in blood vessels （P<0.01） and aggravated the destruction of the inner elastic layer， atrophy of the muscular layer， and hyaline changes in the connective tissue of the medial membrane of the basilar artery wall. Compared with the model group， 2 months of treatment with JTQHX elevated the IL-6 level （P<0.01）， reduced the apoptosis of VSMCs （P<0.01）， decreased the curvature index， elongation length， percentage increase in vessel diameter， and curvature angle （P<0.05， P<0.01）， and down-regulated the protein levels of PI3K and Akt in blood vessels （P<0.01）. In addition， the treatment alleviated the destruction of the inner elastic layer， atrophy of the muscular layer， and hyaline changes in the connective tissue of the medial membrane of the basilar artery wall. ConclusionJTQHX inhibits the elongation， expansion， and curvature of basilar artery vessels and alleviates the pathological changes by reducing the apoptosis of VSMCs and down-regulating the expression of PI3K/Akt pathway.

Electroacupuncture Promotes Functional Recovery after Facial Nerve Injury in Rats by Regulating Autophagy via GDNF and PI3K/mTOR Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway.@*METHODS@#Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR.@*RESULTS@#The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01).@*CONCLUSIONS@#EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism.@*METHODS@#Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 β in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis.@*RESULTS@#DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 β (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01).@*CONCLUSIONS@#DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.

Fangji Fuling Decoction Alleviates Sepsis by Blocking MAPK14/FOXO3A Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments.@*METHODS@#A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed.@*RESULTS@#FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05).@*CONCLUSION@#FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443 / 中国结合医学杂志

OBJECTIVE@#To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration.@*METHODS@#HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR.@*RESULTS@#HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05).@*CONCLUSION@#HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.

Effect of microparticles derived from bone marrow mesenchymal stem cells on cardiomyocyte hypertrophy and its mechanism / 中国生物制品学杂志

@#Objective To investigate the effect of microparticles(MPs)derived from bone marrow mesenchymal stem cells(BMSCs) on myocardial hypertrophy and its mechanism.Methods The osteogenic differentiation and adipogenic differentiation of mesenchymal stem cells(MSCs) were induced. After isolation and purification,the morphological characteristics were observed by transmission electron microscope,and the MPs surface antigen was identified by flow cytometry. Myocardial hypertrophy model was induced by using isoprenaline(ISO)in rats,which were measured for the cardiac structure and function by echocardiography,and then detected for various indexes of the heart and isolated left ventricle. Single ventricular myocytes of rats were acutely isolated and divided into control group(Control group),cardiomyocyte hypertrophy group(ISO group),MPs group(MPs group),and MPs supernatant group(Supernatant group). The mRNA expressions of atrial natriuretic peptide(ANP)and B-type natriuretic peptide(BNP)were detected by qRTPCR. The expression levels of calmodulin-dependent protein kinaseⅡ（CaMKⅡ）and phosphorylated calmodulin-dependent protein kinaseⅡ（p-CaMKⅡ）were detected by ELISA. The L-type calcium current(LCa-L)in single ventricular myocyte of various groups was recorded by whole-cell patch clamp.Results The bone nodules of MSCs osteogenic differentiation turned red after alizarin red staining,and lipid droplets of adipogenic differentiation turned red after oil red O staining;Under transmission electron microscope,MPs membrane had a complete structure,a clear outline and a diameter of about200 nm;The positive rates of CD29 and CD90 on the surface of MPs were(98. 24 ± 0. 82)% and(97. 69 ± 1. 83)%,respectively. Compared with Control group,the left ventricular end diastolic dimension(LVEDD)reduced signifi-cantly(t =5. 065,P < 0. 05),while the interventricular septum end-diastolic dimension(IVSd),left ventricular posterior wall dimension(LVPWd),heart weight to body weight ratio(HW/BW),and heart weight to tibial length ratio(HW/Tibia)significantly increased in ISO group(t = 4. 013,2. 368,4. 392,5. 043 and 6. 120,respectively,each P < 0. 05),indicating that the hypertrophic model was successfully established. The expression levels of ANP and BNP mRNA in hypertrophic cardiomyocytes of rats in ISO group were significantly higher than those in Control group(t = 25. 120 and18. 261,respectively,each P < 0. 01);While the expression levels of ANP and BNP mRNA in MPs group significantly reduced after incubation with 48 μg/mL MPs for 48 h compared with ISO group(t = 12. 110 and 3. 526,respectively,each P < 0. 05);The expression levels of CaMK Ⅱand p-CaMKⅡ in ISO group were significantly higher than those in Control group(t = 3. 278 and 4. 181,respectively,each P < 0. 05),while the expression of p-CaMK Ⅱ in MPs group decreased significantly(t = 5. 420,P < 0. 05);The calcium current density in ISO group was significantly higher than that in Control group(t = 15. 261,P < 0. 01),while that in MPs group was significantly lower than that in ISO group(t =6. 216,P < 0. 05).Conclusion MSC-MPs can significantly inhibit ISO-induced cardiomyocyte hypertrophy in rats,which is related to its down-regulation of cardiomyocyte CaMKⅡ and inhibition of L-type calcium channel.

Efficacy and safety of tyrosine kinase inhibitors in the treatment of HER2-positive breast cancer：a meta-analysis / 中国药房

OBJECTIVE To evaluate the efficacy and safety of tyrosine kinase inhibitors （TKI） in the treatment of HER2- positive breast cancer in order to provide evidence-based evidence for clinical medication. METHODS Retrieved from CNKI， Wanfang database， VIP， PubMed， Cochrane Library， Embase and Web of Science， randomized controlled trial （RCT） about TKI （trial group） versus drugs excluding TKI （control group） in the treatment of HER2-positive breast cancer were collected from the establishment of the database to April 2023. Meta-analysis and sensitivity analysis were performed by using RevMan 5.4.1 and Stata 17 software. RESULTS Total of 24 RCT studies were included， involving 15 538 HER2-positive breast cancer patients. The meta- analysis results showed that compared with the control group， the progression-free survival （PFS） [HR＝0.91， 95%CI （0.80， 1.02）， P＝0.12]， overall survival （OS） [HR＝0.95， 95%CI （0.89， 1.01）， P＝0.11]， objective response rate （ORR） [OR＝1.21， 95%CI （0.86， 1.69）， P＝0.27]， and pathological complete response rate （pCR） [OR＝1.44， 95%CI （0.91， 2.27）， P＝0.12] had no statistically significant difference in the trial group； among the 3/4 grade ADRs， the trial group had a higher incidence of anemia [OR＝1.77， 95%CI （1.16，2.70）， P＝0.008]， rash [OR＝11.26， 95%CI （7.32，17.31）， P＜0.000 01]， paronychia [OR＝8.67， 95%CI（1.62，46.53）， P＝0.01]， diarrhea [OR＝10.17， 95%CI（5.03，20.58）， P＜0.000 01]， oral mucositis inflammation [OR＝ 9.34， 95%CI （3.13， 27.83）， P＜0.000 1]， elevated aspartate aminotransferase [OR＝2.09， 95%CI （1.13，3.84）， P＝0.02]， and hypokalemia [OR＝2.37， 95%CI （1.31，4.30）， P＝0.005] than that of the control group. Subgroup analysis results showed that compared with the placebo group， TKI could improve OS and ORR （P＜0.05）， while compared with trastuzumab， TKI had no advantage in PFS， OS， ORR， and pCR， and TKI combined with trastuzumab could significantly improve PFS， OS， ORR， and pCR compared with the trastuzumab group （P＜ 0.05）. Sensitivity analysis suggested that the results were relatively robust and the risk of publication bias was low. CONCLUSIONS Compared with trastuzumab， TKI has no advantages in PFS， OS， ORR and pCR in the treatment of HER2- positive breast cancer， but TKI combined with trastuzumab can significantly improve PFS， OS， ORR and pCR； TKI can increase the risk of grade 3/4 anemia， rash， paronychia， diarrhea， oral mucositis， elevated aspartate aminotransferase， and hypokalemia.

Treatment of Cerebral Ischemia-reperfusion Injury by Traditional Chinese Medicine Based on PI3K/Akt Signaling Pathway： A Review / 中国实验方剂学杂志

Cerebral ischemia/reperfusion injury （CIRI） is a complex cascade reaction process in which the blood flow and oxygen supply of brain tissue in the infarcted area recover after cerebral ischemia， resulting in secondary injury of ischemic brain tissue. At present， thrombolysis as soon as possible and restoration of cerebral blood supply are still the only strategies for the treatment of stroke， but a considerable number of patients' symptoms will be more serious after reperfusion， making patients face adverse outcomes such as neurological function injury and even death and seriously affecting the quality of life and safety of patients. Therefore， an in-depth exploration of the mechanism and treatment strategy of CIRI has important clinical significance. The phosphatidylinositol 3- kinase （PI3K）/protein kinase B （Akt） signaling pathway is one of the classic anti-apoptosis/reproductive-promoting signal transduction pathways， which is responsible for cell proliferation， growth， and differentiation. It is the key cascade signaling pathway of CIRI， located at the core site in many mechanisms such as mitochondrial disorder， apoptosis， autophagy， oxidative stress， and inflammation. It is closely related to the occurrence and development of CIRI. Traditional Chinese medicine has been used in the clinical treatment of stroke and its complications for thousands of years， and the clinical effect of traditional Chinese medicine in the prevention and treatment of CIRI has been affirmed by a large number of research results in recent years. It is further clarified that the monomers， active components， and their compound prescriptions of traditional Chinese medicine can directly or indirectly regulate the PI3K/Akt signaling pathway by virtue of the biological advantages of multi-targets， multi-components， and multi-pathways and play an overall protective role in CIRI. By analyzing the related research progress of traditional Chinese medicine in China and abroad in recent years， the authors summarized the role and mechanism of regulating the PI3K/Akt signaling pathway in the prevention and treatment of CIRI， so as to provide further theoretical basis for the study of the mechanism of clinical prevention and treatment of CIRI.

Exploring Effect and Mechanism of Astragaloside on Repair and Healing in Chronic Non-healing Wound Rat Model Based on SIRT1 and PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the promotional effect of astragaloside on the repair and healing of chronic non-healing wounds and its mechanism. MethodA total of 60 male SD rats were constructed with full-layer skin defect wounds on the back， and except for the control （Con） group， the rest were constructed with non-healing wounds， which were then randomly divided into the sham-operation （sham） group， the low-dose astragaloside group， the high-dose astragaloside group， the astragaloside + LY294002 ［phosphatidylinositol 3-kinase （PI3K） inhibitor］ group， and the astragaloside + EX527 ［silencing regulatory protein 1 （SIRT1） inhibitor］ group. The percentage of wound area in each group was observed on the 2nd， 4th， 6th， and 8th days after wound molding. Collagen type Ⅰ alpha 1 （COL1A1） and alpha smooth muscle actin （α-SMA） expressions in the wound tissue were detected by immunofluorescence. Hematoxylin and eosin （HE） staining was performed to determine the pathological structure of the wound. The mRNA expression of inflammatory factors in the wound was measured by real-time polymerase chain reaction （Real-time PCR）， and the expression of proteins related to the SIRT1/ nuclear factor （NF）-κB and PI3K/protein kinase B （Akt） signaling pathways in the wound was tested by Western blot. ResultCompared with the sham group， the percentage of postoperative wound area of rats in both low-dose and high-dose astragaloside groups gradually decreased with time， and the efficacy of the high-dose astragaloside group was better. Compared with the Con group， the fluorescence intensity of COL1A1 in wound tissue of the sham group decreased， while the expression of α-SMA increased. The epithelial tissue was severely damaged， with an increase in the thickness， and a large number of inflammatory cells were seen in the infiltration. The mRNA expression of tumor necrosis factor （TNF）-α， interleukin （IL）-1β， IL-6， and inducible nitric oxide synthase （iNOS） was elevated. The protein expression of NF-κB p65， p-PI3K/PI3K， and p-Akt/Akt was elevated， while SIRT1 expression was decreased （P<0.05）. Compared with the sham group， the fluorescence intensity of COL1A1 and α-SMA increased after astragaloside treatment. The number of epithelial cells increased， and the thickness decreased. The inflammatory cells decreased， and the amount of collagen increased. The mRNA expression of TNF-α， IL-1β， IL-6， and iNOS was decreased， and the protein expression of NF-κB p65， p-PI3K/PI3K， and p-Akt/Akt was decreased. SIRT1 was elevated， and the effect was better in the high-dose astragaloside group （P<0.05）. Compared with the high-dose astragaloside group， inhibition of the PI3K/Akt and SIRT1 pathways by LY294002 and EX527 prevented the therapeutic efficacy of astragaloside on chronic non-healing wounds. ConclusionThe topical application of astragaloside significantly promotes the healing of chronic non-healing wounds in rats， and the mechanism may be related to the activation of the PI3K/Akt pathway and the SIRT1/NF-κB pathway.

Inhibition of Oxidative Stress of Sciatic Nerve in Diabetic Rats by Tangbikang Granules Regulating AMPK/PGC-1α/SIRT3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Tangbikang granules on oxidative stress of sciatic nerve in diabetic rats by regulating adenylate activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α/mitochondrial Sirtuins 3 （AMPK/PGC-1α/SIRT3） signaling pathway. MethodThe spontaneous obesity type 2 diabetes model was established using ZDF rats. After modeling， they were randomly divided into high， medium， and low dose Tangbikang granule groups （2.5， 1.25， 0.625 g·kg-1·d-1） and lipoic acid group （0.026 8 g·kg-1·d-1）， and the normal group was set up. The rats were administered continuously for 12 weeks after modeling. The blood glucose of rats was detected before intervention and at 4， 8， 12 weeks after intervention. At the 12th week， motor nerve conduction velocity （MNCV）， sensory nerve conduction velocity （SNCV）， nerve blood flow velocity， mechanical pain threshold， and thermal pain threshold were detected. The sciatic nerve was taken for hematoxylin-eosin （HE） staining to observe the tissue morphology. The ultrastructure of the sciatic nerve was observed by transmission electron microscope. The expression levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） in sciatic nerve were determined by enzyme-related immunosorbent assay （ELISA）. The mRNA expressions of AMPKα， AMPKβ， PGC-1α， and SIRT3 in sciatic nerve were determined by real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the normal group， fasting blood glucose in the model group was increased at each time point （P<0.01）. The mechanical pain threshold was decreased （P<0.05）， and the incubation time of the hot plate was extended （P<0.01）. MNCV， SNCV， and nerve blood flow velocity decreased （P<0.05）. The expression level of SOD was decreased （P<0.01）. The expression levels of MDA， IL-1β， and TNF-α were increased （P<0.01）. The mRNA expression levels of AMPKα， AMPKβ， PGC-1α， and SIRT3 were decreased （P<0.01）. The structure of sciatic nerve fibers in the model group was loose， and the arrangement was disordered. The demyelination change was obvious. Compared with the model group， the fasting blood glucose of rats in the high dose Tangbikang granule group was decreased after the intervention of eight weeks and 12 weeks （P<0.01）. The mechanical pain threshold increased （P<0.05）. The incubation time of the hot plate was shortened （P<0.01）. MNCV， SNCV， and Flux increased （P<0.05）. The expression level of SOD was increased （P<0.01）. The expression levels of MDA， IL-1β， and TNF-α were decreased （P<0.01）. The mRNA expression levels of AMPKα， AMPKβ， PGC-1α， and SIRT3 were increased （P<0.01）. The sciatic nerve fibers in the high-dose Tangbikang granule group were tighter and more neatly arranged， with only a few demyelinating changes. The high， medium， and low dose Tangbikang granule groups showed a significant dose-effect trend. ConclusionTangbikang granules may improve sciatic nerve function in diabetic rats by regulating AMPK/PGC-1α/SIRT3 signaling pathway partly to inhibit oxidative stress.

Wenyang Jieyu Prescription Regulates Hippocampal Neuron Apoptosis and Improves Synaptic Plasticity in Depressed Mice via BDNF/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Wenyang Jieyu prescription in regulating hippocampal neuron apoptosis and improving synaptic plasticity in the mouse model of depression induced by maternal separation combined with restraint stress. MethodThe mice on postnatal day 0 （PD0） were randomly assigned into a control group （n=10） and a modeling group （n=50）. Maternal separation combined with restraint stress was adopted to establish the mouse model of depression， and the modeled mice were randomized into model， Wenyang prescription， Jieyu prescription， Wenyang Jieyu prescription， and fluoxetine groups （n=10） on the weaning day （PD21）. From PD21 to PD111， the mice were fed with the diets mixed with corresponding medicines. The sucrose preference test， open field test， O-maze test， and novel object recognition test were then conducted to evaluate the depression， memory， and learning abilities of mice. Immunohistochemistry （IHC） was employed to measure the atomic absorbance （AA） of postsynaptic density protein 95 （PSD95） in the hippocampus. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling （TUNEL） was employed to detect the apoptosis of hippocampal neurons. Western blot was employed to determine the protein levels of brain-derived neurotrophic factor （BDNF）， phosphorylated tyrosine kinase receptor B/tyrosine kinase receptor B （p-TrkB/TrkB）， phosphorylated protein kinase B/protein kinase B （p-Akt/Akt）， phosphorylated mammalian target of rapamycin/mammalian target of rapamycin （p-mTOR/mTOR）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X （Bax）， cysteinyl aspartate-specific proteinase-3 （Caspase-3）， synaptophysin （Syn）， and PSD95. ResultCompared with the control group， the modeling decreased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.01）. Furthermore， it decreased the expression of PSD95， increased the neuron apoptosis in the hippocampus （P<0.01）， down-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and up-regulated the protein levels of Bax and Caspase-3 （P<0.05） in the hippocampus. Compared with the model group， Wenyang Jieyu prescription and fluoxetine increased the sucrose preference rate， time spent in central zone within 5 min， total movement distance， time spent in the open arm， and cognition index （P<0.05， P<0.01）. Moreover， the drugs increased the expression of PSD95， reduced the neuron apoptosis （P<0.01）， up-regulated the protein levels of BDNF， p-TrkB/TrkB， p-Akt/Akt， p-mTOR/mTOR， Bcl-2， PSD95， and Syn （P<0.01）， and down-regulated the protein levels of Bax and Caspase-3 （P<0.01）. ConclusionWenyang Jieyu prescription outperformed Wenyang prescription and Jieyu prescription in the treatment of the depressive behavior induced by maternal separation combined with restraint stress in mice. It exerted the therapeutic effect by reducing the hippocampal neuron apoptosis and improving the synaptic plasticity via the BDNF/Akt/mTOR pathway.

Network pharmacology and molecular docking reveal the mechanism of resveratrol in oral squamous cell carcinoma treatment / 口腔疾病防治

Objective@#To explore the molecular mechanism of resveratrol (RES) in the treatment of oral squamous cell carcinoma (OSCC) through the use of biological information methods such as network pharmacology and molecular docking and to provide a theoretical reference for the clinical application of RES in the treatment of OSCC.@*Methods@#The Swiss Target Prediction(http://www.swisstargetprediction.ch), SEA (http://sea.bkslab.org)database, and Pharm mapper database(http://lilab-ecust.cn) were used to retrieve RES-related targets, and the DISGENET (www.disgenet.org), OMIM (https://omim.org) and GeneCards (https://www.genecards.org) databases were used to screen OSCC disease targets. The intersection of drugs and disease targets was determined, and Cytoscape 3.7.2 software was used to construct a "drug-diseasetarget pathway" network. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to construct a target protein interaction network, and the DAVID database was used for enrichment analysis of key proteins. Finally, molecular docking validation of key proteins was performed using AutoDock and PyMOL. The enrichment analysis and molecular docking results were integrated to predict the possible molecular mechanisms of RES treatment in OSCC; western blot was used to determine the effect of resveratrol at different concentrations (50, 100) μmol/L on the expression of Src tyrosine kinase (SRC), epidermal growth factor receptor (EGFR), estrogen receptor gene 1 (ESR1), and phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway proteins in OSCC HSC-3 cells.@*Results@#A total of 243 targets of RES drugs and 6 094 targets of OSCC were identified. A total of 116 potential common targets were obtained by intersecting drugs with disease targets. These potential targets mainly participate in biological processes such as in vivo protein self-phosphorylation, peptide tyrosine phosphorylation, transmembrane receptor protein tyrosine kinase signaling pathway, and positive regulation of RNA polymerase Ⅱ promoter transcription, and they interfere with the PI3K/AKT signaling pathway to exert anti-OSCC effects. The docking results of resveratrol with OSCC molecules indicated that key targets, such as EGFR, ESR1, and SRC, have good binding activity. The results of cell-based experiments showed that resveratrol inhibited the protein expression of SRC, EGFR, ESR1, p-PI3K, and p-AKT in HSC-3 cells in a dose-dependent manner.@*Conclusion@#RES can inhibit the expression of its targets EGFR, ESR1, SRC, p-PI3K, and p-AKT in OSCC cells.

Inhibitory Effect of Sesquiterpenoid M36 from Myrrha on Growth of Human Hepatoma Cells / 中国实验方剂学杂志

ObjectiveThe antitumor activity of sesquiterpenoid M36 isolated from Myrrha against human hepatoma HepG2 cells was investigated in this study. MethodHepG2 cells were treated with M36 at different concentrations （0， 2， 4， 6， 8， 10 μmol·L-1）. Firstly， the effects of M36 on the proliferation of human hepatoma HepG2 cells were detected by methyl thiazolyl tetrazolium （MTT）， colony formation assay， and EdU proliferation assay. Hoechst staining， flow cytometry analysis， and Western blot were used to explore the effect of M36 on the apoptosis of human hepatoma HepG2 cells. Acridine orange staining and western blotting were used to examine the effect of M36 on autophagy in HepG2 cells. Finally， Western blot was used to detect protein expression of cancer-related signaling pathways. ResultCompared with the blank group， M36 treatment significantly inhibited the proliferation of human hepatoma HepG2 cells （P<0.01）， and the half inhibitory concentration （IC50） value of M36 for 48 h was 5.03 μmol·L-1， in a dose- and time-dependent manner. M36 was also able to induce apoptosis and autophagy in human hepatoma HepG2 cells. After treatment with 8 μmol·L-1 M36 for 48 hours， the apoptosis rate of HepG2 cells was （42.03±9.65）% （P<0.01）. Compared with the blank group， HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h had a significant increase in cleaved poly ADP-ribose polymerase （cleaved-PARP） protein levels （P<0.01）. Acridine orange staining showed that autophagy was significantly activated in HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h compared with the blank group （P<0.01）， which was further verified by the up-regulation of microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ）. Western blot results showed that compared with the blank group， the levels of phosphorylated extracellular regulated protein kinase （p-ERK）， phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK）， phosphorylated c-Jun N-terminal kinase （p-JNK）， and its downstream nuclear transcription factors c-Jun and p-c-Jun protein were significantly increased in M36 group （P<0.05， P<0.01）. The mechanism may be related to the up-regulation of MAPK signaling pathway. ConclusionThe sesquiterpenoid M36 isolated from Myrrha inhibits the proliferation of human hepatoma HepG2 cells and promotes apoptosis and autophagy， which may be related to the activation of the MAPK signaling pathway.

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

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

Chaihu Longgu Mulitang Relieves Generalized Anxiety Disorder in Rats via p38 MAPK/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo study whether Chaihu Longgu Mulitang can inhibit hypothalamic inflammation， mitigate anxiety-like behavior， and alleviate anxiety symptoms by regulating the p38 mitogen-activated protein kinase/nuclear factor-κB （p38 MAPK/NF-κB） signaling pathway in the rat model of generalized anxiety disorder （GAD）. MethodTwelve out of 74 Wistar rats were randomly selected as the blank group， and the remaining rats were subjected to chronic restraint stress for the modeling of GAD. The open field test （OFT） and elevated Porteus maze test （PMT） were conducted 14 days after modeling to detect the anxiety-like behaviors. Sixty successfully modeled rats were selected and randomized into model， low-， medium-， and high-dose （6， 12， and 24 g·kg-1， respectively） Chaihu Longgu Mulitang， and diazepam （1 mg·kg-1） groups （n=12） and administrated with corresponding drugs for 14 consecutive days. OFT and PMT were then carried out to examine the anxiety-like behaviors of the rats. The levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and interleukin-1β （IL-1β） in the hypothalamus and serum of rats were determined by the enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）was conducted to determine the mRNA levels of p38 MAPK， NF-κB p65， nuclear factor κB inhibitor α （IκBα）， and ionized calcium binding adaptor molecule 1 （Iba-1）. The protein levels of p38 MAPK， phosphorylated （p）-p38 MAPK， NF-κB p65， p-NF-κB p65， and IκBα in the hypothalamus of rats were determined by Western blot. The expression of Iba-1 in the hypothalamic microglia was detected by immunofluorescence assay. ResultCompared with the blank group， the model group had decreased body weight， scattered dark yellow fur， increased irritability， and preference to hibernation in the corner. In addition， the modeled rats showed increased edge movement distance and time in OFT （P<0.01） and decreased movement distance and time and the number of entries in the open arm in PMT （P<0.01）. The modeling increased the fluorescence intensity of Iba-1 in paraventricular nucleus of hypothalamus （P<0.01）， elevated the levels of IL-1β， IL-6， and TNF-α in the serum and hypothalamus （P<0.01）， up-regulated the protein and mRNA levels of p38 MAPK， NF-κB p65， p-p38 MAPK， p-NF-κB p65， and Iba-1 （P<0.05， P<0.01）， and down-regulated the protein and mRNA levels of IκBα （P<0.01） in the hypothalamus. Compared with the model group， medium- and high-dose Chaihu Longgu Mulitang and diazepam increased the body weight， improved the fur and behaviors， decreased the edge movement distance and time in OFT （P<0.05， P<0.01）， and increased the movement distance and time in the open arm in PMT （P<0.05， P<0.01）. Furthermore， they decreased the fluorescence intensity of Iba-1 in hypothalamic microglia （P<0.05， P<0.01）， lowered the levels of IL-1β， IL-6， and TNF-α in the serum and hypothalamic tissue （P<0.05， P<0.01）， down-regulated the mRNA and protein levels of p38 MAPK， NF-κB p65， p-p38 MAPK， p-NF-κB p65， and Iba-1 （P<0.05， P<0.01）， and up-regulated the mRNA and protein levels of IκBα （P<0.05， P<0.01） in the hypothalamus. ConclusionChaihu Longgu Mulitang can mitigate anxiety-like behaviors and relieve anxiety in GAD rats by inhibiting the p38 MAPK/NF-κB signaling pathway and reducing the activation of microglia and the levels of pro-inflammatory cytokines in the hypothalamus.

Intervention Mechanism of Biejiajian Wan on Primary Liver Cancer by Regulating lncRNA SNHG5/miRNA-26a-5p/GSK-3β Signal Axis / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Biejiajian Wan in the intervention of primary liver cancer based on long non-coding RNA SNHG5 （lncRNA SNHG5）/micro RNA-26a-5p （miRNA-26a-5p）/glycogen synthase kinase-3β （GSK-3β） signal axis. MethodDouble luciferase reporting assay was used to verify the targeted interaction between lncRNA SNHG5 and miRNA-26a-5p， miRNA-26a-5p， and GSK-3β in HepG2 cells. Nude-mouse transplanted tumor model of human HepG2 were established and randomly divided into model group， Biejiajian Wan low-dose group （0.5 g·kg-1）， medium-dose group （1.0 g·kg-1）， and high-dose group （2.0 g·kg-1）， and sorafenib group （100 mg·kg-1）， with 10 mice in each group. The mice were given intragastric administration of normal saline or drug for 28 days， and the tumor volume was measured at different time. Hematoxylin-eosin （HE） staining was used to observe the histological changes of tumors. The nucleic acid levels of lncRNA SNHG5， miRNA-26a-5p， GSK-3β， and β-catenin mPNA in tumor tissue were detected by real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of GSK-3β and β-catenin in tumor tissue were detected by western blot. ResultCompared with the SNHG5-WT （wild type） + miRNA NC （negative control） group， the relative luciferase activities of the SNHG5-WT + miRNA-26a-5p mimic group were decreased （P<0.05）. Compared with the GSK-3β-WT + miRNA NC group， the relative luciferase activity of the GSK-3β-WT + miRNA-26a-5p mimic group was decreased （P<0.05）. Compared with the model group， the tumor volume of Biejiajian Wan low-dose， medium-dose， and high-dose groups was significantly decreased （P<0.05， P<0.01）. Compared with the model group， the cells in the tumor tissue of nude mice in each dose group of Biejiajian Wan were sparsely arranged with necrocytosis， which showed concentration-dependent changes. Compared with the model group， the expression levels of lncRNA SNHG5， GSK-3β， and β-catenin were decreased （P<0.05， P<0.01）， while the expression of miRNA-26a-5p was increased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. Compared with the model group， the protein expression levels of GSK-3β and β-catenin were decreased in each dose group of Biejiajian Wan （P<0.05， P<0.01）. ConclusionBiejiajian Wan may affect the necrosis of liver cancer cells through lncRNA SNHG5/miRNA-26a-5p/GSK-3β signal axis and thus play an anti-tumor role. This research will provide more theoretical basis for the clinical application of Biejiajian Wan.

Mechanism of oxymatrine promoting the apoptosis of osteosarcoma MG63 cell line through mitophagy mediated by COX-2/PINK1/Parkin signaling pathway / 中国药房

OBJECTIVE To study the mechanism of oxymatrine inducing apoptosis of osteosarcoma MG63 cell line based on mitophagy mediated by cyclooxygenase-2 （COX-2）/PTEN-induced putative kinase-1 （PINK1）/Parkinson disease protein-2 （Parkin） signaling pathway. METHODS MG63 cells were treated with 2.0， 4.0， 8.0 mg/mL oxymatrine and 6 μmol/L 5-fluorouracil， then the apoptotic rate， the expression of apoptosis-related proteins [B-cell lymphoma-2 （Bcl-2）， Bcl-2 related X protein （Bax）]， the proportion of decrease in mitochondrial membrane potential， the level of mitophagy as well as the protein expressions of PINK1， Parkin， and microtubule-associated protein 1 light chain-3Ⅱ （LC3-Ⅱ） were detected. PINK1 small interfering RNA （siRNA） was adopted to intervene in the expression of PINK1， the cells were divided into control group， PINK1 siRNA group， oxymatrine group， and PINK1 siRNA+oxymatrine group； the protein expressions of PINK1， Parkin， and LC3-Ⅱ， the proportion of decrease in mitochondrial membrane potential （MMP） as well as apoptotic rate were detected. The lentivirus infection technique was used to overexpress COX-2， the cells were divided into control group， oxymatrine group， COX-2 group， and COX-2+oxymatrine group. The protein expressions of COX-2， PINK1， and Parkin， as well as the proportion of decrease in MMP were detected. RESULTS After being treated with oxymatrine， the apoptotic rate， the protein expressions of Bax， PINK1， Parkin， and LC3-Ⅱ， the level of mitophagy as well as the proportion of decrease in MMP were significantly increased （P＜0.05）， while the protein expression of Bcl-2 was significantly decreased （P＜0.05）. Compared with the oxymatrine group， the protein expressions of PINK1， Parkin， and LC3-Ⅱ， apoptotic rate and the proportion of decrease in MMP were significantly decreased in PINK1 siRNA+oxymatrine group （P＜0.05）. Compared with the oxymatrine group， the protein expression of COX-2 in the COX-2+oxymatrine group was increased significantly （P＜0.05）， while the protein expressions of PINK1 and Parkin as well as the proportion of 526087266@qq.com decrease in MMP were decreased significantly （P＜0.05）. CONCLUSIONS Oxymatrine can mediate the overactivity of mitophagy based on the PINK1/Parkin signaling pathway by inhibiting COX-2 expression， thus promoting the apoptosis of the MG63 osteosarcoma cell line.

Mechanism of catalpol regulating Th17 cell differentiation by interfering PKM2/LDHA expression / 中国药房

OBJECTIVE To investigate the mechanism of catalpol affecting the differentiation of helper T cell 17 （Th17） by interfering the expressions of pyruvate kinase M2 （PKM2） and lactate dehydrogenase A （LDHA）. METHODS The naive CD4+ T cells were selected from the spleen of C57BL/6 mice， and were differentiated into Th17 cells by adding directional differentiation stimulants for 72 hours. At the same time， the cells were treated with 0 （directed control）， 20， 40 and 80 μg/mL catalpol. The flow cytometry was used to detect the proportion of Th17 cell differentiation in cells； the colorimetric method was adopted to detect the levels of pyruvate and lactate in cell culture supernatant； mRNA expressions of retinoid-related orphan nuclear receptor gamma t （RORγt）， PKM2 and LDHA were detected by qRT-PCR method； Western blot was used to detect the expression levels of PKM2， LDHA， signal transducer and activator of transcription 3 （STAT3）， and phosphorylated STAT3 （p-STAT3） proteins in cells. RESULTS Compared with the directed control group， after 72 hours of treatment with 20， 40， 80 μg/mL catalpol， the differentiation ratio of Th17 cells were decreased by 6.74%， 8.41%， 9.24%， and the levels of pyruvate and lactate in the cell culture supernatant， the mRNA expressions of PKM2， LDHA and RORγt as well as the protein expressions of PKM2 and LDHA and the phosphorylation of STAT3 were significantly reduced （P＜0.05）. CONCLUSIONS Catalpol can reduce the glycolysis level by down-regulating the expressions of PKM2 and LDHA， thereby inhibiting the differentiation of Th17 cells.