Objective To investigate the intervention effect and mechanism of sinomenine(SIN)on Parkinson's disease(PD)mice based on GSK3 β/Nrf2/HO-1 and NF-κ B signaling pathways.Methods C57BL/6 mice were randomly divided into 6 groups:normal group,model group,positive drug group(Levodopa,75 mg?kg-1)and SIN low-,medium-and high-dose groups(20,40,80 mg?kg-1),with 8 mice in each group.Mice were intraperitoneally injected with 20 mg?kg-1 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)once a day for 5 days.Intragastric administration was performed 1 hour after injection of MPTP,once a day for 12 days.On the day 11 of administration,the mice were subjected to a pole-climbing experiment,and on the day 12,a rotating rod experiment was performed to test the behavioral changes of the mice.The levels of serum tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and IL-6 were detected by ELISA.The mRNA expression levels of TNF-α,IL-1β and IL-6 in brain tissue were detected by RT-qPCR.The protein expression levels of TH,Nrf2,HO-1,p-GSK3β,GSK3β,p-IκB,IκB,p-NF-κB and NF-κB in brain tissue were detected by Western Blot.Results Compared with the normal group,the automatic turning latency(T-turn)of the model group was significantly prolonged(P＜0.05),and the number of falls was significantly increased(P＜0.001).The expression level of TH protein in brain tissue was significantly decreased(P＜0.01),and the mRNA expression levels of IL-1β,TNF-α and IL-6 were significantly increased(P＜0.001).The serum levels of TNF-α,IL-1β and IL-6 were significantly increased(P＜0.05,P＜0.01).The protein expression levels of p-GSK3β/GSK3β,Nrf2 and HO-1 in brain tissue were significantly decreased(P＜0.05,P＜0.001),and the protein expression ratios of p-IκB/IκB and p-NF-κB/NF-κB were significantly increased(P＜0.001).Compared with the model group,the T-turn in the Sin medium-and high-dose groups was significantly shortened(P＜0.05,P＜0.001),the falling latency was significantly prolonged(P＜0.05,P＜0.01),the times of falls was significantly reduced(P＜0.001),the expression level of TH protein in brain tissue was significantly increased(P＜0.01,P＜0.001),and the level of serum TNF-α was significantly decreased(P＜0.05).The mRNA expression levels of IL-1β,TNF-α and IL-6 in brain tissue of mice in each administration group were significantly decreased(P＜0.001),the serum levels of IL-1β and IL-6 were significantly decreased(P＜0.01,P＜0.001),the protein expression levels of p-GSK3β/GSK3β,Nrf2 and HO-1 in brain tissue were significantly increased(P＜0.05,P＜0.01,P＜0.001),and the protein expression ratios of p-IκB/IκB and p-NF-κB/NF-κB were significantly decreased(P＜0.001).Conclusion SIN can enhance anti-oxidative stress and inhibit neuroinflammation by regulating GSK3β/Nrf2/HO-1 and NF-κB pathways in the brain of Parkinson's disease mice,thereby exerting neuroprotective effects.

Objective To investigate the effect of Gegen Qinlian Decoction on liver energy metabolism and free fatty acid(FFA)in rats with insulin resistance(IR).Methods IR rat model was established by feeding 60%fat high-fat diet for 13 consecutive weeks.SD rats were randomly divided into normal group,model group,Rosiglitazone(5 mg·kg-1)group and Gegen Qinlian Decoction low-,medium-and high-dose groups(1.65,4.96,14.86 g·kg-1),with 6 rats in each group.Intragastric administration was given once a day,continuous administration intervention lasted for 16 weeks.Determination of IR-related indicators:serum fasting insulin(FINS),fasting blood glucose(FPG),calculate the IR index;HPLC method was established for the determination of adenosine triphosphate(ATP),adenosine diphosphate(ADP)and adenosine monophosphate(AMP)in rat liver tissue;ELISA was used to determine the content of FFA in rat liver tissue.The contents of serum total cholesterol(TC)and triglyceride(TG)were detected by automatic biochemical analyzer.The pathological changes of liver tissue were observed by HE staining.Results(1)After the model replication,compared with the normal group,the FINS,FPG levels and IR index of the model group were significantly increased(P＜0.05).(2)Compared with the normal group,the levels of FINS,FPG and IR index in the model group were significantly increased(P＜0.01),the contents of ATP,ADP and AMP in liver tissue were significantly decreased(P＜0.01),the content of FFA was significantly increased(P＜0.01),and the levels of TC and TG in serum were significantly increased(P＜0.05).Liver cells arranged in disorder,fatty degeneration,and there are a large number of lipid droplets.Compared with the model group,the FINS level and IR index of rats in each administration group were significantly decreased(P＜0.05,P＜0.01),and the FPG level of rats in the Rosiglitazone group was significantly decreased(P＜0.05).The contents of ADP and AMP in liver tissue of rats in each administration group were significantly increased(P＜0.05,P＜0.01),and the contents of ATP in liver tissue of rats in low-,medium-and high dose-groups of Gegen Qinlian Decoction were significantly increased(P＜0.05,P＜0.01).The content of FFA in liver tissue of rats in Rosiglitazone group and Gegen Qinlian Decoction low-and high-dose groups was significantly decreased(P＜0.01).The serum TC level of rats in the low-and high-dose groups of Gegen Qinlian Decoction was significantly decreased(P＜0.05,P＜0.01),and the serum TG level of rats in the Rosiglitazone group and the low-dose group of Gegen Qinlian Decoction was significantly decreased(P＜0.05,P＜0.01).The steatosis of hepatocytes in rats of each administration group was alleviated to varying degrees,and the lipid droplets were reduced,and the pathological damage was improved.Conclusion Gegen Qinlian Decoction may improve liver lipid metabolism disorder and restore lipid and energy balance by regulating energy metabolism and reducing FFA level,thus improving IR.

Objective To investigate the effect and mechanism of Bushen Huoxue Prescription(Morindae Officinalis Radix,Cuscutae Semen,Astragali Complanati Semen,Codonopsis Radix,Poria,etc.)on endometrial hyperplasia(EH)rats of kidney deficiency and blood stasis type.Methods A total of 90 SD rats were randomly divided into normal group,model group,Mifepristone group(12 mg·kg-1),and low-,medium-,and high-dose Bushen Huoxue Prescription groups(1.2,2.4,4.8 g·kg-1),with 15 rats in each group.The EH rat model of kidney deficiency and blood stasis type was replicated by intragastric administration of Hydroxyurea and Estradiol Valerate combined with tail vein injection of high molecular dextran.After successful modeling,each group was given intragastric administration according to the set dose,once a day for 21 consecutive days.At the end of the experiment,the levels of serum cyclic adenosine monophosphate(cAMP),cyclic guanosine monophosphate(cGMP),cortisol(CORT),adrenocorticotropic hormone(ACTH),triiodothyronine(T3),thyroxine(T4),thyroid stimulating hormone(TSH),whole blood viscosity,hemorheology and coagulation function were detected by biochemical method.The levels of serum estrogen(E2),follicular estrogen(FSH),luteinizing hormone(LH),nitric oxide(NO)and endothelin-1(ET-1)were detected by ELISA.The pathological changes of endometrial tissue were observed by HE staining,and the endometrial thickness was measured.The protein expression levels of matrix metalloproteinase-9(MMP-9)and tissue inhibitor of metalloproteinase-1(TIMP-1)in endometrial tissues were detected by immunohistochemistry.The protein expression levels of MMP-9,TIMP-1,B-cell lymphoma 2(Bcl-2),Bcl-2 related X protein(Bax)and phosphatase and tensin homologue deleted on chromosome 10(PTEN)in endometrial tissue were detected by Western Blot.Results Compared with the normal group,the levels of serum CORT,ACTH,cAMP,T3,T4,TSH,ET-1 and ET-1/NO ratio in the model group were significantly decreased(P＜0.05,P＜0.01),while the levels of cGMP,E2,FSH,LH and NO were significantly increased(P＜0.05,P＜0.01).The indexes of hemorheology and fibrinogen(FIB)content were significantly increased(P＜0.05),and thrombin time(TT),prothrombin time(PT)and activated partial thromboplastin time(APTT)were significantly prolonged(P＜0.05).The uterine index and endometrial thickness were significantly increased(P＜0.01).The protein expressions of MMP-9 and Bcl-2 in endometrial tissue were significantly up-regulated(P＜0.01),and the protein expressions of TIMP-1,PTEN and Bax were significantly down-regulated(P＜0.01).Compared with the model group,the levels of serum CORT,ACTH,cAMP,T3,T4,TSH,ET-1 and ET-1/NO ratio in the Bushen Huoxue Prescription group were significantly increased(P＜0.05,P＜0.01).The levels of cGMP,E2,FSH,LH and NO were significantly decreased(P＜0.05,P＜0.01).The indexes of hemorheology and FIB content were significantly decreased(P＜0.05,P＜0.01),and TT,PT and APTT were significantly shortened(P＜0.05).The uterine index and endometrial thickness were significantly decreased(P＜0.05,P＜0.01).The protein expressions of MMP-9 and Bcl-2 in endometrial tissue were significantly down-regulated(P＜0.05,P＜0.01),and the protein expressions of TIMP-1,PTEN and Bax were significantly up-regulated(P＜0.05,P＜0.01).Conclusion Bushen Huoxue Prescription can improve the symptoms and signs of EH rats with kidney deficiency and blood stasis,protect endometrial vascular endothelial function,and alleviate endometrial hyperplasia.Its mechanism is related to the restoration of MMP-9/TIMP-1 balance,up-regulation of Bax and PTEN expressions,and inhibition of Bcl-2 expression.

Objective To explore the improving effect and mechanism of saikosaponin A on insomnia rats based on cAMP/PKA/CREB pathway.Methods Seventy-five SD rats were randomly divided into blank group,model group,low-dose saikosaponin A group(0.625 mg·kg-1),high-dose saikosaponin A group(2.500 mg·kg-1)and Estazolam group(0.1 mg·kg-1),with 15 rats in each group.Insomnia rat model was established by intraperitoneal injection of Phenylalanine(PCPA,0.1 mg·kg-1).The general condition and circadian rhythm of rats were observed;the sleep latency and sleep duration of rats were measured by pentobarbital sodium righting experiment.The sleep phase of rats was observed,and the duration of slow wave sleep phase 1(SWS1),slow wave sleep phase 2(SWS2),rapid eye movement sleep phase(REMS)and total sleep time(TST)were recorded.The mRNA expression levels of hypothalamic circadian genes Clock,Bmal1 and clock-controlled genes Rev-erbα and Rorα were determined by qRT-PCR.The expression level of NeuN in hippocampus was determined by immunofluorescence.The level of cAMP in brain tissue was determined by ELISA.The expression levels of Clock,Bmal1,Rev-erbα,Rorα and cAMP/PKA/CREB pathway-related proteins in brain tissue were determined by Western Blot.Results Compared with the blank group,the rats in the model group had disordered circadian rhythms,extreme excitement,irritability,and reduced sleep;the sleep latency was significantly prolonged(P＜0.05),and the sleep duration and SWS1,SWS2,REMS and TST were significantly shortened(P＜0.05).The arrangement of neurons was disordered,and the IOD value of NeuN positive neurons was significantly decreased(P＜0.05).The mRNA and protein expression levels of Clock,Bmal1,Rev-erbα and Rorα in brain tissue were significantly decreased(P＜0.05).The expression levels of cAMP,p-PKA/PKA and p-CREB/CREB in brain tissue were significantly decreased(P＜0.05).Compared with the model group,the aggressiveness of the rats in the administration group was significantly weakened,the circadian rhythm was rhythmic,the activity was reduced,and the sleep was increased.The sleep latency was significantly shortened(P＜0.05),and the sleep duration and SWS1,SWS2,REMS and TST were significantly prolonged(P＜0.05).The disorder of neuronal arrangement was restored,and the IOD value of NeuN positive neurons was significantly increased(P＜0.05).The mRNA and protein expression levels of Clock,Bmal1,Rev-erbα and Rorα in brain tissue were significantly increased(P＜0.05).The protein expression levels of cAMP,p-PKA/PKA and p-CREB/CREB in brain tissue were significantly increased(P＜0.05).Conclusion Saikosaponin A may improve the circadian rhythm of insomnia rats by activating cAMP/PKA/CREB pathway.

Objective To investigate the mechanism of Chaihuang Qingyi Huoxue Granules(Bupleuri Radix,unprocessed Rhei Radix et Rhizoma,Paeoniae Radix Rubra,Paeoniae Radix Alba,Cortex Magnoliae Officinalis,etc.)on rats with severe acute pancreatitis(SAP)based on the renin-Ang-Ⅰ otensin system(RAS).Methods Sixty-four SD rats were randomly divided into four groups:sham operation group,model group,Chaihuang Qingyi Huoxue Granules group(4.42 g·kg-1)and Captopril group(5 mg·kg-1).Each group was further divided into 12-hour and 24-hour subgroups,with 8 rats in each group.SAP rat model was replicated by retrograde injection of 3.5％sodium taurocholate into the biliopancreatic duct.The Captopril group was intraperitoneally injected with Captopril(5 mg·kg-1),and the Chaihuang Qingyi Huoxue Granules group was given intragastric administration,once every 6 hours.The serum amylase(AMY)activity was detected by biochemical method at 12 hours and 24 hours after operation.The pathological changes of pancreatic tissue were observed by HE staining.Serum aldosterone(ALD)content was detected by chemiluminescence.Serum Renin,angiotensin converting enzyme(ACE)and angiotensin Ⅱ(Ang-Ⅱ)were detected by ELISA.The expression of AT1R protein in pancreatic tissue was detected by Western Blot.Results In the same subgroup at 12 and 24 hours,compared with the sham operation group,the serum AMY activity of rats in the model group was significantly increased(P<0.05),the pathological score of pancreatic tissue was significantly increased(P<0.05),the levels of serum ALD,Renin,Ang-Ⅱ and ACE were significantly increased(P<0.05),and the expression of AT1R protein in pancreatic tissue was significantly up-regulated(P<0.05).Compared with the model group,the serum AMY activity of rats in Chaihuang Qingyi Huoxue Granules group and Captopril group was significantly decreased(P<0.05),the pathological score of pancreatic tissue was significantly decreased(P<0.05),the levels of serum ALD,Renin,Ang-Ⅱ and ACE were significantly decreased(P<0.05),and the expression of AT1R protein in pancreatic tissue was significantly down-regulated(P<0.05).Compared with the Captopril group,the serum AMY of the rats in the Chaihuang Qingyi Huoxue Granules group was significantly decreased(P<0.05),the pathological score of pancreatic tissue was significantly decreased(P<0.05),and the serum ALD,Renin,Ang-Ⅱ and ACE levels were significantly decreased(P<0.05).Conclusion Chaihuang Qingyi Huoxue Granules may inhibit the production of Renin and ALD by down-regulating the expression of ACE-Ang-Ⅱ-AT1R classical axis,thus exerting a protective effect on SAP rats.

Objective To study the effects of Gouteng Jiangya Jieyu Prescription(Uncariae Ramulus cum Uncis,Gastrodiae Rhizoma,Pheretima,Puerariae Lobatae Radix,Salviae Miltiorrhizae Radix et Rhizoma,etc.)on learning and memory ability,hippocampal inflammatory response and autophagy-related protein expression in rats with hypertension complicated with depression(HD).Methods Thirty spontaneously hypertensive rats(SHR)were randomly divided into model group,positive control group(Levamlodipine Besylate 0.45 mg·kg-1+Fluoxetine Hydrochloride 1.80 mg·kg-1)and Gouteng Jiangya Jieyu Prescription high-,medium-and low-dose groups(25.38,12.69,6.34 g·kg-1).Another 6 SD rats were used as blank control group.The SHR rats were intervened by chronic mild unpredictable stress combined with solitary rearing to replicate the HD rat model.At the same time,intragastric administration was given once a day for 6 weeks.The systolic blood pressure and diastolic blood pressure of rat tail artery were measured by non-invasive sphygmomanometer.The learning and memory ability of rats was detected by Morris water maze test.The ultrastructure of hippocampal neurons was observed by transmission electron microscope.The contents of interleukin-1β(IL-1β),IL-18 and IL-10 in hippocampus were detected by ELISA.The expression of autophagy-related proteins Beclin1 and Bcl-2 in hippocampus was detected by immunohistochemistry.The expression of autophagy-related proteins LC3Ⅰ and LC3Ⅱ in hippocampus was detected by Western Blot.Results Compared with the blank control group,the SBP and DBP of the rats in the model group were significantly increased from week 1-6(P＜0.01).The escape latency was significantly prolonged on the third and fourth day(P＜0.01).The first time of crossing the platform was significantly prolonged(P＜0.01),the times of crossing the platform area was significantly reduced(P＜0.05),and the retention time of the platform area was significantly shortened(P＜0.01).The neuronal cell body was obviously swollen,the ridge was destroyed,the nucleus was shrunk,and a large number of autophagosomes appeared;the contents of IL-1β and IL-18 in hippocampus were significantly increased(P＜0.01).The ratio of LC3Ⅱ/LC3Ⅰ protein expression and the expression of Beclin1 protein in hippocampus were significantly up-regulated(P＜0.05,P＜0.01),and the expression of Bcl-2 protein was significantly down-regulated(P＜0.01).Compared with the model group,the SBP of rats in the low-dose group of Gouteng Jiangya Jieyu Prescription was significantly decreased at the weeks 1,3,4,5,6(P＜0.01),and the DBP was significantly decreased at weeks 1,3,4,5(P＜0.05,P＜0.01).The SBP of the rats in the medium-dose group of Gouteng Jiangya Jieyu Prescription was significantly decreased at weeks 1,5,6(P＜0.01),and the DBP was significantly decreased at week 4(P＜0.05).The SBP of rats in the high-dose group of Gouteng Jiangya Jieyu Prescription was significantly decreased in the first week(P＜0.01).The escape latency of rats in the high-and medium-dose groups of Gouteng Jiangya Jieyu Prescription was significantly shortened on the third day(P＜0.05),and the escape latency of rats in the high-and low-dose groups of Gouteng Jiangya Jieyu Prescription was significantly shortened on the fourth day(P＜0.05).The first crossing platform time of rats in the high-,medium-and low-dose groups of Gouteng Jiangya Jieyu Prescription was significantly shortened(P＜0.01).The times of rats crossing the platform area in the medium-and low-dose groups of Gouteng Jiangya Jieyu Prescription were significantly increased(P＜0.05),and the retention time in the platform area was significantly prolonged(P＜0.05).In the administration group,the degree of hippocampal neuron damage was reduced,the nuclear shrinkage was significantly improved,and the autophagosomes were reduced.The contents of pro-inflammatory factors IL-1β and IL-18 in the hippocampus of rats in the high-and medium-dose groups of Gouteng Jiangya Jieyu Prescription were significantly decreased(P＜0.05,P＜0.01).The content of anti-inflammatory factor IL-10 in the hippocampus of rats in the high-dose group of Gouteng Jiangya Jieyu Prescription was significantly increased(P＜0.01).The protein expression ratio of LC3Ⅱ/LC3Ⅰ in hippocampus of high-,medium-and low-dose groups of Gouteng Jiangya Jieyu Prescription was significantly down-regulated(P＜0.01),and the expression of Bcl-2 protein was significantly up-regulated(P＜0.01).The expression of Beclin1 protein in the hippocampus of the high-and medium-dose groups of Gouteng Jiangya Jieyu Prescription was significantly down-regulated(P＜0.05,P＜0.01).Conclusion Gouteng Jiangya Jieyu Prescription can reduce the tail arterial pressure of HD rats,improve their learning and memory ability,and alleviate hippocampal neuronal damage.The mechanism may be related to reducing the release of inflammatory factors,increasing the level of anti-inflammatory factors,and regulating the expression of hippocampal autophagy-related proteins LC3Ⅱ/LC3Ⅰ,Beclin1 and Bcl-2.

Objective To optimize the extraction process of flavonoids from Plantago asiatica L.rich in plantagoside by response surface methodology,and to explore the protective effect and mechanism of plantain flavonoids on H2O2-damaged L02 cells.Methods The effects of various parameters,including ethanol concentration,extraction temperature and the liquid-solid ratio on the yield of flavonoids from P.asiatica and the transfer rate of plantagoside were studied by single factor experiment.The central composite design(CCD)and response surface methodology(RSM)were employed on this basic to screen the optimized extraction conditions,which yielded the highest extraction rate of flavonoids from P.asiatica and the maximum transfer rate of plantagoside.The protective effect of flavonoids from P.asiatica on H2O2-damaged L02 cells was assessed through the analysis of cell survival rate,apoptosis rate,mitochondrial membrane potential,malondialdehyde(MDA)and glutathione(GSH)levels,oxide dismutase(SOD)concentration,and the expression levels of HO-1,Nrf2,and SOD proteins.Results The results showed that the highest yield of extraction rate of flavonoids from P.asiatica(1.82％)and the maximum transfer rate of plantagoside(35.01％)were achieved under the optimized extraction condition:extraction temperature of 80 ℃,ethanol concentration of 65％and liquid-solid ratio of 15:1.Flavonoids from P.asiatica extracted under these parameters significantly reduced the apoptosis rate(P<0.001)and the degree of mitochondrial membrane potential damage(P<0.001),increased the expressions of SOD and GSH(P<0.001),and reduced the MDA level(P<0.01,P<0.001)in H2O2-damaged L02 cells at concentrations ranging from 25 to 100 μg·mL-1.In addition,100 μg·mL-1 flavonoids from P.asiatica were found to regulate the expressions of HO-1,Nrf2 and SOD in H2O2-damaged L02 cells.These flavonoids decrease the expressions of HO-1 and Nrf2 proteins,while enhance the expression of SOD protein in H2O2-damaged L02 cells(P<0.05,P<0.01).Conclusion The extraction process for flavonoids from P.asiatica,which was optimized using the response surface method,is operationally simple and yields a high transfer rate of the key active component,plantagoside.This approach offers valuable insights for the actual production of flavonoids from P.asiatica.Besides,flavonoids from P.asiatica could reduce oxidative stress level in H2O2-damaged liver cells,which exhibited a certain protective effect on L02 cells against oxidative damage.This study provides a foundational basis for the development and application of flavonoids from P.asiatica as liver-protective health supplements.

Objective To explore the mechanism of Buyang Huanwu Decoction against cerebral ischemia-reperfusion injury in rats by regulating lipid metabolism through the cAMP/PKA/PPARγ pathway.Methods 60 rats were randomly divided into sham operation group(Sham),model group(Model),Buyang Huanwu Decoction low-dose group(BHD-L),Buyang Huanwu Decoction medium-dose group(BHD-M),Buyang Huanwu Decoction high-dose group(BHD-H)and Butylphthalide group(NBP).The cerebral ischemia-reperfusion model was prepared by transient middle cerebral artery embolization.The BHD low-,medium-and high-groups were given different doses of Buyang Huanwu Decoction(6.413,12.825,25.65 g·kg-1)by intragastric administration.The NBP group was administered with Butylphthalide(54 mg·kg-1).The sham operation group and the model group were administered with an equal volume of distilled water,all given for 14 days.The rats were subjected to neurobehavioral scoring.HE staining was used to observe brain pathological changes,and the kit was used to detect the levels of phosphocholine(PC),phosphatidylethanolamine(PE),diacylglycerol(DAG),and free fatty acid(FFA)on the ischemic side.RT-qPCR and Western Blot were applied to detect the mRNA and protein expressions of cyclic adenosine monophosphate(cAMP),protein kinase A(PKA),and peroxisome proliferator-activated receptor γ(PPARγ).Results Compared with the sham group,the neurological deficit score was significantly increased(P＜0.01),pathomorphological damage in ischemic cortex was found,the contents of PC and PE were reduced,the contents of DAG and FFA were increased(P＜0.01),and cAMP mRNA expression increased(P＜0.05)in the model group.Compared with the model group,the neurological deficit score of the BHD-L group was decreased(P＜0.05),and the neurological deficit score of the BHD-M,BHD-H and NBP groups was significantly decreased(P＜0.01),the cells in each treatment group were regularly arranged,the intercellular spaces were reduced,and the normal cells were increased.PC and PE were significantly increased,DAG and FFA were significantly decreased(P＜0.01)in the BHD-M,BHD-H and NBP groups.PC was increased,FFA and DAG were decreased in the BHD-L group(P＜0.05,P＜0.01).The mRNA level of PPARγ was increased in the BHD-L group(P＜0.05),and the mRNA and protein levels of cAMP,PKA,and PPARγ were increased in the other treatment groups(P＜0.05,P＜0.01).Conclusion Buyang Huanwu Decoction has a neuroprotective effect on cerebral ischemia-reperfusion injury rats,and its mechanism may be related to regulating the expression of key factors in the cAMP/PKA/PPARγ signaling pathway and lipid metabolism.

Objective To explore the intervention effect and molecular mechanism of Xinkang Granules on inflammatory factors in rats with chronic heart failure based on cGAS/STING signaling pathway.Methods SD rats were randomly divided into normal group and modeling group.The chronic heart failure model was established by intraperitoneal injection of Doxorubicin Hydrochloride.After successfully modeling,the rats were further divided into model group,Valsartan group and Xinkang Granules group.The model group was treated with distilled water every day,the Valsartan group was treated with Valsartan solution every day,and the Xinkang Granules group was treated with Xinkang Granules every day,all given for 4 consecutive weeks.Echocardiography was used to detect cardiac function,the pathological changes of myocardium were detected by hematoxylin-eosin staining(HE),the ultrastructural changes of myocardium in each group were observed by transmission electron microscope,and the contents of interleukin-1β(IL-1β)and interleukin-6(IL-6)in serum were detected by enzyme-linked immunosorbent assay(ELISA).The mRNA expression levels of mitochondrial transcription factor A(TFAM),cyclic guanosine monophosphate-adenylate synthase(cGAS),interferon-stimulated gene(STING)and IL-6 in myocardial tissue of rats in each group were detected by real-time fluorescence quantitative method(qPCR).The protein expressions of cGAS and STING in rat myocardial tissue were detected by immunohistochemical method.Results Compared with the blank group,the rats in the model group had significant inflammatory cell infiltration and inflammatory edema in myocardial tissue,their cardiac function was significantly reduced(P＜0.05,P＜0.01),and serum inflammatory factors were significantly increased(P＜0.01).The mRNA expression of TFAM in myocardial tissue was significantly reduced(P＜0.01),the mRNA expressions of IL-6,cGAS,and STING were significantly increased(P＜0.01),and the protein expressions of cGAS and STING in the myocardial tissue were significantly increased(P＜0.01).Compared with the model group,the cardiac function of the rats in the Xinkang Granules group was significantly improved(P＜0.05,P＜0.01),the inflammatory infiltration of myocardial cells was reduced,the expression of serum inflammatory factors was significantly reduced(P＜0.01),the mRNA expression of TFAM in myocardial tissue was significantly increased(P＜0.05),and the mRNA expressions of IL-6,cGAS,and STING were significantly decreased(P＜0.01),the protein expressions of cGAS and STING in myocardial tissue were significantly decreased(P＜0.01).Conclusion Xinkang Granules can reduce the expression of inflammatory factors and improve cardiac function in rats with chronic heart failure.Its mechanism may be related to inhibiting the cGAS/STING signaling pathway.

Objective Based on TLR4/NF-κB/MLCK pathway,the therapeutic effect and mechanism of Baihe Dihuang Decoction on insomnia with intestinal flora disturbance in mice induced by p-chlorophenlalanine(PCPA)and multi-factor stimulation were studied.The aim is to provide theoretical basis for clinical use.Methods Eighty-four KM mice were randomly divided into normal group,model group,positive group(Diazepam,1.38 mg·kg-1),Baihe group(2.25 g·kg-1),Dihuang group(2.25 g·kg-1)and Baihe Dihuang Decoction group(4.5 g·kg-1).Insomnia mouse model was established by intraperitoneal injection of PCPA for 2 days combined with 4 weeks of multi-factor stimulation,including stimulating the tail with forceps clip for 2 minutes,reversing day and night for 24 hours,wetting the padding for 24 hours,tilting the cage at 45° for 24 hours,alternating the cages for 24 hours,fasting food for 24 hours,and getting cold bath for 3 minutes,etc.After successfully modeling,corresponding drug treatment was given.The anxiety-like behavior of mice was observed by elevated cross maze system.The latency and duration of sleep were observed by righting reflex experiment.16sRNA sequencing was used to analyze the composition and structure of intestinal flora in mice.The concentration changes of γ-aminobutyric acid(GABA),glutamic acid(Glu),tryptophan(Trp),5-hydroxytryptophan(5-HTP)and 5-hydroxytryptamine(5-HT)in brain and colon were detected by liquid chromatography-mass spectrometry(LC-MS).Immunohistochemical method was used to detect the expressions of zona atresia protein 1(ZO-1)and Occludin in colon.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the genes including interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),ZO-1,Occludin,Toll-like receptor 4(TLR4),nuclear factor κB(NF-κB)and myosin light chain kinase(MLCK)in colonic tissue.Western Blot was used to detect the expressions of TLR4,NF-κB,phosphorylated nuclear factor κB(p-NF-κB),MLCK,myosin light chain(MLC)and phosphorylated myosin light chain(p-MLC)in colonic epithelial tissue.Results Compared with the normal group,the distance of entering the open arm and the duration of stay in the open arm of model group in the elevated cross maze were significantly shortened(P＜0.05).The sleep latency was significantly prolonged,and the sleep duration was significantly shortened(P＜0.05).The richness and uniformity of intestinal flora were decreased(P＜0.05,P＜0.01).The concentration of neurotransmitters GABA,Trp,5-HTP and 5-HT in the brain decreased significantly(P＜0.01),while the concentration of Glu increased significantly(P＜0.01).The concentration of GABA and Glu in colon decreased significantly(P＜0.01),while the concentration of Trp,5-HTP and 5-HT increased significantly(P＜0.01).The expression levels of inflammatory factors IL-1β,IL-6 and TNF-α in colonic tissue were significantly increased(P＜0.01),and the expression levels of ZO-1 and Occludin genes and proteins were significantly decreased(P＜0.01).The gene expression levels of TLR4,NF-κB and MLCK were significantly increased(P＜0.01),and the protein expression levels of TLR4,p-NF-κB,MLCK and p-MLC were significantly increased(P＜0.01).Compared with the model group,Baihe Dihuang Decoction could significantly prolong the distance of entering the open arm and the duration of stay in the open arm of insomnia mice in the elevated cross maze(P＜0.05).The sleep latency was significantly shortened,and the sleep duration was significantly increased(P＜0.05).The richness and uniformity of intestinal flora were increased(P＜0.05,P＜0.01).The concentration of neurotransmitters GABA,Trp,5-HTP and 5-HT in brain was increased(P＜0.05,P＜0.01),and the concentration of Glu was decreased(P＜0.01).The concentration of GABA and Glu in colon was increased(P＜0.01),while the concentration of Trp,5-HTP and 5-HT was decreased(P＜0.05,P＜0.01).The expression levels of IL-1β,IL-6 and TNF-α genes were down-regulated(P＜0.01),the expression levels of ZO-1 and Occludin genes and proteins were up-regulated(P＜0.01),TLR4,NF-κB,MLCK gene expression levels and TLR4,p-NF-κB,MLCK,p-MLC protein expression levels were down-regulated in the pathway(P＜0.01).Conclusion Baihe Dihuang Decoction can effectively treat insomnia with intestinal flora disorders.Its mechanism of action may be related to the regulation of brain and intestinal neurotransmitter disorders,down-regulating TLR4/NF-κB/MLCK signaling pathway,and up-regulating tight junction proteins expression,reducing inflammatory responses,and then repairing the mechanical barrier of intestinal mucosa.