Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

Effect of Qingre Huayu Decoction on Autophagy and Apoptosis of Neurons in Rats with Ischemic Brain Injury by Regulating Ca^(2+)/CaMKKβ-AMPK-mTOR Pathway

[Objectives]To explore the neuroprotective mechanism of Qingre Huayu Decoction on rats with acute cerebral ischemia injury.[Methods]SD rats were divided into sham operation group,ischemia model group,low,medium and high dose groups of Qingre Huayu De-coction,with 10 rats in each group.Referring to the MCAO operation model,both the sham operation group and the model group were given normal saline by gavage,and the Qingre Huayu Decoction group was given different doses of Qingre Huayu Decoction by gavage.After the op-eration,the rats were scored for neurological deficit,neurons were stained with HE,apoptotic cells were detected with TUNEL,and the levels of autophagy and apoptotic proteins in the Ca^(2+)/CaMKKβ-AMPK-mTORpathway in brain tissue were detected with Western-blot.[Results]Compared with the model group,the neurological function score of Qingre Huayu Decoction Group decreased significantly(P<0.05),the pathological damage of neurons in Qingre Huavu Decoction Group decreased.the proportion of apoptosis-positive cells detected by TUNEL de-creased(P<0.05),and the expression of CaMKKβand AMPK increased,expression of mTOR decreased,expression of Beclin-1 and LC3 increased,and expression of Caspase-3decreased in Qingre Huayu Decoction Group(P<0.05).[Conclusions]Qingre Huayu Decoction may play a neuroprotective role by activating Ca^(2+)/CaMKKβ-AMPK-mTOR pathway and regulating the level of apoptosis and autophagy.

Protective effect of Liangxue Huayu decoction on human retinal pigment epithelial cell(ARPE-19)injury induced by hypoxia through autophagy pathway

Background:Exploring the protective mechanism of the Liangxue Huayu(LXHY)decoction on human retinal pigment epithelial(RPE)cells induced by hypoxia through the autophagy pathway.Methods:The appropriate LXHY decoction concentration was determined by CCK-8.ARPE-19 cells were divided into the normal control group(A group),CoCl_(2)group(B group),3-Methyladenine(3-MA)group(treated with 3-MA(the inhibition of autophagy pathway))(C group),blank serum(BS)group(D group),LXHY drug-contained serum(DCS)group(E group),and Rapamycin(RAP)group[treated with LXHY drug-contained serum combined with rapamycin group(the activation of autophagy pathway)](F group).Counting the number of autophagosomes and autolysosomes in each group of cells under transmission electron microscopy.After infection of cells in each group by mRFP-GFP-LC3 fusion protein adenovirus,the strength of autophagic flux was detected.The mRNA expression levels of LC3 and Beclin-1 were detected by Q-PCR.Results:CCK-8 assay results showed that LXHY DCS could inhibit the cell proliferation of ARPE-19 under hypoxia(all P<0.05).As the transmission electron microscopy assay result showed,compared with the normal control group,the number of autolysosomes was significantly increased in the CoCl_(2)group(P<0.05).Compared with CoCl_(2)group,the number of autolysosomes was significantly reduced the 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.001).As autophagic flux assay result showed,compared with the normal control group,the level of autophagosomes and autolysosomes were significantly risen in CoCl_(2)group(all P<0.001).Compared with the CoCl_(2)group,the level of autophagosomes and autolysosomes were significantly fell down in 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.05).The level of autolysosomes in the LXHY drug-contained serum group was lower than in the blank serum group(P<0.05).Compared with the LXHY drug-contained serum group,the levels of autophagosomes and autolysosomes were significantly risen in the LXHY drug-contained serum combined with the rapamycin group(all P<0.05).As the Q-PCR result showed,compared with the normal control group,the expression of LC3 and Beclin-1 mRNA were significantly reduced in the CoCl_(2)group(all P<0.001).Compared with the CoCl_(2)group,the expression of LC3 mRNA were significantly increased in the 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.001).Beclin-1 mRNA expression was increased significantly(all P<0.001)in the blank serum group and the LXHY drug-contained serum group.And Beclin-1 mRNA expression in the LXHY drug-contained serum group was statistically significant increased than blank serum group(P<0.001).In the LXHY drug-contained serum combined with the rapamycin group,the LC3 and Beclin-1 mRNA expression was reduced significantly compared with the LXHY drug-contained serum group(all P<0.001).Conclusion:The LXHY DCS has the ability to protect the human retinal pigment epithelial cell(ARPE-19)damage under hypoxia through the autophagy pathway.

Mechanism of Liangxue Huayu decoction in treatment of age-related macular degeneration based on network pharmacology and clinical validation

Background:This paper investigates the mechanism of Liangxue Huayu decoction treating age-related macular degeneration through network pharmacology and clinical verification.Methods:The potential targets of Liangxue Huayu decoction were retrieved by the BATMAN-TCM database,and age-related macular degeneration-related disease targets were collected using the OMIM,GeneCards,and CTD databases.The intersection of targets for Liangxue Huayu decoction for age-related macular degeneration was performed using the online tool Venny2.1.0.Protein-protein interaction network was drawn using the String database.And core targets were obtained with the CytoNCA plugin.The gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed using the Metascape database.Finally,targets of Liangxue Huayu decoction for age-related macular degeneration were identified in clinical research.Results:In this study,832 potential action targets and 9,475 age-related macular degeneration-related disease targets were screened,and 575 intersection targets of Liangxue Huayu decoction and age-related macular degeneration were obtained.Protein-protein interaction network analysis showed that 15 core targets include chemokine 8,interleukin 1,histone deacetylase 2,estrogen receptor 2,and glutamate ionotropic receptor NMDA type subunit 1,et al.Gene ontology enrichment analysis included 2,727 biological processes,434 molecular functions,and 206 cellular components.Kyoto Encyclopedia of Genes and Genomes enrichment analysis selected 107 signaling pathways.Clinical validation showed that peripheral blood chemokine 8 and interleukin 1 levels were significantly lower after age-related macular degeneration patients compared with before treatment,and the difference was statistically significant(t chemokine 8=12.684,t interleukin 1=11.405,all P<0.001).Conclusion:By treating age-related macular degeneration with multiple targets and multiple pathways,Liangxue Huayu decoction can down-regulate the expression of inflammatory factors and treat age-related macular degeneration,which provides a basis for further studying the action mechanism of regulating age-related macular degeneration.

Evaluating the effect and mechanism of Yiqi Huayu Jiedu decoction combined with FLOT regimen neoadjuvant chemotherapy for the patients with locally advanced gastric cancer:protocol for a prospective,double-arm,randomized controlled clinical trial

Background:Neoadjuvant chemotherapy plays a vital role in the treatment of advanced gastric cancer(GC),however,optimizing its effectiveness remains an important research focus.Traditional Chinese medicine(TCM),a promising adjunctive therapy,has shown enhanced clinical outcomes when combined with postoperative adjuvant chemotherapy.Therefore,this study is designed to evaluate the clinical efficacy of Yiqi Huayu Jiedu decoction combined with neoadjuvant chemotherapy FLOT in the treatment of advanced GC.Methods:This study is a prospective,double-arm,randomized controlled trial.It involves a total of 260 patients diagnosed with advanced GC,who will be randomly assigned to two groups-a TCM treatment group and a control group,each comprising 130 patients.All patients will receive standard FLOT chemotherapy,and patients in the TCM treatment group will additionally receive TCM treatment with Yiqi Huayu Jiedu decoction.After four cycles of chemotherapy,GC D2 radical surgery will be performed.The primary objective is to evaluate the postoperative pathological response rate of the tumor.The secondary objectives include evaluating the perioperative nutritional status,the efficacy of TCM syndrome,and adverse events associated with both chemotherapy and surgery.Discussion:Currently,no trials have investigated the impact of TCM in combination with neoadjuvant chemotherapy on the preoperative treatment in patients with advanced GC.Accordingly,it is imperative to conduct this prospective study to evaluate the clinical efficacy and safety of this regimen,meanwhile providing high-level clinical evidence for TCM combined with neoadjuvant chemotherapy and introducing an innovative regimen for preoperative comprehensive treatment of GC.

Effects of Zhenggan Huayu decoction(正肝化瘀方)combined with entecavir on gut microbiota in patients with chronic hepatitis B fibrosis

OBJECTIVE:To evaluate the effects of Zhenggan Huayu decoction(正肝化瘀方,ZGHY) combined with entecavir(ETV) on the gut microbiota in patients with chronic hepatitis B(CHB) fibrosis.METHODS:A total of 59 CHB-related fibrosis patients were enrolled and treated with ZGHY combined with ETV(ZGHY + ETV) and ETV alone.Fecal samples were collected from patients at weeks 0,12,and 24 after treatment and gut microbiota were analyzed by 16S r RNA gene sequencing.RESULTS:Compared to the ETV group,microbiota diversity in the ZGHY + ETV group was increased after 24 weeks.Some potentially pathogenic bacteria,including Fusobacteriaceae spp.,Acidaminococcus spp.,and Erysipelotrichaceae spp.were reduced in the ZGHY + ETV group,while Eubacteriaceae spp.,Lactococcus spp.,and several other beneficial bacteria were increased.CONCLUSION:Decreases in pathogenic bacteria and increases in probiotics were not always observed in the Traditional Chinese Medicine(TCM) group(e.g.,Rothia was abundant).As an adjuvant TCM formulation for ETV,ZGHY had a positive role in the treatment of CHB patients.

Qushi Huayu Decoction (祛湿化瘀方) Inhibits Protein and Gene Expression of Cathepsin B in HepG2 Cells Induced by Free Fatty Acids

Objective： To study the experimental efficacy of Qushi Huayu Decoction （祛湿化瘀方,QHD） on protein and gene expression of cathepsin B （ctsb） in HepG2 cells induced by free fatty acids （FFAs）.Methods： The model of HepG2 steatosis and tumor necrosis factor-α （TNF-α） secretion was induced by long-chain FFAs.HepG2 cells were divided into 4 groups： control group （group C）,model group （group M）,low-dose QHD group （group L） and high-dose QHD group （group H ）.Long-chain FFAs were added to groups M,L and H.The 10% blank-control serum was added to group C and M,while 5% and 10% QHD-containing sera were added to group L and H,respectively.The levels of serum TNF-α and cellular triglyceride （TG） were detected.Cellular p-IκB and ctsb expression were detected using Western blot and PCR.The expression and distribution of ctsb were observed by immunofluorescence.Results： After incubating with FFA for 24 h,TG deposition in HepG2,TNF-α content in cell supernatant,the protein expression of cellular ctsb and P-IκB,as well as mRNA expression of ctsb increased markedly in group M compared with group C （P〈0.05,P〈0.01）.Compared with group M,TG deposition,the expression of cellular ctsb,P-IκB and ctsb mRNA in groups L and H,as well as TNF-α content in group H,decreased significantly （P〈0.05）.Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA.In this study,these above-mentioned changes were inhibited markedly in groups L and H.Conclusion： QHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNFα pathway of hepatic lipotoxicity.

Serum Pharmacological Effects of Fuzheng Huayu Decoction(扶正化瘀方) on lto Cell Proliferation and Collagen Synthesis in Rats

Objective: To explore mechanism of the effect of Fuzheng Huayu Decoction on Ito cell proliferation and collagen synthesis. Methods: Biological effects of the drug serum taken from rats fed with Fuzheng Huayu decoction （FZHYD） on cell proliferation and collagen synthesis of Ito cell line were observed. Serum collected from rats at various times （1, 2 or 3 hours） after animals were fed with FZHYD once （It） or twice （2t,2nd time repeated with the same dose）, was incubated with Ito cell. Results: The 1t-2h serum in O.46 g/kg FZHYD group, it seems to prevent cell proliferation, but had no significant difference. While all 2t drug serum could inhibit cell proliferation. The 2t-1h drug serum had no obvious effects on Ito cell morphology, but could improve cell viability, its effect on cell proliferation was dose and serum concentration dependent. Also the 2t-1h drug serum in all doses could inhibit Ito cells both intracellular and extracellular collagen production, the drug serum of 0.46 g/kg group had inhibitory effect in serum concentration dependent manner and similar effect as colchicine. Conclusion: Inhibition of Ito cells proliferation and collagen synthesis is perhaps One of main mechanism of FZHYD in antifibrotic action.

Effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis:preliminary experimental study with rats

The aim of this paper is to study the effects of Fuzheng Huayu Decoction on the plasma proteome in cirrhotic rats.Twenty-six male Sprague-Dawley(SD)rats were randomly divided into three groups:cirrhotic model group(n510),treated with CCl_(4)(CCl_(4)/olive oil:v/v51:1);Fuzheng Huayu Decoction intervention group(n510),treated with CCl_(4)+Fuzheng Huayu Decoction;and normal control group(n56),treated with olive oil only.After 8 weeks,blood samples were collected from the inferior vena cava to undergo bi-dimensional electrophoresis(2DE)and analysis by PDQuest 7.3 software.Differential protein spots were cut,enzyme hydrolysis was conducted,and peptide fragments extracted from the mixture underwent mass spectrometry(MS)with MALDI-TOF-TOF-MS.The liver fibrogenesis was assessed using a digital image analysis instrument of Masson’s trichrome stained sections.The fibrosis area of the Fuzheng Huayu Decoction was(8.9±3.7)%,significantly smaller than that of the cirrhotic model group[(12.4±4.7)%,P<0.05].Ten markedly changed protein spots were identified by MALDI-TOFTOF-MS.Eight of the 10 proteins,including plasma glutathione peroxidase,plasma glutathione peroxidase precursor,prealbumin,haptoglobin,apolipoprotein A-IV precursor,complement C4,inter-alpha-inhibitor H4 heavy chain,and serine/threonine-protein kinase microtubuleaffinity regulating kinase 1(MARK1)were expressed very lowly in the cirrhotic model group while they were expressed highly in the Fuzheng Huayu Decoction group.The expression of liver regeneration-related protein LRRG03 and vimentin increased in the cirrhotic model group,and reduced in the FuzhengHuayu Decoction group.Some proteins related to oxidative stress,cell proliferation and transformation have changed in the plasma of cirrhosis induced by CCl_(4).Fuzheng Huayu Decoction promotes protein synthesis and plays an anti-fibrotic role by antioxidation and accommodation of cell proliferation and transformation.

Effect of Huoxue Huayu Lishui Decoction on P65 and TAK1 of acute soft tissue injury in rats

Objective:To explore the mechanism of the action of Huoxue Huayu Lishui prescription on acute ankle joint injury.Methods:Forty-eight SPF rats were randomly divided into blank group,model group,Shujin Huoluo capsule group,low,medium and high dose groups for promoting blood circulation and removing blood stasis.The blank group was not modeled,and the other groups were modeled.Each group of 8 animals,the Shujin Huoluo capsule group was given 0.2g/ml mixed liquid by gavage,and the low,medium and high blood circulation and blood stasis Lishui prescription group was given 1.03g/ml,2.06g/ml,4.12g/ml decoction For the stomach,the normal group was given 1.5ml/100g,once a day for 1 week.The expression of NF-κB P65 and transforming growth factor kinase 1(TAK1)protein in the soft tissue around the ankle joint of rats was detected by sports protein immunoblotting method,and the mRNA expression of NF-κB P65 and TAK1 was detected by PCR technology.Results:One day after modeling,the expressions of P65 and TAK1 mRNA and protein in the model group,the positive drug group,and low,medium and high doses of Huoxue Huayu Lishui Recipe were significantly different from those of the blank control group(P<0.05).After taking the medicine for 7 days,compared with the model group,the expressions of P65 and TAK1 mRNA and protein in the Shujin Huoluo capsule group and the low,medium and high dose Huoxue Huayu Lishui Recipe group were all decreased(P<0.05).After 7 days of taking the medicine,compared with the Shujin Huoxue Capsule group,there was no difference in P65 protein in the low-dose Huoxue Huayu Lishui Recipe group.The expression of P65 protein in the middle and high groups and the Shujin Huoxue Capsule group were all decreased(P<0.05).Compared with the Shujin Huoxue Capsule group,the expressions of TAK1 protein,P65 and TAK1 mRNA in the low,medium,and high groups and the Shujin Huoxue Capsule group were all decreased(P<0.05).The results suggest that Huoxue Huayu Lishui Recipe can inhibit the expression of NF-κB P65 and TAK1 protein in ankle joint injury.Conclusion:Huoxue Huayu Lishui prescription may regulate the expression of NF-κB P65 and TAK1 in ankle joint injury of NF-κB rats,so as to improve the clinical symptoms of local swelling and pain in the ankle joint.