Puerariae lobatae Radix ameliorates chronic kidney disease by reshaping gut microbiota and downregulating Wnt/ß­catenin signaling.

Gut microbiota dysfunction is a key factor affecting chronic kidney disease (CKD) susceptibility. Puerariae lobatae Radix (PLR), a traditional Chinese medicine and food homologous herb, is known to promote the gut microbiota homeostasis; however, its role in renoprotection remains unknown. The present study aimed to investigate the efficacy and potential mechanism of PLR to alleviate CKD. An 8­week 2% NaCl­feeding murine model was applied to induce CKD and evaluate the therapeutic effect of PLR supplementary. After gavage for 8 weeks, The medium and high doses of PLR significantly alleviated CKD­associated creatinine, urine protein increasement and nephritic histopathological injury. Moreover, PLR protected kidney from fibrosis by reducing inflammatory response and downregulating the canonical Wnt/ß­catenin pathway. Furthermore, PLR rescued the gut microbiota dysbiosis and protected against high salt­induced gut barrier dysfunction. Enrichment of Akkermansia and Bifidobacterium was found after PLR intervention, the relative abundances of which were in positive correlation with normal maintenance of renal histology and function. Next, fecal microbiota transplantation experiment verified that the positive effect of PLR on CKD was, at least partially, exerted through gut microbiota reestablishment and downregulation of the Wnt/ß­catenin pathway. The present study provided evidence for a new function of PLR on kidney protection and put forward a potential therapeutic strategy target for CKD.

[Effect and mechanism of Puerariae Lobatae Radix in alleviating insulin resistance in T2DM db/db mice based on intestinal flora].

This study aimed to examine the effect and underlying mechanism of Puerariae Lobatae Radix on insulin resistance in db/db mice with type 2 diabetes mellitus(T2DM) based on the analysis of intestinal flora. Fifty db/db mice were randomly divided into a model group(M group), a metformin group(YX group), a high-dose Puerariae Lobatae Radix group(YGG group), a medium-dose Puerariae Lobatae Radix group(YGZ group), and a low-dose Puerariae Lobatae Radix group(YGD group). Another 10 db/m mice were assigned to the normal group(K group). After continuous administration for eight weeks, body weight and blood sugar of mice were measured. Enzyme linked immunosorbent assay(ELISA) was used to detect glycosylated serum protein(GSP) and fasting serum insulin(FINS), and insulin resistance index(HOMA-IR) was calculated. The histopathological changes in the pancreas were observed by HE staining. Tumor necrosis factor(TNF)-α expression in the pancreas was detected using immunohistochemistry. The structural changes in fecal intestinal flora in the K, M, and YGZ groups were detected by 16S rRNA. Western blot was used to detect the expression of farnesoid X receptor(FXR) and takeda G protein-coupled receptor 5(TGR5) in the ileum, cholesterol 7α-hydroxylase(CYP7A1) and sterol 27α-hydroxylase(CYP27A1) in the liver, and G protein-coupled receptors 41(GPR41) and 43(GPR43) in the colon. Compared with the K group, the M group showed increased body weight, blood sugar, serum GSP, fasting blood glucose(FBG), and FINS, increased HOMA-IR, inflammatory infiltration of islet cells, necrosis and degeneration of massive acinar cells, unclear boundary between islet cells and acinar cells, disturbed intestinal flora, and down-regulated FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43. Compared with the M group, the YX, YGG, YGZ, and YGD groups showed decreased body weight, blood sugar, serum GSP, FBG, and FINS, islet cells with intact and clumpy morphology and clear boundary, necrosis of a few acinar cells, and more visible islet cells. The intestinal flora in the YGZ group changed from phylum to genus levels, and the relative abundance of intestinal flora affecting the metabolites of intestinal flora increased. The protein expression of FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43 increased. The results show that Puerariae Lobatae Radix can improve the inflammatory damage of pancreatic islet cells and reduce insulin resistance in db/db mice with T2DM. The mechanism of action may be related to the increase in the abundance of Actinobacteria, Bifidobacterium, and Bacteroides in the intestinal tract and the protein expression related to metabolites of intestinal flora.

[Hyperspectral imaging technology distinguishes between Puerariae Lobatae Radix and Puerariae Lobatae Caulis].

Puerariae Lobatae Radix, the dried root of Pueraria lobata, is a traditional Chinese medicine with a long history. Puerariae Lobatae Caulis as an adulterant is always mixed into Puerariae Lobatae Radix for sales in the market. This study employed hyperspectral imaging(HSI) to distinguish between the two products. VNIR lens(spectral scope of 410-990 nm) and SWIR lens(spectral scope of 950-2 500 nm) were used for image acquiring. Multi-layer perceptron(MLP), partial least squares discriminant analysis(PLS-DA), and support vector machine(SVM) were employed to establish the full-waveband models and select the effective wavelengths for the distinguishing between Puerariae Lobatae Caulis and Puerariae Lobatae Radix, which provided technical and data support for the development of quick inspection equipment based on HSI. The results showed that MLP model outperformed PLS-DA and SVM models in the accuracy of discrimination with full wavebands in VNIR, SWIR, and VNIR+SWIR lens, which were 95.26%, 99.11%, and 99.05%, respectively. The discriminative band selection(DBS) algorithm was employed to select the effective wavelengths, and the discrimination accuracy was 93.05%, 98.05%, and 98.74% in the three different spectral scopes, respectively. On this basis, the MLP model combined with the effective wavelengths within the range of 2 100-2 400 nm can achieve the accuracy of 97.74%, which was close to that obtained with the full waveband. This waveband can be used to develop quick inspection devices based on HSI for the rapid and non-destructive distinguishing between Puerariae Lobatae Radix and Puerariae Lobatae Caulis.

Gut microbiota-derived melatonin from Puerariae Lobatae Radix-resistant starch supplementation attenuates ischemic stroke injury via a positive microbial co-occurrence pattern.

Ischemic stroke is closely associated with gut microbiota dysbiosis and intestinal barrier dysfunction. Prebiotic intervention could modulate the intestinal microbiota, thus considered a practical strategy for neurological disorders. Puerariae Lobatae Radix-resistant starch (PLR-RS) is a potential novel prebiotic; however, its role in ischemic stroke remains unknown. This study aimed to clarify the effects and underlying mechanisms of PLR-RS in ischemic stroke. Middle cerebral artery occlusion surgery was performed to establish a model of ischemic stroke in rats. After gavage for 14 days, PLR-RS attenuated ischemic stroke-induced brain impairment and gut barrier dysfunction. Moreover, PLR-RS rescued gut microbiota dysbiosis and enriched Akkermansia and Bifidobacterium. We transplanted the fecal microbiota from PLR-RS-treated rats into rats with ischemic stroke and found that the brain and colon damage were also ameliorated. Notably, we found that PLR-RS promoted the gut microbiota to produce a higher level of melatonin. Intriguingly, exogenous gavage of melatonin attenuated ischemic stroke injury. In particular, melatonin attenuated brain impairment via a positive co-occurrence pattern in the intestinal microecology. Specific beneficial bacteria served as leaders or keystone species to promoted gut homeostasis, such as Enterobacter, Bacteroidales_S24-7_group, Prevotella_9, Ruminococcaceae and Lachnospiraceae. Thus, this new underlying mechanism could explain that the therapeutic efficacy of PLR-RS on ischemic stroke at least partly attributed to gut microbiota-derived melatonin. In summary, improving intestinal microecology by prebiotic intervention and melatonin supplementation in the gut were found to be effective therapies for ischemic stroke.

Analysis of Chemical Constituents in Puerariae Lobatae Radix Dispensing Granules by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

ObjectiveA rapid method for identification of chemical constituents in Puerariae Lobatae Radix dispensing granules was established in order to clarify the material basis. MethodThe chemical constituents of Puerariae Lobatae Radix dispensing granules was qualitatively analyzed by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） under positive and negative ion modes， and the chromatographic conditions were on an ACQUITY UPLC HSS T3 column（2.1 mm×100 mm， 1.8 μm） with 0.1% formic acid aqueous solution（A）-0.1% formic acid acetonitrile solution（B） as mobile phase for gradient elution（0-4 min， 5%-10%B； 4-10 min， 10%-15%B； 10-20 min， 15%-16%B； 20-27 min， 16%-31%B； 27-33 min， 31%-59%B； 33-42 min， 59%-95%B； 42-42.1 min， 95%-5%B； 42.1-45 min， 5%B）， the flow rate was 0.35 mL·min-1， the column temperature was 40 ℃， the injection volume was 5 μL， and electrospray ionization（ESI） was selected. Then these chemical constituents were comprehensively identified based on PeakView 1.2， PubChem， ChemicalBook， ChemSpider， comparative control profiles and literature information. ResultA total of 128 chemical constituents were identified from the dispensing granules， including 60 flavonoids， 26 organic acids， 7 glycosides， 6 coumarins， 3 nucleosides and 26 other compounds. By focusing on the cleavage patterns of flavonoids， organic acids， glycosides， coumarins， nucleosides and other compounds， 12 compounds that have not been reported in Puerariae Lobatae Radix species were identified from the dispensing granules. ConclusionThe established method can systematically and rapidly identify the chemical constituents in Puerariae Lobatae Radix dispensing granules， and cleared it composition is mainly flavonoids and organic acids. Laying a foundation for the study of the material basis， mechanism of action and clinical application of the dispensing granules.

Hyperspectral imaging technology distinguishes between Puerariae Lobatae Radix and Puerariae Lobatae Caulis / 中国中药杂志

Puerariae Lobatae Radix, the dried root of Pueraria lobata, is a traditional Chinese medicine with a long history. Puerariae Lobatae Caulis as an adulterant is always mixed into Puerariae Lobatae Radix for sales in the market. This study employed hyperspectral imaging(HSI) to distinguish between the two products. VNIR lens(spectral scope of 410-990 nm) and SWIR lens(spectral scope of 950-2 500 nm) were used for image acquiring. Multi-layer perceptron(MLP), partial least squares discriminant analysis(PLS-DA), and support vector machine(SVM) were employed to establish the full-waveband models and select the effective wavelengths for the distinguishing between Puerariae Lobatae Caulis and Puerariae Lobatae Radix, which provided technical and data support for the development of quick inspection equipment based on HSI. The results showed that MLP model outperformed PLS-DA and SVM models in the accuracy of discrimination with full wavebands in VNIR, SWIR, and VNIR+SWIR lens, which were 95.26%, 99.11%, and 99.05%, respectively. The discriminative band selection(DBS) algorithm was employed to select the effective wavelengths, and the discrimination accuracy was 93.05%, 98.05%, and 98.74% in the three different spectral scopes, respectively. On this basis, the MLP model combined with the effective wavelengths within the range of 2 100-2 400 nm can achieve the accuracy of 97.74%, which was close to that obtained with the full waveband. This waveband can be used to develop quick inspection devices based on HSI for the rapid and non-destructive distinguishing between Puerariae Lobatae Radix and Puerariae Lobatae Caulis.

Effect and mechanism of Puerariae Lobatae Radix in alleviating insulin resistance in T2DM db/db mice based on intestinal flora / 中国中药杂志

This study aimed to examine the effect and underlying mechanism of Puerariae Lobatae Radix on insulin resistance in db/db mice with type 2 diabetes mellitus(T2DM) based on the analysis of intestinal flora. Fifty db/db mice were randomly divided into a model group(M group), a metformin group(YX group), a high-dose Puerariae Lobatae Radix group(YGG group), a medium-dose Puerariae Lobatae Radix group(YGZ group), and a low-dose Puerariae Lobatae Radix group(YGD group). Another 10 db/m mice were assigned to the normal group(K group). After continuous administration for eight weeks, body weight and blood sugar of mice were measured. Enzyme linked immunosorbent assay(ELISA) was used to detect glycosylated serum protein(GSP) and fasting serum insulin(FINS), and insulin resistance index(HOMA-IR) was calculated. The histopathological changes in the pancreas were observed by HE staining. Tumor necrosis factor(TNF)-α expression in the pancreas was detected using immunohistochemistry. The structural changes in fecal intestinal flora in the K, M, and YGZ groups were detected by 16S rRNA. Western blot was used to detect the expression of farnesoid X receptor(FXR) and takeda G protein-coupled receptor 5(TGR5) in the ileum, cholesterol 7α-hydroxylase(CYP7A1) and sterol 27α-hydroxylase(CYP27A1) in the liver, and G protein-coupled receptors 41(GPR41) and 43(GPR43) in the colon. Compared with the K group, the M group showed increased body weight, blood sugar, serum GSP, fasting blood glucose(FBG), and FINS, increased HOMA-IR, inflammatory infiltration of islet cells, necrosis and degeneration of massive acinar cells, unclear boundary between islet cells and acinar cells, disturbed intestinal flora, and down-regulated FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43. Compared with the M group, the YX, YGG, YGZ, and YGD groups showed decreased body weight, blood sugar, serum GSP, FBG, and FINS, islet cells with intact and clumpy morphology and clear boundary, necrosis of a few acinar cells, and more visible islet cells. The intestinal flora in the YGZ group changed from phylum to genus levels, and the relative abundance of intestinal flora affecting the metabolites of intestinal flora increased. The protein expression of FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43 increased. The results show that Puerariae Lobatae Radix can improve the inflammatory damage of pancreatic islet cells and reduce insulin resistance in db/db mice with T2DM. The mechanism of action may be related to the increase in the abundance of Actinobacteria, Bifidobacterium, and Bacteroides in the intestinal tract and the protein expression related to metabolites of intestinal flora.

Geju Hugan Tablets Ameliorate Alcohol-induced Liver Injury in Mice by Regulating NF-κB and Bcl-2/Bax Signaling Pathways / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of Geju Hugan tablets on the liver of mice with alcohol-induced liver injury， and explore the underlying mechanism based on nuclear factor-κB p65 （NF-κB p65） and B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax） signaling pathways. MethodAccording to the body weight， 60 SPF-grade male ICR mice were randomized into normal， model， Compound Yiganling tablets （0.16 g·kg-1）， and low-， medium-， and high-dose （0.2， 0.4， 0.8 g·kg-1， respectively） Geju Hugan tablets groups. The drugs were administrated at the corresponding doses by gavage， and the normal and model groups with equal volume of pure water once a day for 28 consecutive days. On day 29， the mice in other groups except the normal group were administrated with liquor （53% Vol） by gavage twice a day at the doses of 20， 10 mL·kg-1 and with the interval of 6 h. Samples were harvested on day 30. The histopathological changes in the liver were observed by hematoxylin-eosin （HE） staining， and the ultrastructural changes in hepatocytes were observed by transmission electron microscopy. The enzyme-linked immunosorbent assay was employed to measure the levels of malonaldehyde （MDA）， reduced glutathione （GSH）， and triglycerides （TG） in the liver tissue and the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum. Western blotting was employed to determine the protein levels of NF-κB p65， phosphorylated p-inhibitor kappa B alpha （p-IκBα）， Bcl-2， and Bax in the liver tissue. ResultCompared with the normal group， the model group showed increases in the ALT， AST， MDA， and TG levels， a decrease in the GSH level， and increases in the liver injury scores evaluated based on the HE， oil red O， and transmission electron microscopy （P<0.01）. Moreover， the model group showed up-regulated expression of NF-κB， p-IκBα， and Bax （P<0.05， P<0.01） and down-regulated expression of Bcl-2 （P<0.05） in the liver tissue. Compared with the model group， Geju Hugan tablets of all the doses lowered the ALT， AST， MDA， and TG levels and elevated the GSH level （P<0.01）. The liver injury scores assessed based on HE staining and transmission electron microscopy in the medium- and high-dose Geju Hugan tablets groups were lower than those in the model group （P<0.01）. Compared with the model group， medium- and high-dose Geju Hugan tablets down-regulated the protein levels of NF-κB， p-IκBα， and Bax （P<0.01） and all doses of Geju Hugan tablets up-regulated the protein level of Bcl-2 （P<0.01）. ConclusionGeju Hugan tablets protect mice from alcohol-induced liver injury by down-regulating NF-κB signaling pathway to alleviate inflammation in the liver tissue and down-regulating the expression of Bax and up-regulating the expression of Bcl-2 to inhibit hepatocyte apoptosis.

[Mechanism of Puerariae Lobatae Radix against lung cancer by inhibiting histone demethylase LSD1].

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.

Puerariae lobatae Radix Alleviates Pre-Eclampsia by Remodeling Gut Microbiota and Protecting the Gut and Placental Barriers.

Pre-eclampsia (PE) is a serious pregnancy complication, and gut dysbiosis is an important cause of it. Puerariae lobatae Radix (PLR) is a medicine and food homologous species; however, its effect on PE is unclear. This study aimed to investigate the efficacy of PLR in alleviating PE and its mechanisms. We used an NG-nitro-L-arginine methyl ester (L-NAME)-induced PE mouse model to examine the efficacy of preventive and therapeutic PLR supplementation. The results showed that both PLR interventions alleviated hypertension and proteinuria, increased fetal and placental weights, and elevated the levels of VEGF and PlGF. Moreover, PLR protected the placenta from oxidative stress via activating the Nrf2/HO-1/NQO1 pathway and mitigated placental damage by increasing intestinal barrier markers (ZO-1, Occludin, and Claudin-1) expression and reducing lipopolysaccharide leakage. Notably, preventive PLR administration corrected gut dysbiosis in PE mice, as evidenced by the increased abundance and positive interactions of beneficial bacteria including Bifidobacterium, Blautia, and Turicibacter. Fecal microbiota transplantation confirmed that the gut microbiota partially mediated the beneficial effects of PLR on PE. Our findings revealed that modulating the gut microbiota is an effective strategy for the treatment of PE and highlighted that PLR might be used as an intestinal nutrient supplement in PE patients.

Daidzein is the in vivo active compound of Puerariae Lobatae Radix water extract for muscarinic receptor-3 inhibition against overactive bladder.

Background: In the previous study, Puerariae Lobatae Radix (named Gegen in Chinese) water extract attenuated M3 receptor agonist carbachol-induced detrusor contraction after 3-week oral administration in a hypertension-associated OAB (overactive bladder) model. This research aimed to investigate the active ingredients from Gegen water extract against OAB. Methods: Bioassay-guided fractionation was performed by using preparative HPLC for fast isolation of fractions followed by screening their ex vivo activity through carbachol-induced bladder strip contraction assay. Chemicals in each active fraction were analyzed by HPLC-UV. Urine metabolites were quantified by LC-MS/MS after sub-acute administration. Thermal shift assay with the recombinant human M3 receptor protein was performed, and molecular docking analysis was used for molecular modelling of M3 receptor inhibition. Results: Bioassay-guided fractionation results for isolating M3 receptor inhibitors indicated that four compounds were identified as active ingredients of Gegen water extract, and their inhibition potency on carbachol-induced detrusor contraction was ranked in descending order according to their inhibition concentrations as follows: genistein > daidzein > biochanin A >> puerarin. Daidzein in urine reached an ex vivo effective concentration to inhibit detrusor contraction, but others did not. Daidzein concentration-dependently increased the melt temperature (Tm) of recombinant human M3 receptor protein with a positive binding (ΔTm = 2.12 °C at 100 µg/ml). Molecular docking analysis showed that daidzein can potently bind to the ligand binding pocket of the M3 receptor via hydrogen bonding. Conclusion: Puerarin and its derivatives were pro-drugs, and daidzein was their in vivo active form via M3 receptor inhibition for treating OAB.

[Comparison of major chemical components in Puerariae Thomsonii Radix and Puerariae Lobatae Radix].

For further development and utilization of the germplasm resources of Puerariae Thomsonii Radix and Puerariae Lobatae Radix, this study developed the ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) method, high performance liquid chromatography(HPLC) method, and anthrone colorimetry to detect the content of 23 flavonoids, cellulose, hemicellulose, lignin, soluble sugar, and starch in Puerariae Thomsonii Radix and Puerariae Lobatae Radix. The content differences of various chemical components were analyzed. The methodological test of the established UPLC-MS/MS method for the determination of flavonoids showed that each component had satisfactory linearity within the corresponding linear range(R~2≥0.995), and the average spiked recoveries were 94.48%-105.5%. With this method, 17 flavonoids in Puerariae Lobatae Radix and Puerariae Thomsonii Radix were detected. Based on HPLC and anthrone colorimetry, the determination methods of lignocellulose, soluble sugar, and starch were established. According to the determination results, the content of cellulose in Puerariae Thomsonii Radix was significantly lower than that in Puerariae Lobatae Radix, and the content of starch was significantly higher than that in Puerariae Lobatae Radix. The content of hemicellulose, lignin, and soluble sugar showed no significant difference between the two medicinals, and the content of soluble sugar was in highly significantly negative correlation with that of starch. The established methods are simple, rapid, accurate, and sensitive. The results can lay a basis for the evaluation, and comprehensive development and utilization of the germplasm resources of Puerariae Thomsonii Radix and Puerariae Lobatae Radix.

[Mechanism of Astragali Radix-Puerariae Lobatae Radix combination in regulating type 2 diabetes mellitus through AMPK signaling pathway: based on network pharmacology and experimental verification].

This study aims to explore the mechanism of Astragali Radix-Puerariae Lobatae Radix(AP) combination in the treatment of type 2 diabetes mellitus(T2 DM) based on network pharmacology and experiment. The effective components and targets of the pair were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and targets of T2 DM from each disease database. On this basis, the common targets of the medicinals and the disease were screened out. The protein-protein interaction(PPI) network was established based on STRING. Then Cytoscape 3.7.1 was employed for visualization of the common targets and the network topology analysis of key targets, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of core targets by DAVID. Thereby, the possible molecular mechanism was unveiled. High-fat diet was combined with streptozotocin(STZ, injected into tail vein) for T2 DM rat modeling. Rats were classified into the normal group, model group, positive control group(metformin hydrochloride), AP high-dose, medium-dose, and low-dose groups. After 4 weeks of intragastric administration, serum fasting blood glucose(FBG), fasting insulin(FINS), aspartate aminotransferase(AST), alanine aminotransferase(ALT), triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), interleukin(IL)-6, and tumor necrosis factor(TNF)-α of rats in each group were measured. The expression of insulin receptor substrate-2(IRS-2), adenosine monophosphate-activated protein kinase(AMPK), phosphorylated AMPK(p-AMPK), glucose 6 phosphatase(G6 Pase), and phosphoenolpyruvate carboxy kinase(Pepck) in rat liver was detected by Western blot. A total of 131 core targets of the combination in the treatment of T2 DM were screened out, among which protein kinase B(AKT) 1, mitogen-activated protein kinase(MAPK) 1, TNF-α, IL-6 were more critical. KEGG enrichment analysis suggested that the combination decreased blood glucose mainly through PI3 K/AKT signaling pathway, AMPK signaling pathway, TNF signaling pathway, and MAPK signaling pathway. The levels of FBG and FINS were lower and the glycogen level was higher in the AP high-dose and medium-dose groups than in the model group. The levels of AST, ALT, TG, and LDL-C in the three AP groups and the level of TC in AP high-dose and low-dose groups decreased compared with those in the model group. Levels of IL-6 and TNF-α were lower in AP high-dose and medium-dose groups than in the model group. The expression of IRS-2, AMPK, and p-AMPK was higher and that of G6 Pase and Pepck was lower in AP high-dose group than in the model group. Thus, the combination had multi-component, multi-target, and multi-pathway characteristics in the treatment of T2 DM. It may regulate AMPK signaling pathway through IL-6 and TNF-α to influence insulin resistance, glycogen synthesis, gluconeogenesis, islet ß cell transport, and inflammatory response, thereby exerting therapeutic effect on T2 DM.

[Comparative study of rat serum pharmacochemistry between Puerariae Lobatae Radix and Puerariae Thomsonii Radix based on UPLC-Q-TOF-MS].

UPLC-Q-TOF-MS and serum pharmacochemistry were employed to study the migrating components in rat sera after intragastric administration of the water extracts of Puerariae Lobatae Radix(PLR) and Puerariae Thomsonii Radix(PTR). After the respective intragastric administration of PLR and PTR extracts, blood samples were collected from the orbital vein. The serum samples were treated by protein precipitation method with methanol and acetonitrile at a ratio of 1∶1 and then passed through Agilent ZORBAX RRHD SB-C_(18) column(3 mm×100 mm, 1.8 µm) and Agilent SB-C_(18) pre-column(3 mm×5 mm, 1.8 µm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as the mobile phase. The elution was performed at the flow rate of 0.25 mL·min~(-1), the column temperature of 40 ℃, and the injection volume of 2 µL. By comparison of the total ion chromatogram and secondary fragment ion information of PLR and PTR water extracts, PLR-and PTR-containing sera, and blank serum, we found 42 migrating components(including 17 prototype components and 25 metabolites) in the sera of rats treated with PLR and 35 migrating components(including 15 prototype components and 20 metabolites) in the sera of rats treated with PTR. Thirty-three common components were shared by the two treatments, including 13 prototype components and 20 metabolites. The differences of migrating components in the PLR-and PTR-treated rat sera provide a scientific basis for further study of the active components and quality markers of PLR and PTR.

Puerariae lobatae radix protects against UVB-induced skin aging via antagonism of REV-ERBα in mice.

Puerariae lobatae radix (PLR) is a wildly used herbal medicine. Here we aimed to assess the PLR efficacy against UVB (ultraviolet-B)-induced skin aging and to determine the mechanisms thereof. We found a significant protective effect of PLR (topical application) on UVB-induced skin aging in mice, as evidenced by reduced skin wrinkles, epidermal thickness, and MDA (malondialdehyde) content as well as increased levels of HYP (hydroxyproline) and SOD (superoxide dismutase) in the skin. In the meantime, Mmp-1, p21 and p53 levels were decreased in the skin of PLR-treated mice. Anti-aging effects of PLR were also confirmed in L929 cells. Furthermore, PLR up-regulated skin expression of BMAL1, which is a known regulator of aging by promoting Nrf2 and antioxidant enzymes. Consistently, Nrf2 and several genes (i.e., Prdx6, Sod1, and Sod2) encoding antioxidant enzymes in the skin were increased in PLR-treated mice. Moreover, based on Gal4 chimeric assay, Bmal1 reporter gene and expression assays, we identified PLR as an antagonist of REV-ERBα that can increase Bmal1 expression. Intriguingly, loss of Rev-erbα protected mice against UVB-induced skin aging and abrogated the protective effect of PLR. In conclusion, PLR acts as an antagonist of REV-ERBα and promotes the expression of BMAL1 to protect against skin aging in mice.

Protective Effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix Extract on SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.

Protective Effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix Extract on SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo explore the protective effect of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix （SP） extract on oxygen-glucose deprivation/reoxygenation （OGD/R）-injured SH-SY5Y cells based on oxidative stress and apoptosis. MethodThe extracts of the two medicinal materials mixed in different ratios were prepared. Human neuroblastoma SH-SY5Y cells were cultured in vitro and the injury was induced by OGD/R. Cell counting kit-8 （CCK-8） assay was used to screen the optimal ratio of the two medicinals and then the extract was used for further experiment. SH-SY5Y cells were classified into normal control group， OGD/R group， and low-， medium-， and high-dose SP （2∶1） extract groups （10， 30， 100 mg·L-1， respectively）. Cells in the groups， except the normal control group， were rapidly reoxygenated for 12 h after 4 h OGD for modeling. Then cell viability was detected by CCK-8 and cell morphology was observed under the microscope. The release rate of lactate dehydrogenase （LDH）， superoxide dismutase （SOD） activity， and content of glutathione （GSH） and malondialdehyde （MDA） were determined by spectrophotometry. The level of reactive oxygen species （ROS） was detected with 2，7-dichlorodihydrofluorescein diacetate （DCFH-DA） and mitochondrial membrane potential with JC-1 assay. The nuclear morphology was observed based on Hoechst 33342 staining， and apoptosis was examined by flow cytometry combined with Annexin V-FITC/PI staining. ResultThe viability of the cells was highest in the presence of the extract of the two medicinals mixed at the ratio of 2∶1. Compared with normal control group， OGD/R group showed damaged cell morphology， high release rate of LDH and levels of ROS and MDA （P<0.01）， low SOD activity and GSH level （P<0.01）， low mitochondrial membrane potential， and high apoptosis rate （P<0.01）. Compared with OGD/R group， SP extract improved cell viability and cell morphology and reduce cell LDH release rate in a concentration-dependent manner （P<0.01）. In addition， SP extract at 30， 100 mg·L-1 reduced the level of intracellular ROS and increased SOD activity and GSH level （P<0.05， P<0.01）， and SP extract at 100 mg·L-1 decreased the content of MDA （P <0.05）. Moreover， SP extract increased mitochondrial membrane potential， and SP extract at 30， 100 mg·L-1 lowered the apoptosis rate （P<0.01）. ConclusionThe extract of Salvia miltiorrhiza Bunge and Radix Puerariae mixed at 2∶1 shows better protective effect on OGD/R-injured SH-SY5Y cells. The mechanism is the likelihood that it alleviates oxidative damage of cells and inhibits cell apoptosis.

Regulatory Effect of Herbal Pair Astragali Radix-Puerariae Lobatae Radix on PI3K/Akt/FoxO1 Signaling Pathway in Rats with T2DM Based on Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of herbal pair Astragali Radix-Puerariae Lobatae Radix （AR-PLR） against type 2 diabetes mellitus （T2DM） based on network pharmacology and experimental verification. MethodThe active ingredients and targets of AR and PLR were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. The related targets of T2DM were retrieved from disease databases and the common targets of drugs and diseases were extracted. The protein-protein interaction （PPI） network was analyzed and constructed by STRING and the network topology of key targets was analyzed by Cytoscape 3.7.1. Then gene ontology（GO） and Kyoto encyclopedia of genes and genomes（KEGG） enrichment analyses of core targets were carried out by DAVID to explore its possible molecular mechanism. The T2DM model was induced in rats by the high-fat diet combined with tail intravenous injection of streptozocin. The rats were divided into a normal group，a model group，a metformin group，and high-，medium- and low-dose AR-PLR groups. After four weeks of intragastric administration，the serum levels of fasting blood sugar （FBS），fasting insulin（FINS），aspartate aminotransferase（AST），alanine aminotransferase（ALT），triglyceride（TG），total cholesterol（TC），low-density lipoprotein cholesterin（LDL-C），high-density lipoprotein cholesterin （HDL-C），interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） of rats in each group were measured. The protein expression of insulin receptor substrate-2（IRS-2），phosphatidylinositol 3-kinase（PI3K），protein kinase B（Akt）， and forkhead box transcription factor O1（FoxO1） in rat liver was detected by Western blot. ResultA total of 131 core targets of AR-PLR in the treatment of T2DM were screened out by network pharmacology， where Akt1，mitogen-activated protein kinase 1 （MAPK1），TNF-α，and IL-6 were critical. As revealed by KEGG enrichment analysis， AR-PLR exerted the hypoglycemic effect mainly through the PI3K/Akt，TNF， and FoxO signaling pathways. Compared with the model group，the high- and medium-dose AR-PLR groups showed reduced FBS and FINS levels and increased glycogen level （P<0.05，P<0.01），all the AR-PLR groups showed decreased levels of AST，ALT，TG， and LDL-C （P<0.05，P<0.01）， the high- and low-dose AR-PLR groups showed decreased TC levels （P<0.05）. Compared with the model group， the high- and medium-dose AR-PLR groups showed reduced levels of IL-6 and TNF-α（P<0.05，P<0.01）， and the high-dose AR-PLR group showed increased expression of IRS-2， Akt， p-Akt， PI3K， and p-PI3K， and decreased expression of FoxO1 protein（P<0.05）. ConclusionAR-PLR has the characteristics of multi-component，Multi-target and multi-pathway in the treatment of T2DM. This herbal pair may regulate the PI3K/Akt/FoxO1 signaling pathway through IL-6， TNF-α， and other targets to affect insulin resistance， glycogen synthesis， gluconeogenesis， glucose transport， inflammation， immune response， and other processes， thereby treating T2DM.

Comparative study of rat serum pharmacochemistry between Puerariae Lobatae Radix and Puerariae Thomsonii Radix based on UPLC-Q-TOF-MS / 中国中药杂志

UPLC-Q-TOF-MS and serum pharmacochemistry were employed to study the migrating components in rat sera after intragastric administration of the water extracts of Puerariae Lobatae Radix(PLR) and Puerariae Thomsonii Radix(PTR). After the respective intragastric administration of PLR and PTR extracts, blood samples were collected from the orbital vein. The serum samples were treated by protein precipitation method with methanol and acetonitrile at a ratio of 1∶1 and then passed through Agilent ZORBAX RRHD SB-C_(18) column(3 mm×100 mm, 1.8 μm) and Agilent SB-C_(18) pre-column(3 mm×5 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as the mobile phase. The elution was performed at the flow rate of 0.25 mL·min~(-1), the column temperature of 40 ℃, and the injection volume of 2 μL. By comparison of the total ion chromatogram and secondary fragment ion information of PLR and PTR water extracts, PLR-and PTR-containing sera, and blank serum, we found 42 migrating components(including 17 prototype components and 25 metabolites) in the sera of rats treated with PLR and 35 migrating components(including 15 prototype components and 20 metabolites) in the sera of rats treated with PTR. Thirty-three common components were shared by the two treatments, including 13 prototype components and 20 metabolites. The differences of migrating components in the PLR-and PTR-treated rat sera provide a scientific basis for further study of the active components and quality markers of PLR and PTR.

[Effect of active component compound of Epimedii Folium,Astragali Radix,and Puerariae Lobatae Radix on expression of ADAM17 in HT22 cells by mediating hepcidin].

Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of ß amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aß25-35 induction(Aß) group, hepcidin-siRNA(siRNA) group, Aß25-35 + hepcidin-siRNA(Aß + siRNA) group, Aß25-35+YHG(Aß+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aß25-35+hepcidin-siRNA+YHG(Aß+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aß group, siRNA group, and Aß+siRNA group than in the control group(P<0.05) and the expression was lower in the Aß+siRNA group(P<0.05) and higher in the Aß+YHG group(P<0.05) than in the Aß group. Moreover, the ADAM17 protein expression was lower in the Aß+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aß+siRNA+YHG group than in the Aß+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.