Huangqin decoction attenuates spared nerve injury (SNI)-induced neuropathic pain by modulating microglial M1/M2 polarization partially mediated by intestinal nicotinamide metabolism.

BACKGROUND: The incidence of neuropathic pain is progressively increasing over time. The activation of M1-type microglia plays a crucial role in the initiation and progression of neuropathic pain. Huangqin Decoction (HQD) is traditionally used to alleviate dysentery and abdominal pain. However, it remains unclear whether HQD can effectively mitigate neuropathic pain and the underlying mechanisms. PURPOSE: The present study aims to investigate the impact of HQD on neuropathic pain induced by spared nerve injury (SNI) in mice, and to elucidate whether the analgesic effect of HQD is associated with microglia polarization. METHODS: The analgesic effect of HQD on SNI mice was investigated through assessments of mechanical pain threshold, thermal pain threshold, cold pain threshold, and motor ability. We elucidated the molecular mechanisms of HQD in alleviating SNI-induced neuropathic pain by focusing on microglia polarization and intestinal metabolite abnormalities. The expression levels of markers associated with microglia polarization (Iba-1, CD68, CD206, iNOS) was detected by immunofluorescence and Western blot, and the levels of inflammatory factors (IL-4, IL-10, IL-6, TNF-α) were assessed by ELISA. UPLC-QTOF-MS metabolomics was utilized to identify differential metabolites in the intestines of SNI mice. We screened the differential metabolites related to microglial polarization by correlation analysis, subsequently nicotinamide was selected for validation in LPS-induced BV-2 cells. RESULTS: Our findings demonstrated that HQD (20 g/kg) significantly enhanced the mechanical pain threshold, thermal pain threshold, and cold pain threshold, and protected the injured DRG neurons of SNI mice. Moreover, HQD (20 g/kg) obviously suppressed the expression of microglia M1 polarization markers (Iba-1, CD68, iNOS, IL-6, TNF-α), and promoted the expression of microglia M2 polarization markers (CD206, IL-10, IL-4) in the spinal cord of SNI mice. Additionally, HQD (20 g/kg) prominently ameliorated intestinal barrier damage by upregulating Claudin 1 and Occludin expression in the colon of SNI mice. Furthermore, HQD (20 g/kg) rectified 19 metabolite abnormalities in the intestine. Notably, nicotinamide (100 µM), an amide derivative with anti-inflammatory property, effectively suppresses microglia activation and polarization in LPS-induced BV-2 cells by downregulating IL-6 level and CD68 expression while upregulating IL-4 level and CD206 expression. CONCLUSION: In summary, HQD alleviates neuropathic pain in SNI mice by regulating the activation and polarization of microglia, partially mediated through intestinal nicotinamide metabolism.

The Beneficial Effects of Dietary Astragali Radix Are Related to the Regulation of Gut Microbiota and Its Metabolites.

Astragali Radix (AR) or its extract has been used as an herbal medicine and dietary supplement in China, Europe, and the United States. The gut microbiota could provide new insights for exploring dietary supplements' underlying mechanism on organisms. However, no reports have focused on the regulatory effect of AR on the gut microbiota as a dietary supplement. In this study, healthy ICR mice of either sex were divided into AR and control (CON) groups and given AR water extract (4.55 mg/kg·day-1) or saline by gavage for 14 days, respectively. Then 16S rRNA gene sequencing and ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry-based fecal metabolomics were integrated to investigate the benefits of dietary AR. Weighted gene coexpression network analysis was also introduced to investigate the metabolites with highly synergistic changes. AR supplementation influenced the structure of intestinal microflora, especially enriching short-chain fatty acid-producing bacteria g_Coprobacillus, g_Prevotella, and g_Parabacteroides. AR also significantly altered the fecal metabolome, mainly related to amino acid metabolism, nucleotide metabolism, and bile acid (BA) metabolism. Moreover, the increased secondary BAs and BA-sulfates might closely relate to intestinal microflora. These findings provide valuable insights for future research of dietary AR as a functional food.

The ethanol extract of Scutellaria baicalensis Georgi attenuates complete Freund's adjuvant (CFA)-induced inflammatory pain by suppression of P2X3 receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis Georgi (SBG) is a perennial herb with anti-inflammatory, antibacterial, and antioxidant activities, which is traditionally used to treat inflammation of respiratory tract and gastrointestinal tract, abdominal cramps, bacterial and viral infections. Clinically, it is often used to treat inflammatory-related diseases. Research has shown that the ethanol extract of Scutellaria baicalensis Georgi (SGE) has anti-inflammatory effect, and its main components baicalin and baicalein have analgesic effects. However, the mechanism of SGE in relieving inflammatory pain has not been deeply studied. AIM OF THE STUDY: This study aimed to evaluate the analgesic effect of SGE on complete Freund's adjuvant (CFA)-induced inflammatory pain rats, and to investigate whether its effect on relieving inflammatory pain is associated with regulation of P2X3 receptor. MATERIALS AND METHODS: The analgesic effects of SGE on CFA-induced inflammatory pain rats were evaluated by measuring mechanical pain threshold, thermal pain threshold, and motor coordination ability. The mechanisms of SGE in relieving inflammatory pain were explored by detecting inflammatory factors levels, NF-κB, COX-2 and P2X3 expression, and were further verified by addition of P2X3 receptor agonist (α, ß me-ATP). RESULTS: Our results revealed that SGE can notably increase the mechanical pain threshold and thermal pain threshold of CFA-induced inflammatory pain rats, and markedly alleviate the pathological damage in DRG. SGE could suppress the release of inflammatory factors including IL-1ß, IL-6, TNF-α and restrain the expression of NF-κB, COX-2 and P2X3. Moreover, α, ß me-ATP further exacerbated the inflammatory pain of CFA-induced rats, while SGE could markedly raise the pain thresholds and relieve inflammatory pain. SGE could attenuate the pathological damage, inhibit P2X3 expression, inhibit the elevation of inflammatory factors caused by α, ß me-ATP. SGE can also inhibit NF-κB and ERK1/2 activation caused by α, ß me-ATP, and inhibit the mRNA expression of P2X3, COX-2, NF-κB, IL-1ß, IL-6 and TNF-α in DRG of rats induced by CFA coupled with α, ß me-ATP. CONCLUSIONS: In summary, our research indicated that SGE could alleviate CFA-induced inflammatory pain by suppression of P2X3 receptor.

A unique insight for Xiaoyao San exerts antidepressant effects by modulating hippocampal glucose catabolism using stable isotope-resolved metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM) theory, depression is an emotional disease, which is thought to be related to stagnation of liver qi and dysfunction of the spleen in transport. Xiaoyao San (XYS) is considered to have the effects of soothing liver-qi stagnation and invigorating the spleen. The spleen has the function to transport and transform nutrients. The liver has also termed the center of energy metabolism in the body. Therefore, exploring the antidepressant effects of XYS from the perspective of energy metabolism may reveal new findings. AIM OF THE STUDY: Glucose catabolism is an important part of energy metabolism. In recent years, several researchers have found that XYS can exert antidepressant effects by modulating abnormalities in glucose catabolism-related metabolites. The previous research of our research group found that the hippocampus glucose catabolism was disordered in depression. However, the antidepressant potential of XYS through modulating the disorders of hippocampal glucose catabolism and the specific metabolic pathways and targets of XYS action were still unknown. The aim of this study was to address the above scientific questions. MATERIALS AND METHODS: In this research, the CUMS (chronic unpredictable mild stress) model was used as the animal model of depression. The antidepressant effect of XYS was evaluated by behavioral indicators. The specific pathways and targets of XYS modulating the disorders of glucose catabolism in the hippocampus of CUMS rats were obtained by stable isotope-resolved metabolomics. Further, the isotope tracing results were also verified by molecular biology and electron transmission electron microscopy. RESULTS: The results demonstrated that XYS pretreatment could significantly improve the depressive symptoms induced by CUMS. More importantly, it was found that XYS could modulate the disorders of glucose catabolism in the hippocampus of CUMS rats. Stable isotope-resolved metabolomics and enzyme activity tests showed that Lactate dehydrogenase (LDH), Pyruvate carboxylase (PC), and Pyruvate dehydrogenase (PDH) were targets of XYS for modulating the disorders of glucose catabolism in the hippocampus of CUMS rats. The Succinate dehydrogenase (SDH) and mitochondrial respiratory chain complex V (MRCC-Ⅴ) were targets of XYS to improve abnormal mitochondrial oxidative phosphorylation in the hippocampus of CUMS rats. XYS was also found to have the ability to improve the structural damage of mitochondria and nuclei in the hippocampal caused by CUMS. CONCLUSIONS: This study was to explore the antidepressant effect of XYS from the perspective of glucose catabolism based on a strategy combining stable isotope tracing, molecular biology techniques, and transmission electron microscopy. We not only obtained the specific pathways and targets of XYS to improve the disorders of glucose catabolism in the hippocampus of CUMS rats, but also revealed the specific targets of the pathways of XYS compared with VLF.

[Toxicity comparison of raw and vinegar-processed Bupleuri Radix based on ~1H-NMR metabolomics].

This study compared the toxicity of raw Bupleuri Radix(BR) and vinegar-processed Bupleuri Radix(VPBR) based on proton nuclear magnetic resonance(~1H-NMR), and explored the mechanism of toxicity. Thirty-two male Sprague-Dawley(SD) rats were randomly divided into four groups: a control group(distilled water), a raw BR group(15 g·kg~(-1)·d~(-1)), a rice VPBR(R-VPBR) group(15 g·kg~(-1)·d~(-1)), and a shanxi VPBR(S-VPBR) group(15 g·kg~(-1)·d~(-1)). After administration for 30 d, pathological sections were treated and observed, and biochemical indexes related to liver and renal function were determined. The serum, liver, and kidney of rats were collected and analyzed by ~1H-NMR. The principal component analysis(PCA) and orthogonal partial least squares-discrimination analysis(OPLS-DA) were performed. The results showed that, as compared with the control group, alanine aminotransferase(ALT), aspartate aminotransferase(AST), and alkaline phosphatase(ALP) in the raw BR group were increased significantly, while ALT and ALP in the R-VPBR and S-VPBR groups were significantly decreased(P<0.05), which indicated that BR showed certain hepatotoxicity, and vinegar processing reduced its hepatotoxicity. No significant difference of blood urea nitrogen(BUN) and creatinine(CREA), the biochemical indexes related to renal function, was observed in the control group and administration groups, indicating that BR had less effect on the renal function. The results of multivariate statistical analysis showed that the biomarkers of BR affecting liver metabolism were methionine, glutamine, and glutamic acid, and affecting kidney metabolism were taurine, ornithine, and inosine. These biomarkers were mainly involved in amino acid metabolism, energy metabolism, lipid metabolism, and taurine metabolism. VPBR alleviated the effect on the biomarkers, and S-VPBR had smaller effect than R-VPBR. Combining the results of biochemical indexes and metabolomics analysis, both raw BR and VPBR showed toxic effect on rats, whereas vinegar processing reduced its toxicity. S-VPBR has smaller effect on kidney and liver metabolism than R-VPBR, which indicates that the vinegar used for processing has certain effect on the toxicity of BR.

Huangqin Decoction Exerts Beneficial Effects on Rotenone-Induced Rat Model of Parkinson's Disease by Improving Mitochondrial Dysfunction and Alleviating Metabolic Abnormality of Mitochondria.

Parkinson's disease (PD) is a common neurodegenerative disease, and the pathogenesis of PD is closely related to mitochondrial dysfunction. Previous studies have indicated that traditional Chinese medicine composition of Huangqin Decoction (HQD), including Scutellariae Radix, licorice, and Paeoniae Radix Alba, has therapeutic effects on PD, but whether HQD has a therapeutic effect on PD has not been reported. In this study, the protective effects of HQD on rotenone-induced PD rats were evaluated by behavioral assays (open field, rotating rod, suspension, gait, inclined plate, and grid) and immunohistochemistry. The mechanisms of HQD on attenuation of mitochondrial dysfunction were detected by biochemical assays and mitochondrial metabolomics. The results showed that HQD (20 g/kg) can protect rats with PD by improving motor coordination and muscle strength, increasing the number of tyrosine hydroxylase (TH)-positive neurons in rats with PD. Besides, HQD can improve mitochondrial dysfunction by increasing the content of adenosine triphosphate (ATP) and mitochondrial complex I. Mitochondrial metabolomics analysis revealed that the ketone body of acetoacetic acid (AcAc) in the rotenone group was significantly higher than that of the control group. Ketone bodies have been known to be used as an alternative energy source to provide energy to the brain when glucose was deficient. Further studies demonstrated that HQD could increase the expression of glucose transporter GLUT1, the content of tricarboxylic acid cycle rate-limiting enzyme citrate synthase (CS), and the level of hexokinase (HK) in rats with PD but could decrease the content of ketone bodies [AcAc and ß-hydroxybutyric acid (ß-HB)] and the expression of their transporters (MCT1). Our study revealed that the decrease of glucose metabolism in the rotenone group was parallel to the increase of substitute substrates (ketone bodies) and related transporters, and HQD could improve PD symptoms by activating the aerobic glycolysis pathway.

[Molecular mechanism underlying difference of astragaloside â £ content in imitating wild and cultivated Astragalus mongholicus].

The difference of astragaloside Ⅳ content and the expression of its biosynthesis related genes in imitating wild Astragalus mongolicus(IWA) and cultivated A.mongolicus(CA) under different growth years were systematically compared and analyzed.Then the key enzyme genes affected the difference of astragaloside Ⅳ content in the above two A.mongolicus were screened.High-perfo-rmance liquid chromatography(HPLC)was used to determine the content of astragaloside Ⅳ in A.mongolicusunderthe above two diffe-rent growth patterns.Based on the Illumina HiSeq and PacBio high-throughput sequencing platforms, thesecond-and third-generation transcriptome sequencing(RNA-Seq)databaseof the two A.mongolicuswas constructed.The related enzyme genes in the biosynthetic pathway of astragaloside Ⅳ were screened and verified byquantitative reverse transcriptase polymerase chain reaction(RT-qPCR).The RNA-sequencing(RNA-Seq) and RT-qPCR data of each gene were subjected to correlation analysis and trend analysis.The results showed that the variation trend of astragaloside Ⅳ contentby HPLC wasthe same as that of genes by RNA-Seq and RT-qPCR in 1-4 year IWA and 1-2 year CA.The trend level of astragaloside Ⅳ contentwas lower in 2-year IWA than 1-year IWA.Compared with 2-year IWA, 3-year IWA had an upward trend, while 4-year IWA hada downward trend versus 3-year IWA.Additionally, 1-year CA had increased trendthan 2-year CA.However, the content of astragaloside Ⅳ in 5-year IWA was higher than that of 6-year IWA, which wasinconsistent with the findings of RNA-Seq and RT-qPCR.This study preliminarily clarifiedthat the difference of astragaloside Ⅳ contentin 1-4 year IWA and 1-2 year CA wasclosely related to the expression of the upstream and midstream genes(MVK, CMK, PMK, MVD, SS) in the biosynthetic pathway.The results facilitate the production and planting of Radix Astragali seu Hedysari.

[Quality evaluation methods and quality transfer law of Mori Cortex, fried Mori Cortex and its standard decoction].

This study aims to establish a method for the component content determination and fingerprint evaluation of Mori Cortex, fried Mori Cortex and its standard decoction, and to reveal the quality transfer law among the three based on transfer rate, extraction rate, and fingerprint similarity.Fifteen representative batches of Mori Cortex decoction pieces were collected to prepare fried Mori Cortex and its standard decoction.UPLC-PDA was employed to establish the content determination method and fingerprint.The established UPLC method and fingerprint could be applied to the detection of Mori Cortex, fried Mori Cortex and its standard decoction.The UPLC fingerprints of the 15 batches of Mori Cortex and fried Mori Cortex had good similarity(>0.9) and the same common peaks.However, only one characteristic peak(mulberroside A) could be observed in the fingerprint of fried Mori Cortex standard decoction, which indicated that the corresponding components of other common peaks in the fingerprint of Mori Cortex had low content in the water extract.The extraction rates of mulberroside A from Mori Cortex, fried Mori Cortex and its standard decoction were 1.49%-2.00%, 1.62%-2.27% and 0.75%-1.29%, respectively.Mulberroside A showed the transfer rate of 103.7%-116.3% from Mori Cortex to fried Mori Cortex and 45.7%-56.9% from fried Mori Cortex to its standard decoction.The extraction rates of the 15 batches of fried Mori Cortex standard decoctions were 14.7%-19.5%.All the above indicators were within±30% of the mean value.This study established a method for the determination of mulberroside A content and fingerprint of Mori Cortex, fried Mori Cortex and its standard decoction, and clarified the quality transfer law among the three.It established the method for quality evaluation of Mori Cortex and fried Mori Cortex and can provide reference for the whole-process quality control in the preparation of the agents containing fried Mori Cortex.

Investigating the inter-individual variability of Astragali Radix against cisplatin-induced liver injury via 16S rRNA gene sequencing and LC/MS-based metabolomics.

BACKGROUND: Cisplatin (CDDP), one of the widely used chemotherapeutic drugs, can induce a series of side effects, such as hepatotoxicity and gastrointestinal toxicity. Astragali Radix (AR) is widely used as the tonic herbal medicine in traditional Chinese medicine (TCM). However, there was no report about the hepatoprotective effect of AR against the cisplatin-induced hepatic damage. PURPOSE: This study aimed to investigate the protective effect and potential mechanism of AR water extract against the cisplatin-induced liver injury. METHODS: Cisplatin was utilized to induce the liver injury using ICR mice, and the protective effect of AR was evaluated by serum biochemistry indices and liver histopathology. Then UHPLC Q-TOF-MS/MS-based untargeted serum metabolomics approach combined with 16S rRNA-based microbiota analysis was used to explore the underlying biomarkers and mechanism about the liver-protective effect of AR. RESULTS: AR could decrease the serum AST and ALT, ameliorate hepatic pathological damages caused by cisplatin. Serum metabolomics indicated AR could regulate the biosynthesis of unsaturated fatty acids, arachidonic acid metabolism, purine metabolism, and fatty acid biosynthesis. In addition, 16S rRNA gene sequencing analysis showed that AR could regulate cisplatin-induced gut microbiota disorder, especially the inflammation-related bacteria (p_Deferribacteres, g_Enterococcus, and g_Alistipes, etc.), and the short chain fatty acids (SCFAs)-producing bacteria (g_Alloprevotella, g_Intestinimoas, and g_Flavonifractor). Moreover, 7 mice (AR-7) showed better liver protective effect than the other 3 mice (AR-3), and their regulatory effect on the gut microbiota and serum metabolites were also different, indicating the presence of inter-individual variability for the liver protective effect of AR. CONCLUSIONS: This study revealed the protective effect and the potential mechanisms of AR against cisplatin-induced liver injury, and found that inter-individual variability of the liver protective effect of AR was related to the host microbiome and metabolome. These findings provided new insight into the health effect of dietary AR as a functional food for cisplatin-based chemotherapy.

Naoluo Xintong Decoction Ameliorates Cerebral Ischemia-Reperfusion Injury by Promoting Angiogenesis through Activating the HIF-1α/VEGF Signaling Pathway in Rats.

Background: Naoluo Xintong decoction (NLXTD) is a traditional Chinese medicine (TCM) formula which has been used to improve neuronal functional recovery after cerebral ischemic stroke. However, the molecular mechanism underlying NLXTD's amelioration of ischemic stroke remains unclear. The present study was designed to explore the effect and mechanism of NLXTD on brain angiogenesis in a rat model with cerebral ischemia-reperfusion (I/R) injury targeting the hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway. Materials and Methods: Cerebral I/R model was established by the classical middle cerebral artery occlusion (MCAO) method. Sprague-Dawley (SD) male rats (n = 80) were randomly divided into the sham-operation group, the model group, the HIF-1α inhibitor 2-methoxyestradiol (2ME2) group, the 2ME2 with NLXTD group, and the NLXTD group. Neurological deficit test, TTC staining, H&E staining, TUNEL staining, immunohistochemistry (IH), immunofluorescence (IF), western blot, and quantitative RT-PCR were performed to evaluate the effect of NLXTD after MCAO. Results: Administration of NLXTD significantly decreased neuron deficiency scores, reduced brain infarct volume, and lowered damaged and apoptotic cells after brain I/R injury in rats. Meanwhile, NLXTD had a protective effect on angiogenesis by increasing the MVD and the expressions of BrdU and CD34, which enhanced the number of endothelial cells in the ischemic penumbra brain. NLXTD treatment significantly raised the protein and mRNA levels of HIF-1α, VEGF, VEGFR2, and Notch1 compared with the model treatment. In contrast, a specific HIF-1α inhibitor, 2ME2, inhibited the improvement of neurological function and angiogenesis in NLXTD-induced rats with cerebral I/R injury, suggesting that NLXTD played a positive role in ischemic brain injury by activating the HIF-1α/VEGF signaling pathway. Conclusions: NLXTD exerts neuroprotection targeting angiogenesis by upregulating the HIF-1α/VEGF signaling pathway on cerebral I/R injury rats.

Unveiling the anti-senescence effects and senescence-associated secretory phenotype (SASP) inhibitory mechanisms of Scutellaria baicalensis Georgi in low glucose-induced astrocytes based on boolean network.

BACKGROUND: Astrocytes senescence has been demonstrated in the aging brain and Alzheimer's disease (AD). Moreover, lower glucose metabolism has been confirmed in the early stage of AD. However, whether low glucose could induce astrocytes senescence remain ambiguous. Studies have shown that the ethanol extracts of Scutellaria baicalensis Georgi (SGE) exert neuroprotective and anti-aging effects, while whether SGE could delay astrocytes senescence was unclear. PURPOSE: This study investigated the anti-senescence effect of SGE in low glucose-induced T98G cells and primary astrocytes, and explored the possible mechanisms based on boolean network. METHODS: The neuroprotective effects of SGE in low glucose-induced T98G cells were evaluated by measurement of cell viability, LDH, ROS and ATP. The anti-senescence effects of SGE were investigated by detection of senescence-associated ß-galactosidase (SA-ß-Gal), senescence-associated secretory phenotype (SASP), cell cycle and senescence-related markers. The possible mechanisms of SGE in delaying astrocytes senescence were discovered through integrating transcriptomics with boolean network, and validation experiments were further performed. RESULTS: Our results revealed that low glucose could induce astrocytes senescence, and SGE could delay astrocytes senescence by decreasing the staining rate of SA-ß-gal, reducing secretions of SASP factors (IL-6, CXCL1, MMP-1), alleviating cell cycle arrest in G0/G1 phase, decreasing the formation of punctate DNA foci and down-regulating the expression of p16INK4A, p21 and γH2A.X. Transcriptomics and further verification results showed that SGE could markedly inhibit the mRNA expression levels of SASP factors (CXCL10, CXCL2, CCL2, IL-6, CXCR4, CCR7). Moreover, C-X-C motif chemokine 10 (CXCL10) was predicted to be the key SASP factor affecting the network stability by using boolean network. Further experiments validated that SGE could markedly reduce CXCL10 level, decrease the secretion of IL-6 and inhibit cell migration in CXCL10 induced primary astrocytes. CONCLUSION: In summary, our research unmasks that the anti-senescence effects of SGE were highly correlated with the suppression of SASP secretions, and CXCL10 mediated the SASP inhibition effect of SGE in low glucose-induced astrocytes. Our study highlights that the delay of astrocytes senescence and the inhibition of SASP might be a new mechanism of SGE for alleviating neurodegenerative diseases such as AD.

Branched-Chain Amino Acids Catabolism Pathway Regulation Plays a Critical Role in the Improvement of Leukopenia Induced by Cyclophosphamide in 4T1 Tumor-Bearing Mice Treated With Lvjiaobuxue Granule.

Background: Cyclophosphamide is a common tumor chemotherapy drug used to treat various cancers. However, the resulting immunosuppression leads to leukopenia, which is a serious limiting factor in clinical application. Therefore, the introduction of immunomodulators as adjuvant therapy may help to reduce the hematological side effects of cyclophosphamide. Lvjiaobuxue granule has been widely used in the clinical treatment of gynecological diseases such as anemia and irregular menstruation. Recently, it has been found to increase the function of white blood cells, but its mechanism of action is still unclear. We aimed to reveal the mechanisms of Lvjiaobuxue granule against acute leukopenia by an integrated strategy combining metabolomics with network pharmacology. Methods: Subcutaneously inoculated 4T1 breast cancer cells to prepare tumor-bearing mice, intraperitoneal injection of cyclophosphamide to establish a 4T1 tumor-bearing mice leukopenia animal model, using pharmacodynamic indicators, metabolomics, network pharmacology and molecular biology and other technical methods. To comprehensively and systematically elucidate the effect and mechanism of Lvjiaobuxue granule in improving cyclophosphamide-induced leukopenia in 4T1 tumor-bearing mice. Results: Lvjiaobuxue granule can improve the blood routine parameters and organ index levels of the leukopenia model of 4T1 tumor-bearing mice. Metabolomics studies revealed that 15 endogenous metabolites in the spleen of mice were considered as potential biomarkers of Lvjiaobuxue granule for their protective effect. Metabonomics and network pharmacology integrated analysis indicated that Lvjiaobuxue granule exerted the leukocyte elevation activity by inhibiting the branched-chain amino acids (BCAAs) degradation pathway and increasing the levels of valine, leucine and isoleucine. The results of molecular biology also showed that Lvjiaobuxue granule can significantly regulate the key enzymes in the catabolism of BCAAs, which further illustrates the importance of BCAAs in improving leukopenia. Conclusion: Lvjiaobuxue granule exerts obvious pharmacological effects on the leukopenia model of 4T1 tumor-bearing mice induced by cyclophosphamide, which could be mediated by regulating the branched-chain amino acid degradation pathway and the levels of valine, leucine and isoleucine.

Compound Kushen Injection intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating ß-catenin/c-Myc signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most extensive and most deadly cancers worldwide. The invasion and metastasis characteristics of HCC dramatically affect the prognosis and survival of HCC patients. Compound Kushen Injection (CKI) is a GMP produced, proverbially applied traditional Chinese medicine formula in China to treat cancer-associated pains, and used as an adjunctive therapy for HCC. Until so far, whether CKI could suppress the metastasis of HCC through regulation of epithelial-mesenchymal transition or metabolic reprogramming is still ambiguous. PURPOSE: In this study, the anti-metastasis effects of CKI were clarified and its pharmacological mechanisms were systematically explored. METHODS: Cell invasion and cell adhesion assay were performed in SMMC-7721 cells to assess the anti-metastasis role of CKI, and the histopathological evaluation and biochemical detection were utilized in DEN-induced HCC rats to verify the anti-HCC effect of CKI. Serum and liver samples were analyzed with 1H NMR metabolomics approach to screen the differential metabolites and further target quantification the content of key metabolites. Finally, western blotting and immunofluorescence assay were applied to verify the crucial signaling pathway involved in metabolites. RESULTS: CKI markedly repressed the invasion and adhesion in SMMC-7721 cells and significantly improved the liver function of DEN-induced HCC rats. CKI significantly regulated the expression of epithelial-mesenchymal transition (EMT) markers (Vimentin and E-cadherin). Metabolomics results showed that CKI regulated the metabolic reprogramming of HCC by inhibiting the key metabolites (citrate and lactate) and enzymes (HK and PK) in glycolysis process. Importantly, we found that c-Myc mediates the inhibitory effect of CKI on glycolysis. We further demonstrated that CKI inhibits c-Myc expression through modulating Wnt/ß-catenin pathway in SMMC-7721 cells and DEN-induced HCC rats. Furthermore, through activating Wnt/ß-catenin pathway with LiCl, the inhibitory effects of CKI on HCC were diminished. CONCLUSION: Together, this study reveals that CKI intervenes metabolic reprogramming and epithelial-mesenchymal transition of HCC via regulating ß-catenin/c-Myc signaling pathway. Our research provides a new understanding of the mechanism of CKI against invasion and metastasis of HCC from the perspective of metabolic reprogramming.

[Mechanism of Xiaoyao San in treatment of depression,breast hyperplasia,and functional dyspepsia based on network pharmacology].

This study aimed to explore the mechanism of Xiaoyao San(XYS) in the treatment of three diseases of liver depression and spleen deficiency, ie, depression, breast hyperplasia, and functional dyspepsia, and to provide a theoretical basis for the interpretation of the scientific connotation of "treating different diseases with the same method" of traditional Chinese medicines. Traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP) was used to screen the active components of XYS which underwent principal component analysis(PCA) with the available drugs for these three diseases to determine the corresponding biological activities. The targets of XYS on depression, breast hyperplasia, and functional dyspepsia were obtained from GeneCards, TTD, CTD, and DrugBank databases. Cytoscape was used to plot the "individual herbal medicine-active components-potential targets" network. The resulting key targets were subjected to Kyoto encyclopedia of genes and genomes(KEGG) pathway analysis and gene ontology(GO) enrichment analysis. A total of 121 active components of XYS and 38 common targets in the treatment of depression, breast hyperplasia, and functional dyspepsia were collected. The key biological pathways were identified, including advanced glycation and products(AGEs)-receptor for advanced glycation and products(RAGE) signaling pathway in diabetic complications, HIF-1 signaling pathway, and cancer-related pathways. The key targets of XYS in the treatment of depression, breast hyperplasia, and functional dyspepsia included IL6, IL4, and TNF, and the key components were kaempferol, quercetin, aloe-emodin, etc. As revealed by the molecular docking, a strong affinity was observed between the key components and the key targets, which confirmed the results. The therapeutic efficacy of XYS in the treatment of diseases of liver depression and spleen deficiency was presumedly achieved by reducing the inflammatory reactions. The current findings are expected to provide novel research ideas and approaches to classify the scientific connotation of "treating different diseases with the same method" of Chinese medicines, as well as a theoretical basis for understanding the mechanism of XYS and exploring its clinical applications.

[Research progress of circadian rhythm].

Circadian rhythm disorder is a common society issue caused by jet lag,shift work,sleep disruption and changes in food consumption. Light is the major factor affecting the circadian rhythm system. Disruption of the circadian rhythm system can cause damage to the body,leading to some diseases. Maintaining a normal circadian system is of great importance for good health. Ideal therapeutic effect can not only alleviate symptoms of the diseases,but also recovery the disturbed circadian rhythm to normal. The paper summarizes the modeling methods of animal circadian rhythm disorder,diseases of circadian rhythm abnormality,regulation of circadian clock genes and medicine which are related to circadian rhythm to diseases of circadian rhythm disorder.

Hepatic metabolomics of the compatibility effect of Xiaoyaosan on CUMS-induced depression based on the TCM theory of "Treating Diseases via Regulating the Liver's Function".

This study aimed to demonstrate the scientific connotations and compatibility effects of Xiaoyaosan (XYS) based on the theory of "Treating Diseases via Regulating the Liver's Function" by hepatic metabolomics. XYS was divided into two efficacy groups, i.e. the Shugan (SG) and the Jianpi (JP) groups, according to the strategy of "Efficacy Compositions". The chronic unpredictable mild stress (CUMS) depression model was constructed. A 1H NMR-based hepatic metabolomics approach coupled with multivariate data (MVD) analysis was performed. Meanwhile, relative distance (RD) and Efficacy Index (EI) were calculated. XYS and its efficacy groups significantly reversed the abnormality of behavior and hepatic metabolomics of depression rats, but to different degrees. The results of ethology and metabolomics showed the same order, i.e. XYS > JP > SG. Two metabolites, i.e. tyrosine and malate, were regulated by all the treatment groups. Four metabolites were significantly regulated only by XYS group. Of note, the results showed the two efficacy groups of XYS exhibited synergistic anti-depression effects, and glutamate, malate and taurine could be the key hepatic metabolites for these synergistic effects. The current study not only complements and consummates the mechanisms of depression and the anti-depression effects of XYS from the perspective of hepatic metabolomics, but also lays a solid foundation for comprehensively and deeply understanding the compatibility effects of XYS against depression, especially from the points of view of compatibility in Traditional Chinese medicine (TCM) theory and synergism in modern medicine theory.

Comprehensive Analysis Strategy of Nervous-Endocrine-Immune-Related Metabolites to Evaluate Arachidonic Acid as a Novel Diagnostic Biomarker in Depression.

Depression is one of the most complex multifactorial diseases affected by genetic and environmental factors. The molecular mechanism underlying depression remains largely unclear. To address this issue, a novel nervous-endocrine-immune (NEI) network module was used to find the metabolites and evaluate the diagnostic ability of patients with depression. During this process, metabolites were acquired from a professional depression metabolism database. Over-representation analysis was performed using IMPaLA. Then, the metabolite-metabolite interaction (MMI) network of the NEI system was used to select key metabolites. Finally, the receiver operating characteristic curve analysis was evaluated for the diagnostic ability of arachidonic acid. The results show that the numbers of the nervous system, endocrine system, and immune system pathways are 10, 19, and 12 and the numbers of metabolites are 38, 52, and 13, respectively. The selected shared metabolite-enriched pathways can be 97.56% of the NEI-related pathways. Arachidonic acid was extracted from the NEI system network by using an optimization formula and validated by in vivo experiments. It was indicated that the proposed model was good at screening arachidonic acid for the diagnosis of depression. This method provides reliable evidences and references for the diagnosis and mechanism research of other related diseases.

A metabolic data-driven systems pharmacology strategy for decoding and validating the mechanism of Compound Kushen Injection against HCC.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Kushen Injection (CKI) is a widely used TCM formula for treatment of carcinomatous pain and tumors of digestive system including hepatocellular carcinoma (HCC). However, the potential mechanisms of CKI for treatment of HCC have not been systematically and deeply studied. AIM OF STUDY: A metabolic data-driven systems pharmacology approach was utilized to investigate the potential mechanisms of CKI for treatment of HCC. MATERIALS AND METHODS: Based on phenotypic data generated by metabolomics and genotypic data of drug targets, a propagation model based on Dijkstra program was proposed to decode the effective network of key genotype-phenotype of CKI in treating HCC. The pivotal pathway was predicted by target propagation mode of our proposed model, and was validated in SMMC-7721 cells and diethylnitrosamine-induced rats. RESULTS: Metabolomics results indicated that 12 differential metabolites, and 5 metabolic pathways might be involved in the anti-HCC effect of CKI. A total of 86 metabolic related genes that affected by CKI were obtained. The results calculated by propagation model showed that 6475 shortest distance chains might be involved in the anti-HCC effect of CKI. According to the results of propagation mode, EGFR was identified as the core target of CKI for the anti-HCC effect. Finally, EGFR and its related pathway EGFR-STAT3 signaling pathway were validated in vivo and in vitro. CONCLUSION: The proposed method provides a methodological reference for explaining the underlying mechanism of TCM in treating HCC.

Identification and activity evaluation of Astragalus Radix from different germplasm resources based on specific oligosaccharide fragments.

Objective: Based on trifluoroacetic acid (TFA) hydrolysis, polyacrylamide gel electrophoresis (PAGE) and high performance thin layer chromatography (HPTLC) analysis, the carbohydrate responsible for immunomodulatory activity are used as quality indicators for Astragalus Radix (AR). Methods: In this study, 24 batches of AR from different germplasm resources were selected as the research object, and AR polysaccharides were extracted. PAGE and HPTLC methods were used to analyze the partial acid hydrolyzate of AR polysaccharides and obtain a series of saccharide fingerprints. The data were analyzed by principal component analysis to obtain the difference between AR from different germplasm resources. Results: The results showed that trisaccharide and tetrasaccharide could be used as differential fragments to distinguish AR of different cultivation methods; Disaccharides and trisaccharides can be used as differential fragments to distinguish different species of AR. The immunological activity analysis of the specific oligosaccharide fragment of AR showed that the specific oligosaccharide fragment of AR could promote the secretion of TNF-α, IL-1ß, IL-6, and NO in THP-1 cells in a concentration-dependent manner. Conclusion: Both PAGE and HPTLC methods can be used to evaluate AR from different germplasm resources. This study laid the foundation for the quality evaluation of AR medicinal herbs.

Integrated network pharmacology and metabolomics to dissect the combination mechanisms of Bupleurum chinense DC-Paeonia lactiflora Pall herb pair for treating depression.

ETHNOPHARMACOLOGICAL RELEVANCE: The compatibility of Bupleurum chinense DC (Chaihu)-Paeonia lactiflora Pall (Baishao) is one of the most accepted herb pairs in traditional Chinese medicine (TCM) prescriptions for treating depression. However, the combination mechanisms of this herb pair for anti-depression remain unclear. MATERIALS AND METHODS: In this study, the combined effect of Chaihu-Baishao was evaluated by the chronic unpredictable mild stress (CUMS) rat model. Secondly, network pharmacology was constructed to dissect the united mechanisms. Based on the results of network pharmacology analysis, plasma metabolomics based on ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was performed to discover the collaborative effect on metabolite regulation. Furthermore, the targets from network pharmacology and the metabolites from metabolomics were jointly analyzed to select crucial metabolism pathways by MetaScape. Finally, the key metabolic enzymes and metabolites were experimentally validated by ELISA. RESULTS: The antidepressant effect of Chaihu-Baishao herb pair was significantly better than Chaihu or Baishao in sucrose preference test (SPT), open-field test (OFT), and forced swim test (FST). In network pharmacology, herb pair played synergetic effect through regulating shared pathways, such as MAPK signaling pathway and arachidonic acid metabolism, etc. Besides, by metabolomics, the herb pair improved more metabolites (14) than a single herb (10 & 9) and has a stronger regulation effect on metabolites. Correspondingly, herb pair adjusted more metabolism pathways (5) than individual herb (4 & 4). Furthermore, the arachidonic acid metabolism was selected as crucial metabolism pathways by a joint analysis of 199 targets and 14 metabolites. The results showed that herb pair regulated arachidonic acid metabolism by synergetic reducing the level of arachidonic acid, and inhibiting the enzyme activity of prostaglandin-endoperoxide synthase 1 (PTGS1) and prostaglandin-endoperoxide synthase 2 (PTGS2). CONCLUSIONS: This work provided an integrated strategy for revealing the combination mechanisms of Chaihu-Baishao herb pair for treating depression, and also a rational way for clarifying the composition rules of TCM.