Utilizing network pharmacology and experimental validation to investigate the underlying mechanism of Denglao Qingguan decoction against HCoV-229E.

Background: Denglao Qingguan decoction (DLQGD) has been extensively utilized for the treatment of colds, demonstrating significant therapeutic efficacy. Human Coronavirus 229E (HCoV-229E) is considered a crucial etiological agent of influenza. However, the specific impact and underlying mechanisms of DLQGD on HCoV-229E remain poorly understood. Methods: Active ingredients and targets information of DLQGD were collected from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), literature search, and Swiss ADEM database. The Genecard database was used to collect HCoV-229E related targets. We built an "ingredient-target network" through Cytoscape. Protein - Protein interaction (PPI) networks were mapped using the String database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were enriched using the DAVID database. Then, we used molecular docking techniques to verify the binding activity between the core compounds and the core gene targets. Finally, in vitro experiments were conducted to validate DLQGD's antiviral activity against HCoV-229E and assess its anti-inflammatory effects. Results: In total, we identified 227 active components in DLQGD. 18 key targets involved in its activity against HCoV-229E. Notably, the core active ingredients including quercetin, luteolin, kaempferol, ß-sitosterol, and apigenin, and the core therapeutic targets were CXCL8, RELA, MAPK14, NFKB1, and CXCL10, all associated with HCoV-229E. KEGG enrichment results included IL-17 signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and so on. The core active ingredients and the core therapeutic targets and Human Aminopeptidase N (ANPEP) all showed good binding activity by molecular docking verification. In vitro, DLQGD exhibited anti-HCoV-229E activity and anti-inflammatory effects. Conclusion: Our study suggests that DLQGD has both effects of anti-HCoV-229E and anti-inflammatory. The core active ingredients (quercetin, luteolin, kaempferol, ß-sitosterol, apigenin) and the core therapeutic targets (CXCL8, RELA, MAPK14, NFKB1, CXCL10) may play key roles in the pharmacological action of DLQGD against HCoV-229E.

Low-level laser therapy induces human umbilical vascular endothelial cell proliferation, migration and tube formation through activating the PI3K/Akt signaling pathway.

Low-level laser therapy (LLLT) has been recognized as a light therapy that may be used for tissue regeneration, inflammation reduction, and pain relief. We intended to evaluate the effects of LLLT on the proliferation, migration, and tube formation of HUVECs as well as their related mechanisms. HUVECs were exposed to laser irradiation under different laser parameters (irradiation dose, interval and power intensity) in order to choose the optimal parameters, which were determined by the increase in proliferation of HUVECs as follows: irradiation dose of 4.0 J/m2, interval time of 12 h and 6 times in total. The HUVEC proliferation, migration, and tube formation, and levels of angiogenesis-related genes (HIF-1α, eNOS and VEGFA) were examined following LLLT. As suggested by the obtained data, LLLT (1.0, 2.0 and 4.0 J/m2) increased the HUVEC proliferation, migration, and tube formation in dose-and time-dependent manner, accompanied with increases in the levels of HIF-1α, eNOS, and VEGFA. Furthermore, the regulatory mechanism regarding the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was explored, phosphorylation levels of PI3K and Akt proteins were assessed by Western blot assay, which showed the enhancement of phosphorylation of PI3K, Akt, and mTOR by LLLT. The inhibitor for the PI3K/Akt axis was used to verify the involvement of PI3K/Akt signaling pathway. The obtained results suggested that the inhibition of the PI3K/Akt signaling pathway attenuated the effects of LLLT on proliferation, migration, and angiogenesis of HUVECs. In conclusion, LLLT promotes the proliferation, migration, and angiogenesis of HUVECs via activation of the PI3K/Akt signaling pathway.

Ginsenoside 20(S)-protopanaxadiol inhibits triple-negative breast cancer metastasis in vivo by targeting EGFR-mediated MAPK pathway.

Metastasis is the primary cause of cancer recurrence and cancer related mortality in triple-negative breast cancer (TNBC). EGFR overexpression is in 50-75% TNBC and EGFR-mediated signaling has potential as an attractive therapeutic target in some specific subtypes of breast cancer due to its significant association with tumor metastasis and poor prognosis. Therefore, identification of promising therapeutic strategies targeting EGFR with higher specificity toward cancer metastasis is urgently needed. 20(S)-protopanaxadiol (PPD), one of the major active metabolites from Panax ginseng, has been widely reported to possess pleiotropic anticancer activities in various cancers. In this study, we investigated the effect of PPD against cancer metastasis and the related molecular mechanisms in TNBC in vitro and in vivo. PPD (>30 µM) suppressed cell proliferation by arresting cell cycle in G0/1 phase and triggering cells apoptosis as shown by cell viability assay, flow cytometry analysis and colony formation assay, whereas lower dose of PPD (<20 µM) decreased metastatic potential of MDA-MB-231 and SUM159 cells through direct inhibition of cell adhesion, motility and invasiveness. In TNBC xenograft and syngeneic models, PPD treatment markedly decreased tumor growth and lung metastasis. PPD reversed epithelial-mesenchymal transition (EMT), decreased the expression and activity of matrix metalloproteinases (MMPs) while increased the expression of tissue inhibitors of metalloproteinases (TIMPs) as shown by Western blot and gelatin zymography. Cell signaling pathways that control the expression or activation of these processes were investigated by Western blot and ELISA assay. PPD treatment reduced the phosphorylation of EGFR and down-regulated the activation ERK1/2, p38 and JNK signaling, which was further validated by using the agonists or inhibitors of EGFR and MAP kinases family. Collectively, these findings suggest that PPD holds therapeutic potential against the tumor metastasis of TNBC via targeting EGFR-mediated MAPK pathway.

[Study on main pharmacodynamics and underlying mechanisms of 999 Ganmaoling].

To observe synergistic effects of 999 Ganmaoling (GML) and its Chinese/Western materia medica (CMM and WMM) on pharmacodynamic action and to study underlying mechanisms, their anti-inflammatory, antipyretic effects were compared by assaying the increased capillary permeability induced by glacial acetic acid in mice, ear swelling induced by Xylene in mice, non-specific pleurisy induced by carrageenan in rats, and yeast induced fever in rats. Crystal violet (CV) and microbial activity (XTT) assay were used to evaluate the inhibition of GML and its CMM and WMM on KPN biofilm formation, and scanning electron microscopy (SEM) was applied for observing KPN biofilm morphology changes. The results showed that compared with control group, GML could reduce exudation amount of Evans-Blue and the degree of Ear swelling significantly, and CMM and WMM have no significant effects. The concentration of TNF-α and IL-1ß of rat pleural effusion in GML, CMM and WMM group decreased significantly. The concentration of TNF-α, IL-1ß and IL-8 in GML group, TNF-α, IL-8 in WMM group and IL-8 in CMM in rats serum decreased significantly. The body temperature in rats decreased significantly in GML and WMM group after 4-8 h of administration. CMM group showed no significant difference in rat body temperature compare with control. Compared with control group, GML (55-13.75 g•L⁻¹) could inhibit KPN biofilm formation and reduce number of viable cells in the KPN biofilm. CMM (45-22.5 g•L⁻¹) and WMM (10 g•L⁻¹) could also inhibit KPN biofilm formation and reduce number of viable cells (P<0.01). Result of SEM also showed that GML (55 g•L⁻¹) and its CMM (45 g•L⁻¹) and WMM (10 g•L⁻¹) could interfere the bacterial arrangement of KPN biofilm and extracellular matrix. GML and its CMM & WMM could inhibit the formation of KPN biofilm, CMM & WMM in GML showed synergism and complementation in inhibit KPN biofilm. Results showed that GML had obvious anti-inflammatory and antipyretic effects and could destruct KPN mature biofilm. WMM and CMM showed obvious synergistic effect against inflammation and inhibition of KPN biofilm formation and reduction of number of viable cells but no same effects against fever.

Pharmacokinetics of 8-O-acetylharpagide and harpagide after oral administration of Ajuga decumbens Thunb extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga decumbens Thunb is a medicinal plant native to China popularly used to treat chronic pelvic inflammation and hysteromyoma. Its main bioactive components are iridoid glycosides, such as 8-O-acetylharpagide and harpagide that had presented antibacterial, anti-inflammatory, and antiviral activities. AIM OF THE STUDY: To establish a sensitive LC-MS/MS method and compare the pharmacokinetics of 8-O-acetylharpagide and harpagide in rats after oral administration of their pure forms and from compounds obtained from Ajuga decumbens extract. MATERIALS AND METHODS: Rats received orally 15 mg/kg (equivalent of 6 mg/kg 8-O-acetylharpagide and 1.5mg/kg harpagide), 30 mg/kg and 60 mg/kg of Ajuga decumbens Thunb extract and were compared to animals that received 12 mg/kg of 8-O-acetylharpagide or 3mg/kg of harpagide p.o. Concentrations of 8-O-acetylharpagide and harpagide in plasma were determined by LC-MS/MS method at different time points and all pharmacokinetic parameters were estimated by non-compartmental analysis. RESULTS: Results showed that the iridoid glycosides were quickly absorbed by oral route and showed a dose-dependence profile. Pharmacokinetic parameters of both glycosides were essentially the same except Tmax when dosed as the extract or pure forms. CONCLUSION: 8-O-acetylharpagide was metabolized to harpagide, which affected the pharmacokinetic profiles of harpagide when dosed as the extract. This pharmacokinetic study seems to be useful for a further clinical study of Ajuga decumbens Thunb extract.

[Study on mercury accumulation in rats induced by badu shengji san].

OBJECTIVE: To study the mercury accumulation in injured skin rats induced by Badu Shengji San (BDSJS), a traditional Chinese medicine preparation for external use. METHOD: Injured skin rats were treated with BDSJS for consecutively 4 weeks. During the 4 weeks and the following 4 weeks after the drug withdrawal, samples were collected for determining mercury contents in blood, urine and kidney, with urinary N-acetyl-beta-D-glucosaminidase(NAG) and beta2-microglobulin (beta2-MG) as indicators of renal toxicity and serum biochemical indicators of hepatic and renal functions. Additionally, activated partial thromboplastin time (APTT), prothrombin time (PT) and kidney and renal pathological changes were also observed. RESULT: Compared to injured skin rats, mercury contents of blood, urine and kidney were increased significantly in low, middle and high-dose BDSJS groups administered for consecutive 4 weeks. The levels of mercury showed decreases in urine (89%, 78%, 93%) and kidney (55%, 51%, 57%), and blood mercury concentration recovered to the normal range in low, middle and high-dose BDSJS groups after the drug withdrawal for 4 weeks. Kidney coefficient and beta2-MG were remarkably increased and renal tubular epithelial cell swelling could be found in the high-dose group, and kidney coefficient, beta2-MG and renal morphology basically recovered to the normal levels after the drug withdrawal for 4 weeks. CONCLUSION: The administration of BDSJS for consecutively 4 weeks can cause mercury accumulation in blood and mainly in kidney. Once the accumulated mercury concentration of kidney reaches a certain level, renal tubular epithelial cells would be injured. 1.1 mg x cm(-2) of BDSJS is proved to be safe and 2.2 mg x cm(-2) can cause mild but reversible injury in the function of kidney which can be recovered after drug withdrawal for 4 weeks.

[Comparison of effects of badu shengji san on rats with different injured skins].

OBJECTIVE: To compare the effects of Badu Shengji San (BDSJS) on rats with different injured skins. METHOD: The injured and ulcerous skin rat model was established to observe the renal injury induced by BDSJS, a mercury-containing external preparation of Chinese medicine, with urinary N-acetyl-beta-D-glucosaminidase (NAG) and retinol binding protein (RBP) as indicators of renal toxicity. RESULT: Compared to injured skin rats with the same dose, both of high and low-dose ulcerous skin groups showed obvious increase in urinary RBP and kidney coefficients, significant pathomorphological changes in renal tubules and notable epithelial cytopathic effects. In terms of NAG, the high-dose ulcerous skin group saw no significant increase, but the low-dose group recorded sharp rise. CONCLUSION: The renal toxicity induced by BDSJS in ulcerous skin rats was more toxic than that in injured skin ones.

[Comparative study on external use of mercury-containing preparation badu shengji san in sensitive monitoring indicators of induced early renal injury].

OBJECTIVE: To compare the sensitivity of early renal injury induced by mercury-containing medicine in rats, including urinary N-acetyl-beta-D-glucosdminidase (NAG), beta2-microglobulin (beta2-MG), retinol binding protein (RBP) and clusterin (CLU). METHOD: Badu Shengji San(BDSJS), a mercury-containing preparation of traditional Chinese medicine, was adopted as the mercury contact drug. The lowest effective toxic dose was used to observe its effect on serum creatinine (SCr), blood urea nitrogen (BUN), and such early renal injury indicators as NAG, RBP, beta2-MG and CLU and compare the sensitivity of tested indicators. RESULT: Compared to the broken skin group, groups with administration of 60 and 120 mg x kg(-1) doses of BDSJS showed no obvious difference in SCr and BUN when kidney indicators is remarkably increased and obvious pathological changes were found in kidney tubules but with significant increase in the urinary level of CLU and the levels of NAG and RBP. H&E staining of renal tubule showed that exposure of 30 mg x kg(-1) BDSJS had no significant morphological changes, but at the same concentrations, the level of RBP was markedly increased. Urinary beta2-MG levels were markedly decreased in BDSJS 30, 60 mg x kg(-1) group rats, whereas 120 mg x kg(-1) dose group showed no obvious change in urinary beta2-MG levels. CONCLUSION: Urinary RBP, NAG and CLU were more sensitive than SCr and BUN as indicators for early renal injury in the order of RBP > NAG > CLU, and urinary RBP, NAG would increase earlier than beta2-MG.

[Effect of zhuhong ointment on renal antioxidant capability in skin ulcer model rats].

OBJECTIVE: To study the effect of repeated administration of Zhuhong ointment on renal antioxidant capability of ulcerous skin in rats, in order to further discuss the mechanism of mercury contained in Zhuhong ointment on the antioxidant capability of kidney in skin ulcer rats. METHOD: Eighty SD rats were randomly divided into eight groups: Zhuhong ointment A, B, C, D, E (1.219, 0.609, 0.305, 0.152, 0.76 g x kg(-1)) groups, the vaseline group, the ulcer model group and the impairment control group. The levels of NAG and RBP of toxicity for early kidney tubular injury and T-AOC, SOD, GSH-PX and GSH in kidney were determined after consecutive administration for 14 days. RESULT: Compared with ulcer model group, the levels of RBP in groups A, B, C and D increased, while the levels of NAG increased only in the group A. The level of T-AOC increased in groups A, B and C. The level of T-SOD increased in the group E, while it dropped down greatly in the group A. The level of GSH-PX increased in groups A, B and C. The content of GSH increased in every dose groups. CONCLUSION: Antioxidant capacity in rats can be increased in a reasonable dose of Zhuhong ointment, but some antioxidant activity can be notably inhibited by with the increase of dose.

[Effects of zhuhong ointment on mercury cumulation and renal organization modality in skin-impaired model rat].

OBJECTIVE: To study the effects of Zhuhong ointment on accumulation in the body of mercury and the pathological morphology changes of kidney, via the measurement of related indicators of the skin-impaired model rat. METHOD: Eighty-eight SD rats were randomly divided into the impairment control group, and high-, middle-, low-dose Zhuhong ointment groups. Each group was treated by corresponding methods for 4 weeks, and recovering for 4 weeks. Urinary potein (PRO), pH, Beta N-acetyl aminoglycosidase enzymes (NAG) and beta2-microglobulin (beta2-MG) contents in urine were taken as monitoring indexes, blood urea nitrogen (BUN) and serum creatinine (SCr) in blood and the levels of mercury in urine, blood and kidney were tested, and the pathological morphology changes of kidney were observed. RESULT: After treatment for 4 weeks, compared with impairment control group, the levels of mercury in urine, blood and kidney in every dose group increased significantly (P < 0.01). And the relation exists between toxicity and dose on Zhuhong ointment. After recovery for 4 weeks, the levels of mercury in urine and blood in every dose group restore normal, while the level of mercury in kidney in high- dose group still increased (P < 0.01). The level of NAG increased only in high-dose group. There was no significant difference in NAG contents between Zhuhong ointment groups and the impairment control group (P < 0.05). CONCLUSION: Excess using Zhuhong ointment repeatedly may lead to accumulation of mercury and pathological morphology changes of kidney. So the levels of mercury in the body and related indicators of renal functions should be tested in clinical when long-term using Zhuhong ointment.

[Effect of chlorogenic acid on degranulation in mast cell RBL-2H3].

OBJECTIVE: To provide evidences for evaluating the role of chlorogenic acid (CA) on the adverse reaction of traditional Chinese medicine injection and promoting clinical rational usage of CA, the effect of CA and chlorogenic acid-HSA(CA-HSA) on the degranulation in mast cell RBL-2H3 were compared and the allergenic effect and its mechanism were investigated. METHOD: The unsensitized and sensitized RBL-2H3 cells were used. The releasing rate of histamine and beta-hexosaminidase was detected by colormetric assays. The degranulating rate was detected by neutral red staining and Annexin V positive cell rate was detected by flow cytometry. RESULT: CA and CA-HSA could not induce degranulation in unsensitized RBL-2H3 cells. CA and CA-HSA could significantly increase the release of histamine and beta-hexosaminidase, degranulating rate and Annexin V positive cell rate. CONCLUSION: CA has strong allergenicity after combination with serum proteins. As an active ingredient of Shuanghuanglian injection, CA is a kind of possible allergen which caused hypersensitivity reactions induced by Shuanghuanglian injection.

[Correlation between antimetastatic action of Ajuga decumbens and expression of MMPs and TIMPs].

OBJECTIVE: To investigate the anticancer and anti-metastatic effect of Ajuga decumbens extraction (HBG) on breast cancer and to clarify the effect of HBG on MMPs and TIMPs. METHOD: The antitumor and antimetastic effect of HBG was determined using orthotopic 4T1 breast cancer mouse model. Western blot analysis was employed to detect the expression of associated proteins in breast cancer metastasis. RESULT: Administration with 50-200 mg x kg(-1) doses of HBG significantly reduced the tumor weight, tumor volume and numbers of lung tumor nodules in a dose-dependent manner. Tumor metastasis correlated proteins were altered following HBG treatment, MMP-2 and MMP-9 were down-regulated while TIMP-1 and TIMP-2 were up-regulated. CONCLUSION: HBG showed anticancer and antimetastatic effect towards breast cancer through regulating the expression of MMPs and TIMPs. These data sustain our contention that HBG might be used as a potential therapeutic agent.