Identification of Andrographolide as a novel FABP4 inhibitor for osteoarthritis treatment.

BACKGROUND AND PURPOSE: Fatty acid binding protein 4 (FABP4) has been identified as a contributor to cartilage degradation in osteoarthritis (OA) patients, and inhibiting FABP4 using small molecules has emerged as a promising approach for developing OA drugs. Our previous research showed that Andrographis paniculata, a medicinal plant, strongly inhibits FABP4 activity. This led us to hypothesize that Andrographis paniculata ingredients might have protective effects on OA cartilage through FABP4 inhibition. METHODS: We analyzed scRNA-seq data from joint tissue of OA patients (GSE152805; GSE145286) using Scanpy 1.9.1 and Single Cell Portal. We conducted docking analysis of FABP4 inhibitors using Autodock Vina v.1.0.2. We evaluated the anti-FABP4 activity using a fluorescence displacement assay and measured the fatty acid oxidation (FAO) activity using the FAOBlue assay. We used H2DCF-DA to measure reactive oxygen species (ROS) levels. We studied signaling pathways using bulk RNA sequencing and western blot analysis in human C28/I2 chondrocytes. We evaluated anti-OA activity in monosodium iodoacetate (MIA)-induced rats. RESULTS: We identified Andrographolide (AP) as a novel FABP4 inhibitor. Bulk RNA-sequencing analysis revealed that FABP4 upregulated FAO and ROS in chondrocytes, which was inhibited by AP. ROS generation activated the NF-κB pathway, leading to overexpression of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), which is a responsible factor for cartilage degradation in OA patients. AP inhibited FABP4, thereby reducing the overexpression of ADAMTS4 by inhibiting the NF-κB pathway. In MIA rats, AP treatment reduced the overexpression of ADAMTS4, repaired cartilage and subchondral bone, and promoted cartilage regeneration. CONCLUSION: Our results indicate that the inhibition of FABP4 activity by AP explains the anti-OA properties of Andrographis paniculata by protecting against cartilage degradation in OA patients. Additionally, our findings suggest that AP may be a promising therapeutic agent for OA treatment due to its ability to alleviate cartilage damage and bone erosion.

Understanding Public Perspectives on Fracking in the United States using Social Media Big Data.

People's attitudes toward hydraulic fracturing (i.e., "fracking") to extract fossil fuels can be shaped by factors associated with socio-demographics, economic development, social equity and politics, environmental impacts, and fracking-related information obtainment. Existing research typically conducts surveys and interviews to study public attitudes toward fracking among a small group of individuals in a specific geographic area, where limited samples may introduce bias. Here, we compiled geo-referenced social media big data from Twitter during 2018-2019 for the entire United States to present a more holistic picture of people's attitudes toward fracking. We used a multiscale geographically weighted regression (MGWR) to investigate county-level relationships between the aforementioned factors and percentages of negative tweets concerning fracking. Results clearly depict spatial heterogeneity and varying scales of those associations. Counties with higher median household income, larger African American populations, and/or lower educational level are less likely to oppose fracking, and these associations show global stationarity in all contiguous U.S. counties. Eastern and Central U.S. counties with higher unemployment rate, counties east of the Great Plains with less fracking sites nearby, and Western and Gulf Coast region counties with higher health insurance enrollments are more likely to oppose fracking activities. These three variables show clear East-West geographical divides in influencing public perspective on fracking. In counties across the southern Great Plains, negative attitudes toward fracking are less often vocalized on Twitter as the share of Republican voters increases. These findings have implications for both predicting public perspectives and needed policy adjustments. The methodology can also be conveniently applied to investigate public perspectives on other controversial topics.

Astragaloside IV as a novel CXCR4 antagonist alleviates osteoarthritis in the knee of monosodium iodoacetate-induced rats.

BACKGROUND AND PURPOSE: C-X-C chemokine receptor type 4 (CXCR4) inhibition protects cartilage in osteoarthritis (OA) animal models. Therefore, CXCR4 has becoming a novel target for OA drug development. Since dietary and herbal supplements have been widely used for joint health, we hypothesized that some supplements exhibit protective effects on OA cartilage through inhibiting CXCR4 signaling. METHODS: The single-cell RNA sequencing data of OA patients (GSE152805) was re-analyzed by Scanpy 1.9.0. The docking screening of CXCR4 antagonists was conducted by Autodock Vina 1.2.0. The CXCR4 antagonistic activity was evaluated by calcium response in THP-1 cells. Signaling pathway study was conducted by bulk RNA sequencing and western blot analysis in human C28/I2 chondrocytes. The anti-OA activity was evaluated in monosodium iodoacetate (MIA)-induced rats. RESULTS: Astragaloside IV (ASN IV), the predominate phytochemical in Astragalus membranaceus, has been identified as a novel CXCR4 antagonist. ASN IV reduced CXCL12-induced ADAMTS4,5 overexpression in chondrocytes through blocking Akt signaling pathway. Furthermore, ASN IV administration significantly repaired the damaged cartilage and subchondral bone in MIA-induced rats. CONCLUSION: The blockade of CXCR4 signaling by ASN IV could explain anti-OA activities of Astragalus membranaceus by protection of cartilage degradation in OA patients. Since ASN IV as an antiviral has been approved by China National Medical Products Administration for testing in people, repurposing of ASN IV as a joint protective agent might be a promising strategy for OA drug development.

Overexpression of fucosyltransferase 8 reverses the inhibitory effect of high-dose dexamethasone on osteogenic response of MC3T3-E1 preosteoblasts.

BACKGROUND: Core fucosylation catalyzed by FUT8 is essential for TGF-ß binding to TGF-ß receptors. METHODS: Indirect TGF-ß1 binding assay was used to evaluate the ability of TGF-ß1 to bind to TGFBRs, Alizarin red and alkaline phosphatase staining were used to detect osteogenic differentiation and mineralization ability , western blot and quantitative RT-PCR were used to measure the differential expression of osteogenesis-related proteins and genes. Plasmid-mediated gain-of-function study. The scale of core fucosylation modification was detected by Lectin-blot and LCA laser confocal. RESULTS: Our results showed that compared with vehicle treatment, high-dose (10-6 and 10-5 M) dexamethasone significantly inhibited cell proliferation, osteogenic differentiation, and FUT8 mRNA expression while promoting mRNA expression of adipogenesis-related genes in MC3T3-E1 cells, suggesting that downregulation of FUT8 is involved in the inhibitory effect of high-dose dexamethasone on osteogenesis. Overexpression of FUT8 significantly promoted osteogenic differentiation and activated TGF-ß/Smad signaling in MC3T3-E1 cells in the presence of high-dose dexamethasone, suggesting that FUT8 reverses the inhibitory effect of high-dose dexamethasone on osteogenesis. In addition, lectin fluorescent staining and blotting showed that overexpression of FUT8 significantly reversed the inhibitory effects of high-dose dexamethasone on core fucosylation of TGFBR1 and TGFBR2. Furthermore, indirect TGF-ß1 binding assay showed that overexpression of FUT8 remarkably promoted TGF-ß1 binding to TGFBRs in MC3T3-E1 cells in the presence of high-dose dexamethasone. CONCLUSIONS: Taken together, these results suggest that overexpression of FUT8 facilitates counteracting the inhibitory effect of dexamethasone on TGF-ß signaling and osteogenesis.

[Concentration of Nitrate in Main Anoxic Stage and PHA, TP Metabolism for Nitrogen and Phosphorus Removal in Single Sludge System with Continuous Flow].

Based on test results and mass balance, PHA, TP metabolic regularity was revealed under different nitrate nitrogen concentrations in main anoxic stage [c(NO3)] for nitrogen and phosphorus removal in single sludge system with continuous flow, then the effectiveness of using c(NO3) as control parameter was proved from the perspective of the reaction mechanism. During experiment period, the influent COD, total nitrogen (TN), and total phosphorus (TP) concentrations were stabilized at (285.78±18.19), (58.13±3.79), and(7.14±0.51) mg·L-1, respectively. The experiment was carried out under the condition that the c(NO3) values were 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg·L-1 based on the feedback control structure using PLC automatic control system to control the nitrifying liquid flow with the water quality. The sludge load of COD was (0.253±0.071)kg·(kg·d)-1, the sludge load of TP in anaerobic stage was (0.006±0.001) kg·(kg·d)-1, the sludge load of TN in aerobic stage was (0.049±0.006) kg·(kg·d)-1, the hydraulic retention time (HRT) in bioreactor was 9h, the sludge recycle flow was 0.5, and the mixed liquor recycle was 1.0. The results showed that effect of c(NO3) value on PHA synthesis and storage rate in the ANS was conspicuous, and the percentage of PHA storage occupied 74% of COD removal when c(NO3) value was 2.5 mg·L-1.The impact of c(NO3) value on PHA degraded in the main anoxic stage was great, and the percentage of PHA degradation in the main anoxic stage occupied 55% of total PHA degradation when c(NO3) value was 2.5 mg·L-1. The phosphorus released in anaerobic stage changed along with increasing c(NO3), and the amount of phosphorus released obtained the maximum value 6.16 g·d-1 when c(NO3) value was 2.5 mg·L-1. In addition, under c(NO3) value of 2.5 mg·L-1, the amount of total phosphorus uptake and anoxic phosphorus uptake obtained the maximum values of 8.04 g·d-1 and 3.67 g·d-1, respectively. Then it was confirmed thatc(NO3) could serve as a run controlling parameter with the best value of 2.5 mg·L-1 from the perspective of PHA and TP metabolic mechanism.

[ORP in the Main Anoxic Stage as the Control Parameter for Nitrogen and Phosphorus Removal in the Single Sludge System with a Continuous Flow].

To optimize the performance of nitrogen and phosphorus removal, based on test results and mass balance, the feasibility of control for nitrogen and phosphorus removal in the single sludge system with a continuous flow using ORP in the main anoxic stage (ORPm) was investigated, meanwhile, the objective laws of conversion for nitrogen and phosphorus under different ORPm were expounded. During the experiments, nitration liquid internal circulation flow rate was controlled as the variable. The OPRm was controlled by PLC automatically, and the other operation parameters remained unchanged. The experiments tested six different ORPs in main anoxic stage affecting nitrogen and phosphorus removal, i.e., -143, -123, -105, -95, -72, and -57 mV. The ammonia concentration changed a little in effluent under the condition of different ORP.s, however, the TN and TP concentrations changed obviously. When the ORPm was controlled as -95 mV, the active sludge reached the maximal nitrogen and phosphorus removal with the continuous flow. According to mass balance calculation, when ORPm increased from -143 mV to -57 mV, (1) In the main anoxic stage, nitrate nitrogen reaction rates were 214. 40, 235. 16, 241. 16, 244. 02, 240. 90 and 233. 65 mg.h-1, respectively; the amount of total nitrogen conversions were 244. 92, 255. 85, 328. 04, 347. 45, 336. 42 and 320. 60 mg.h-1, respectively; both reaction rates reached the peak at the ORPm of -95 mV; (2)Phosphorus release rates in anaerobic stage were -214. 12, -228. 64, -259. 26, -264.54, -256.92 and -252.84 mg.h-1, respectively; total phosphorus absorption rates were 252. 15, 275.85, 332. 25, 338. 10, 336. 15 and 324. 30 mg.h-1, respectively, and phosphorus absorption rates were 30. 27, 62. 14, 124. 58, 154. 41, 150. 41 and 138. 30 mg.h-1, respectively, in the main anoxic stage; phosphorus absorption rates reached the peak when ORPm was -95 mV. The experiments revealed that ORPm could be used as the control parameter of nitrogen and phosphorus removal in single sludge system with a continuous flow.