Xuebijing injection and its bioactive components alleviate nephrotic syndrome by inhibiting podocyte inflammatory injury.

Xuebijing injection (XBJ) is widely used to treat nephrotic syndrome (NS) in clinic, but its bioactive components and therapeutic mechanism are still unclear. In this study, the bioactive components of XBJ were determined by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). The therapeutic effect of XBJ on NS was evaluated in BALB/c mice induced by adriamycin (ADR, 10 mg/kg) via a single tail vein. The protective effect of XBJ and its bioactive components on podocytes was demonstrated using mouse podocytes (MPC-5) induced by lipopolysaccharide (LPS, 4 µg/mL). The results show that 33 components of XBJ were identified. Furthermore, 12 bioactive components were detected in blood, including protocatechuic acid, salvianolic acid C, benzoyloxypaeoniflorin, danshensu, salvianolic acid A, salvianolic acid B, catechin, caffeic acid, galloylpaeoniflorin, oxypaeoniflorin, hydroxysafflor yellow A, rosmarinic acid. The relative content (%) of the bioactive components were 59.32, 16.01, 9.97, 9.73, 8.72, 8.31, 7.92, 6.54, 1.54, 1.30, 0.68 and 0.59 in this order. After XBJ treatment, the renal function, hyperlipidemia and renal pathological damage were improved in NS model mice. Moreover, the levels of nephrin and desmin which are functional proteins in podocytes were reversed, and the levels of pro-inflammatory factors were reduced by XBJ. Interestingly, protocatechuic acid and salvianolic acid C also showed good protective effects on podocyte function and reduced the level of inflammation in LPS-induced MPC-5. The study is the first time to elucidate the bioactive components of XBJ and its potential therapeutic mechanism for treating NS by protecting podocyte function.

Paeoniflorin suppresses kidney inflammation by regulating macrophage polarization via KLF4-mediated mitophagy.

BACKGROUND: Macrophages M1 polarization involved in the process of renal inflammatory injury, is a well-established hallmark of chronic kidney disease (CKD). Paeoniflorin (PF), a water-soluble monoterpene glycoside extracted from Paeonia lactiflora, revealed renal anti-inflammatory activities in our previous study. However, the potential molecular mechanism of PF on CKD remains unknown. PURPOSE: The present study aims to investigate the regulation of PF on macrophage polarization in CKD. METHODS: A CKD model was established by cationic bovine serum albumin and a murine macrophage cell line RAW264.7 induced with lipopolysaccharide (LPS) were used to clarify the underlying mechanisms of PF in CKD. RESULTS: Results showed that PF exhibited favorable protective effects on CKD model mice by promoting renal function, ameliorating renal pathological injury and podocyte damage. Furthermore, PF inhibited the infiltration of M1 macrophage marker CD68 and iNOS in kidney tissue, but increased the proportion of M2 macrophage marker CD206. In RAW264.7 cells stimulated with LPS, the levels of cytokines including IL-6, IL-1ß, TNF-α, MCP-1 were lessened under PF treatment, while the levels of Arg1, Fizz1, IL-10 and Ym-1 were augmented. These results indicated that PF promoted macrophage polarization from M1 to M2 in vivo and in vitro. More importantly, PF repaired the damaged mitochondria through increasing mitochondrial membrane potential and reducing ROS accumulation. The mitophagy-related proteins PINK1, Parkin, Bnip3, P62 and LC3 were up-regulated by PF, accompanied by the incremental expressions of Krüppel-like transcription factor 4 (KLF4). Moreover, the promotion of mitophagy and inhibition of M1 macrophage polarization owing to PF were reversed by mitophagy inhibitor Mdivi-1 or silencing KLF4. CONCLUSION: Overall, PF suppressed renal inflammation by promoting macrophage polarization from M1 to M2 and inducing mitophagy via regulating KLF4. It is expected to provide a new strategy for exploring the effects of PF in treating CKD.

First Report of Anthracnose on Chili Pepper Caused by Colletotrichum sojae in Hebei Province, China.

China is the largest chili pepper producing country, and Hebei Province stands out as the forth with planting area at about 1500 km2 in China. Pepper (Capsicum annuum L.) is susceptible to Colletotrichum spp. infection during its growth, which seriously affects production yield and quality. In September 2020, widespread anthracnose was observed on pepper in Hebei (115.48° N, 38.77° E), China. Necrotic lesions on pepper fruits were suborbcular, sunken, with acervuli arranged in the middle of lesion (e-Xtra 1A). To perform fungal isolation, small tissue with 0.3 cm2 in size at the symptomatic tissue margin was surface disinfested with 75% ethanol for 10 s, and 0.1% HgCl2 for 40 s, then washed three times with sterile ddH2O. Fragments were placed on potato dextrose agar (PDA) amended with 100 mg·L-1 chloramphenicol and incubated at 28 ºC under darkness for 4 days. One of the strains of Colletotrichum spp., named HQY157, was purified by single-spore isolation, then used for morphological characterization, phylogenetic analysis, and pathogenicity tests. Colonies presented light grey aerial mycelium, occasionally mixed with gray-black strips, and the reverse was similar to the surface on PDA (e-Xtra 1B). Conidia were smooth-walled, aseptate, straight with obtuse to slightly rounded ends, 17.3-28.5 × 3.1-7.4 µm (n=50) (e-Xtra 1C). For molecular identification, the internal transcribed spacer (ITS) region, partial sequences of actin (ACT), ß-tublin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS) were sequenced using the specific primers (Weir et al. 2012). Sequences were deposited in GenBank with the following accession numbers OM317600-OM317604. A Maximum-Likelihood phylogenetic tree was constructed, based on the concatenated sequences (ACT, CHS, GAPDH, TUB, and ITS) of HQY157 and other closely matching Colletotrichum species obtained from GenBank, by using MEGA-X. It showed that HQY157 was grouped with the C. sojae with bootstrap values of 100% (e-Xtra 2). To confirm the pathogenicity, surface-sterilized healthy pepper fruits and healthy fruits with wounds (deal with a sterile toothpick after surface-sterilized) were then inoculated with 2 µL of conidial suspension (106 conidia/mL). The fruits inoculated with 2 µL sterile distilled water were taken as negative controls. After inoculation, the fruits were kept in a plastic box with sterilized filter paper moistened with sterilized water, and maintained at 25°C in the dark. The experiment was repeated three times. Anthracnose symptoms were observed 7 days after inoculation on the wounded pepper fruits, whereas the unwounded and negative control fruits remained symptomless (e-Xtra 1D). Colletotrichum sojae was re-isolated from the infected pepper fruits and identified by morphological and molecular analysis, fulfilling Koch's postulates. Colletotrichum sojae occurs mainly on Fabaceae plants such as Glycine max, Medicago sativa, Phaseolus vulgaris, and Vigna unguiculata (Damm et al. 2019, Talhinhas and Baroncelli, 2021), and Panax quinquefolium (Guan et al. 2021). To our knowledge, this is the first report of C. sojae causing anthracnose on pepper in China. This study provided crucial information for epidemiologic studies and appropriate control strategies for this chili pepper disease.

Danshen injection induces autophagy in podocytes to alleviate nephrotic syndrome via the PI3K/AKT/mTOR pathway.

BACKGROUND: Danshen injection (DSI) is an agent extracted from the Salvia miltiorrhiza Bunge, a natural drug commonly used to alleviate kidney diseases. However, the material basis and therapeutic effects of DSI on nephrotic syndrome (NS) remain unclear. PURPOSE: To investigate the material basis of DSI and the therapeutic effects and underlying mechanisms of NS. METHODS: NS models were established using adriamycin-induced BALB/c mice and lipopolysaccharide-induced mouse podocytes (MPC-5). Following DSI and prednisone administration, kidney coefficients, 24 h urine protein, blood urea nitrogen, and serum creatinine levels were tested. Histomorphology was observed by periodic acid-Schiff staining and hematoxylin and eosin staining of the kidney sections. The glomerular basement membrane and autophagosomes of the kidneys were observed using transmission electron microscopy. Nephrin and desmin levels in the glomeruli were tested using immunohistochemistry. The viability of MPC-5 cells was tested using cell counting kit-8 after chloroquine and rapamycin administration in combination with DSI. The in vivo and in vitro protein levels of phosphatidylinositol 3-kinase (PI3K), AKT, phosphorylated AKT (Ser473), mammalian target of rapamycin (mTOR), microtubule-associated protein light chain 3 (LC3), beclin1, cleaved caspase-3, and caspase-3 were detected using western blotting. RESULTS: Our results showed that DSI contained nine main components: caffeic acid, danshensu, lithospermic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, salvianolic acid C, salvianolic acid D, and 3, 4-Dihydroxybenzaldehyde. In in vivo studies, the NS mice showed renal function and pathological impairment. Podocytes were damaged, with decreased levels of autophagy and apoptosis, accompanied by inhibition of the PI3K/AKT/mTOR signaling. DSI administration resulted in improved renal function and pathology in NS mice, with the activation of autophagy and PI3K/AKT/mTOR signaling in the kidneys. Additionally, podocytes were less damaged and intracellular autophagosomes were markedly increased. In vitro studies have shown that DSI activated MPC-5 autophagy and reduced apoptosis via the PI3K/AKT/mTOR pathway. CONCLUSION: Collectively, this study demonstrated that DSI activated podocyte autophagy and reduced apoptosis via the PI3K/AKT/mTOR signaling, ultimately attenuating NS. Our study clarified the main components of DSI and elucidated its therapeutic effects and potential mechanisms for NS, providing new targets and agents for the clinical treatment of NS.

TaNAC35 acts as a negative regulator for leaf rust resistance in a compatible interaction between common wheat and Puccinia triticina.

NAC (NAM, AFAT1/2, and CUC2) transcription factors play important roles in plant growth and in resistance to abiotic and biotic stresses. Here, we show that the TaNAC35 gene negatively regulates leaf rust resistance in the wheat line Thatcher + Lr14b (TcLr14b) when challenged with a virulent isolate of Puccinia triticina (Pt). The TaNAC35 gene was cloned from this line, and blastp results showed that its open reading frame (ORF) was 96.16% identical to the NAC35-like sequence reported from Aegilops tauschii, and that it encoded a protein with 387 amino acids (aa) including a conserved NAM domain with 145 aa at the N-terminal alongside the transcriptional activation domain with 220 aa in the C-terminal. Yeast-one-hybrid analysis proved that the C-terminal of the TaNAC35 protein was responsible for transcriptional activation. A 250-bp fragment from the 3'-end of this target gene was introduced to a BSMV-VIGS vector and used to infect the wheat line Thatcher + Lr14b (TcLr14b). The BSMV-VIGS/TaNAC35-infected plant material showed enhanced resistance (infection type "1") to Pt pathotype THTT, which was fully virulent (infection type "4") on BSMV-VIGS only infected TcLr14b plants. Histological studies showed that inhibition of TaNAC35 reduced the formation of haustorial mother cells (HMC) and mycelial growth, implying that the TaNAC35 gene plays a negative role in the response of TcLr14b to Pt pathotype THTT. These results provide molecular insight into the interaction between Pt and its wheat host, and identify a potential target for engineering resistance in wheat to this damaging pathogen.

A sustainable process for the recovery of volatile constituents from Gracilaria lemaneiformis in agar production and evaluation of their antioxidant activities.

BACKGROUND: Gracilaria lemaneiformis is a common red alga used as a raw material source for the agar industry. Its extract is rich in natural volatile constituents (VCs) having antioxidant activities. Herein, a sustainable method was used to recover VCs from the alga. The chemical composition of VCs present in the n-hexane fraction was analyzed by gas chromatography-mass spectroscopy (GC-MS) and the antioxidant potential was measured using a series of in vitro biochemical assays, including DPPH, hydroxyl, and superoxide radical scavenging assays. RESULTS: The recovery yield of the VCs was 0.823 wt% of the dry mass of G. lemaneiformis. A total of 25 VCs were successfully identified, comprising approximately 99.94% of the total volume. The major component was n-hexadecanoic acid (38.57%), followed by oleic acid (25.48%), arachidonic acid (12.84%), and tetradecanoic acid (2.52%). In addition, The VCs displayed strong free radical scavenging activity in the DPPH (IC50 = 21.56 mg/L), hydroxyl (IC50 = 18.34 mg/L), and superoxide (IC50 = 391.12 mg/L) radical scavenging assays. The antioxidant activities of the VCs exhibited a dose-dependence at concentrations ranging from 5 to 200 mg/L. CONCLUSION: The results indicated that the sustainable process improved the agar quality and that the extract contained many natural VCs with antioxidant activities, which have the potential to be used in functional food and cosmetics instead of as a discarded byproduct of the agar industry.

Optimization of decolorization process in agar production from Gracilaria lemaneiformis and evaluation of antioxidant activities of the extract rich in natural pigments.

Gracilaria lemaneiformis is mainly used as a raw material source for agar industry, and its extract is rich in natural pigments with antioxidant activities. In this study, a solvent reflux extraction method for decolorization of G. lemaneiformis has been developed in agar production. The extraction conditions were optimized as follows: solvent-to-material ratio, 50:1; ethanol concentration, 70%; number of extractions, 3; extraction time, 0.5 h, under which the total antioxidant yield of the extract reached 2.89 ± 0.88 mg/g dried seaweeds. Their IC50 values of DPPH radical scavenging activity, hydroxyl radical scavenging activity and superoxide anion scavenging activity were 21.91 ± 1.8 mg/L, 40.59 ± 1.5 mg/L and 160.87 ± 2.8 mg/L, respectively. Further isolation and spectroscopic analysis of natural pigments suggested the strong antioxidant capacities were attributed to chlorophyll derivatives. The results indicate that the decolorization process was able to improve the agar quality, and the extract containing lots of natural pigments had antioxidant activities which may be used in functional food and cosmetics.

[Arthropod communities in jujube-grain intercropping system: composition and time series dynamics].

In order to reveal the correlations between the arthropod communities in two sub-systems (jujube yard and grain field) of jujube-grain intercropping system, an investigation was conducted on the composition and time series dynamics of the communities in the two sub-systems. A total of 14 936 arthropod individuals belonging to 14 orders and 96 families were found, among which, 5992 individuals of 14 orders and 82 families were in jujube yard and 8971 of 14 orders and 80 families were in grain field, and some differences were observed in the arthropod community structure between the two sub-systems. The time series dynamics of the arthropod communities could be divided into four periods, according to the optimal cut-apart of time series, i.e., decline period in grain field sub-system, species number increase period in whole system, steady period in whole system, and decline period in whole system. The primary natural enemies in the system showed a characteristic of transferring between the two sub-systems in the four periods, and the parasitical natural enemies in a sub-system could impact the pest insects in another sub-system. The species transferring and mutual effect of the arthropod communities between the two sub-systems could benefit the stability of the jujube-grain intercropping system, and the capability of the system in controlling pest insects.

[Community diversity of soil arthropods in forest-steppe ecotone].

An investigation on the community diversity of soil arthropods in the forest-steppe ecotone of north Hebei Province was conducted. A total of 10 420 individuals of soil arthropods were collected, which belonged to 25 groups, 6 classes and 24 orders. Acarina and Collembola were the dominant orders, and there were 8 groups of frequent orders and 15 groups of rare orders. The diversity index (H'), DG index, and evenness of soil arthropod community were relatively higher in forest zone, but lower in meadow-steppe zone. Soil pH had a higher degree of interconnection with the numbers of soil arthropod groups, while soil temperature and moisture content had a higher degree of interconnection with the numbers of soil arthropod individuals.