Artemisia judaica ethanolic extract (ArEx) as well as its phenolic fraction (ArPh), and terpenoid fraction (ArT) against Cryptosporidium parvum (C. parvum) oocysts.This study investigates the toxic activity of in vitro antioxidant activity of ArEx, ArPh, and ArT; evaluation of ArEx, ArPh, and ArT toxic activity against C. parvum oocysts using MTT assay; parasitological analysis on ArPh-treated C. parvum oocysts and comet assay were performed both in vitro and in vivo (infectivity).Over a 4 months period, estimation of the total phenolic (TPC), total flavonoids (TFC), and total terpenoids contents (TTC) in ArEx; investigation of the in vitro antioxidant activity, the ArPh exhibited the highest antioxidant activity compared to the ArEx and ArT. The ArPh showed promising free radical scavenging activity of DPPH and ABTS•+ with IC50 values of 47.27 ± 1.86 μg/mL and 66.89 ± 1.94 μg/mL, respectively. Moreover, the FRAP of ArPh was 2.97 ± 0.65 mMol Fe+2/g while its TAC was 46.23 ± 3.15 mg GAE/g. The ArPh demonstrated toxic activity against C. parvum oocysts with a potent IC50 value of 31.6 μg/mL compared to ArT (promising) and ArEx (non-effective). ArPh parasitological analysis demonstrated MIC90 at 1000 μg/ml and effective oocysts destruction on count and morphology. ArPh fragmented oocysts nuclear DNA in comet assay. Beginning at 200 μg/mL, ArPh-treated oocysts did not infect mice.The ArEx TPC, TFC, and TTC was 52.6 ± 3.1 mgGAE/g, 64.5 ± 3.1 mg QE/g, and 9.5 ± 1.1 mg Linol/g, respectively. Regarding the phytochemical C. parvum infection, the phenolic fraction of A. judaica L. shows promise as an adjuvant therapy or as a source of potentially useful lead structures for drug discovery.To combat  Around 905 waterborne and 25 foodborne outbreaks are reported by Cryptosporidium sp. worldwide (The waterborne pathogen orldwide .Cryptosporidium sp. have been identified (Cryptosporidium hominis (C. hominis) and Cryptosporidium parvum (C. parvum) being the two most notable species  is considered one of the most prominent and widely distributed plant families . In Egyprtemisia  which isdaica L. . Plants daica L. . These bdaica L. .A. judaica (Asteraceae) is a perennial aromatic shrub, wildly distributed in Egypt, particularly in Sinai, Gabal Elba, Mediterranean, and Red Sea regions  in vitro experiments, (b) acting mechanism, (c) in vivo bioassay to demonstrate the infectivity of the treated oocysts. regions  and has research . Due to 2.2.1.Cryptosporidium sp. oocysts were collected from a farm with a known history of cryptosporidiosis in its newborn calves   for subsequent oocysts processing at 4°C.Approximately 2 mL of the watery fecal specimens were immediately transferred into 2-mL Eppendorf tubes and stored at minus 20°C for further molecular identification and confirmation. The remaining part of fecal samples containing oocysts were filtered through four layers of gauze and stored in potassium dichromate   (0.324 g KH pH 7.4) .Each sample’s 3 mL final volume of concentrated sediment was mixed in potassium dichromate at 4°C until the purification process commenced.2.1.1.To purify oocysts from previously concentrated fecal samples, discontinuous gradient sucrose flotation was applied . On a diSolution A  and solution B  were prepared by diluting a stock solution of modified Sheather’s sugar   with PBS. In a 15-mL conical tube, 5 mL of solution A was poured first, followed by 5 mL of solution B.Cryptosporidium sp. Purified oocysts were stored in a solution of 2.5% potassium dichromate 1:4 v/v at 4°C for use in subsequent research within 4 months period.Oocysts were previously condensed into a suspension (3 mL), which was then carefully poured over the gradients. For 30 min, the gradient tubes were centrifuged at 1200 g . After the second layer from the top had been separated, it was washed in saline and finally PBS. Decanting the supernatant allowed the sediments (about 1 mL/tube) to be examined in wet mount (WM) and mZN stain for oocysts of 2.1.2.Cryptosporidium used were determined using molecular identification. Frozen fecal samples were thawed in cold PBS and filtered through two layers of gauze. PBS washed and centrifuged at least three times. The Qiagen DNA stool-mini kit were used to extract DNA from 200 μL of collected silt, per manufacturer directions . The Qiagen method was modified by raising the water bath temperature to 90°C and storing a 100 μL DNA aliquot at – 20°C for PCR amplification.The species/genotypes of Nested PCR primers CPr I (5’-AAACCCCTTTACAAGTATCAATTGGA-3′) and CPr II (5’-TTCCTATGTCTGGACCTGGTGAGTT-3′), CPr III (5’-TGCTTAAAGCAGGCATATGCCTTGAA-3′) and CPr IV (5’-AACCTCCAATCTCTAGTTGGCATAGT-3′) were employed to amplify the small subunit ribosomal RNA gene of the four calves’ fecal samples . With pr2.2.2.2.1.A. judaica aerial parts  was prepared by macerating 0.2 Kg of the plant twice with 95% ethyl alcohol  at 25°C. The combined extracts were vacuum - concentrated to obtain A. judaica crude extract (17 g), which was then kept at 4°C.Aerial parts of ntioned, , the cru2.2.2.A. judaica was prepared according to a previously described method (A. judaica extract (5 g) was extracted with chloroform (3 × 25 mL). The chloroform extracts were filtered, combined then concentrated under reduced pressure to afford 0.45 g of the terpenoid fraction of A. judaica (ArT).The terpenoid fraction of d method  with fewA. judaica was prepared according to a previously mentioned method (A. judaica crude extract was suspended in aqueous solution of 5% Na2CO3 before being extracted chloroform (3 × 50 mL). The aqueous extract lift was then acidified with HCl and fractionated successively with chloroform, ethyl acetate, and n-butanol. These obtained extracts were combined and vacuum dried to yield approximately 8.5 g of the phenolic fraction of A. judaica (ArPh).The phenolic fraction of d method . An amou2.2.3.Total phenolics content (TPC) in ArEx was quantified spectrophotometrically by the Folin–Ciocalteu assay as previously described , 2022 wh3 method, as previously stated  was quantified by AlCly stated . Quercet2.2.4.A. judaica was determined as linalool equivalents per gram of dry extract (mg Linol/g).The total terpenoids content (TTC) in ArEx was analyzed quantitatively using Salkowski test described by 2.3.2.3.1.2.3.1.1.The ArEx as well as its ArPh and ArT fractions were investigated for their scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical using the procedure previously detailed , 2022. AIn triplicate, the absorbances at λ 515 nm were recorded against blank by Milton Roy Spectronic 1,201 UV/Vis Spectrophotometer . The DPPH radical quenching the sample of was calculated applying this equation: PI = [{(AC − AT)/AC} × 100].50 of DPPH was estimated from the dose/response curve created by Graphpad Prism software .Where PI = percent inhibition, AC = control absorbance and AT = absorbance of DPPH + sample mixture. The IC2.3.1.2.•+ radical cation  was assayed using the procedure described in detail by The ability of ArEx, ArT, and ArPh to capture and neutralize ABTS•+ neutralizing activity of a sample was estimated using the following equation: PI = 100 [(Control – Sample)/Control].The ABTS50 was obtained from the dose/response curve obtained by Graphpad Prism software .The IC2.3.1.3.The reducing power of ArEx, ArPh, and ArT was estimated spectrophotometrically by FRAP assay mentioned before , 2022. T+2 equivalents per gram of dry sample (mMol Fe+2/g).At λ700 nm, the absorbances were measured against a blank (in triplicate) by Milton Roy Spectronic 1,201 UV/Vis Spectrophotometer . Ascorbic acid and BHT were utilized as standards. Results were represented in terms of m Mol Fe2.3.1.4.+6 to Mo+5 in an acidic media by an antioxidant substance, resulting in a green phosphate/Mo+5 complex.TAC of ArEx, ArT, and ArPh were estimated using the phosphomolybdenum spectrophotometric assay according to the method previously described , 2022. TThe absorbances were recorded in triplicate at λ 695 nm against blank by Milton Roy Spectronic 1,201 UV/Vis Spectrophotometer . The standards were ascorbic acid and BHT. Data were obtained as mg equivalents of gallic per gram of dry extract (mg GAE/g).2.3.2.A. judaica L  against C. parvum oocysts. In a 96-well plate, C. parvum oocysts-PBS suspension were plated in triplicate at a density of 1 × 104 oocysts. The oocysts were treated with ArEx, ArPh, and ArT at concentrations of . These concentrations were produced in a double-fold manner. Oocysts viability was assessed after 48 h using the MTT assay kit “Promega, New York, NY, USA”  values were computed from the relative viability using GraphPad prism 7. A blank treatment of PBS and ethanol was applied to three wells, and untreated C. parvum oocysts were utilized as a control in another three wells. Based on the results of MTT assay, ArPh was used in the subsequent experiments.Using an ELISA microplate reader, the absorbance was determined to be 570 nm . Half-maximal inhibitory concentration (IC2.3.3.2.3.3.1.Within 2 weeks of preparation of the previously prepared suspension (section 2.1.1), the concentration of cryptosporidial oocysts was determined using a Neubauer hemocytometer. After multiple washing with PBS (to remove potassium dichromate), the stock suspension was diluted by a factor determined by the mean of four hemocytometer counts .5/mL) was stored. An antibiotic suspension was added at room temperature .Between the hemocytometer cover slide and counting chambers, oocyst suspension in a 10-μL aliquot was pipetted. Counting oocysts were performed under a 400x dry microscopic objective. A final concentration of oocysts/PBS suspension  was used for comparison with ArPh-treated oocysts (ArPh-TO). Assessments of the cytotoxic effects of ArPh on oocysts’ number, viability, and shape were made after 2, 24, and 48 h of incubation. For each time period, the findings were compared to a control group, and the differences between the two groups were computed.To generate a total volume of 1 mL, various quantities of ArPh  were added to the estimated oocysts solution. A control of untreated oocysts (With the aid of a viability dye [0.4% Trypan Blue (TB)], the number of oocysts/μL was calculated after measuring the oocysts distress in count on a Neubauer hemocytometer of The lethal dose was calculated by applying the destruction rate equation (A-B/A) and multiplying it by 100. In this equation, A represents the mean number of intact oocysts found in the control tube, and B represents the mean number of intact oocysts found in the ArPh-TO tube .in vitro exposure experiment was carried out with three separate replicates for each different concentration of ArPh. Based on preliminary trials, the different ArPh concentrations were chosen .Alterations in morphological size were captured on camera with the use of an Olympus bright field 1,000x oil immersion lens and an ocular micrometer. The 2.3.3.2.C. parvum oocysts exposed to varying doses of ArPh based on v/v), and 75 μL of this mixture was immediately imbedded in a CometSlide (frosted slide) placed flat at 4°C in the dark for 10 min.The neutral comet assay was applied to The slides were then placed in lysis buffer  at pH 10) with the coverslips removed. To remove the lysis solution from the slide, it was placed twice for 5 min at room temperature in electrophoresis buffer . The slide was then placed in a horizontal electrophoresis tank, and 20 min of electrophoresis at 28 V  was conducted. After removing the excess electrophoresis buffer from the slide, it was gently rinsed in distilled water. The gel was then fixed by immersion in 70% ethanol for 5 min and air-dried for 15 min at room temperature. The slide was stained with 50 μL of 20 μg/mL ethidium bromide for 1 h. The migrating DNA (comet) was spotted at 400x magnification using an Epifluorescence optika microscope.C. parvum untreated oocysts in PBS.Using an automatic image analysis system (Comet score V software), DNA damage was determined by measuring the tail moment, which is defined as the product of the amount of DNA in the tail and the displacement between the center of mass of the comet head and the center of mass of the tail (tail length) . The oli2.4.2.4.1.ad libitum. Even though the mice were guaranteed to be free of pathogens when they were acquired, WM and mZN were used to check their feces for three successive days , and maintained under a regular day/night cycle and sanitary conditions. The animals were pathogen-free, as evidenced by a sanitary certificate. Before the experiment, the mice were acclimatized to the experimental environment for 10 days and basal diet and water were administered ive days .2.4.2.C. parvum oocysts .Group 2: Positive control .Groups 3–7: Infected animals with the five concentrations of ArPh-TO.In the animal house of the Faculty of Medicine at Suez Canal University, every mouse was kept individually and in accordance with the established protocols for animal care.2.4.3.Cryptosporidium oocysts and was graded as positive or negative  . All thenegative .2.5.50 was expressed with a non-linear regression curve fit using Microsoft Excel software. The statistical significance was calculated using an unpaired t-test. One-way analysis of variance (ANOVA) test was used to determine the statistical significance of differences between treatments and the control group in the hemocytometer and comet assays. The p values of ≤ 0.05 were considered significant. IBM SPSS Statistics V26.0 was used for all statistical analyzes . The charts were made with GraphPad Prism 7. .Three independent experiments with three replicates per test sample were used to conduct the MTT assay. The mean absorbance values were represented as a percentage of the values of the untreated control oocysts standard error of the mean. The IC3.3.1.Cryptosporidium species in the examined samples was determined using a C. parvum-specific nested PCR technique. C. parvum was present in all the calves’ fecal samples. The four employed samples’ amplified products of 285 bp were successfully electrophoresed , respectively.As shown in +2 /g were demonstrated in +2 /g, compared with ArEx (2.34 ± 0.42 mMol Fe+2 /g) and the ArT (1.38 ± 0.26 mMol Fe+2 /g). Ascorbic acid (7.41 ± 0.57 mMol Fe+2 /g) and BHT (2.86 ± 0.38 mMol Fe+2 /g) served as positive controls.The ferric ion reducing power of ArEx, ArT, and ArPh expressed in terms of mMol FeThe obtained results  revealed3.3.3.3.1.50 values of the ArEx, ArPh, and ArT. The ArPh displayed significant toxicity against C. parvum oocysts, with an IC50 value of 31.6 μg/mL  caused “visible stress” on the total number of C. parvum oocysts compared to control  was determined to be 1,000 μg/mL. The highest rate of oocysts destruction (100%) was recorded 48 h later at a concentration of 2000 μg/mL. The data revealed a statistically significant difference (p < 0.05) in the concentrations of ArPh measured at various time intervals throughout the experiment.3.3.2.2.Cryptosporidium are typically brightly colored, range in size from 4 to 6 micrometers, and consist of a center globule, one to four sporozoites, and eccentric black granules. TB and mZN stain were used to track the morphological changes of oocysts. When ArPh was applied to oocysts at varying concentrations and exposure times, the oocysts exhibited cascades of death beginning with inflating, filling with single/multiple vacuoles, granulation of content, cracking, expulsion of content, and shrinkage  increased considerably . The rest of the treated groups exhibited no infectiousness .Plant phenolics are a diverse, widely distributed group of phytochemicals. They comprise stilbenes, tannins, coumarins, phenolic acids and flavonoids . PolypheA. judaica was estimated spectrophotometrically, where linalool was employed as a standard. Our findings demonstrated that A. judaica contained a considerable amount of terpenoids (9.5 ± 1.1 mg Linol/g).On the other hand, terpenes and terpenoids are a large, chemically, and functionally diverse group of secondary metabolites, among which several members exhibited cytotoxic, antimicrobial, and antiparasitic effects . Thus, i•+ radical quenching effects  as well as the FRAP and TAC . The ArEx showed noticeable anti-free radical effects against DPPH and ABTS•+ , FRAP (2.34 ± 0.42 mMol Fe+2/g) and TAC (40.91 ± 2.63 mg GAE/g) compared to ArT .Plant antioxidants, among them polyphenolics (phenolic acids and flavonoids) and terpenoids exert their effect through various processes, including metal reduction, chelation, hydrogen transfer, electron transfer, and singlet oxygen quenching . In the Leishmania amazonensis amastigotes  and have a low infectious dose (1–10 oocysts); it is reported that they can survive in water for 6–12 months or longer and can produce epidemics even with the drinking of a treated water . MTT assay is the gold standard for cytotoxicity testing and promptly determines the viability of microorganisms following drug treatment . As a result, the parasite solely depends on anaerobic respiration (the glycolytic pathway) to generate ATP, which is essential for the parasite’s survival and the pathogenesis it causes in the host  of ArPh in vitro. Similar reported data were obtained from in vivo experiments with Cryptosporidium sp. at 20 mg/mL, A. spicigera possessed significant anti-Cryptosporidium efficacy in mice  pomace, after oil pressing and phenol recovery, reduced C. parvum development in a reproducible manner. Curcuma longa L. extract significantly impacted Cryptosporidium oocyst shedding in in vivo research .SA and PK contributed to conception and design of the study. SA, EE, SE, MN, and AM organized the database. SA, EE, SE, and MN performed the statistical analysis. SA wrote the first draft of the manuscript. EE, SE, and MN wrote sections of the manuscript. SA, EE, MN, and AM funded resources of the experiment. SE funding of publication. All authors contributed to manuscript revision, read, and approved the submitted version.This research work was funded by Institutional Fund Projects under grant no. (IFPIP:1929-166-1443). The authors gratefully acknowledge technical and financial support provided by the Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Plantago lanceolata and Sida ovata have been used as medicinal plants for centuries to cure numerous diseases. This study aimed to evaluate antifungal activity of P. lanceolata and S. ovata leaf extracts against dermatomycotic fungi. Crude extracts from leaves of both plants were prepared using methanol and ethyl acetate. Phytochemical screening of both plants leaves was performed. Antifungal activity of crude extracts was evaluated against three dermatomycotic fungi . In addition, minimum inhibitory concentration (MIC) of the extracts was determined by microbroth dilution method. Maximum inhibition zone of 32.00 ± 11.64 mm was exhibited when combined ethyl acetate extract of both plants was applied against M. globosa. Best effect of MIC was demonstrated by ethyl acetate extract against most tested dermatomycotic fungi. Average MIC of ethyl acetate and methanol extracts ranged as follows: (0.19 ± 0.00 to 0.65 ± 0.00 mg/mL and 0.19 ± 0.00 to 0.52 ± 0.22 mg/mL) and (0.65 ± 0.22 to 1.56 ± 0.00 mg/mL and 0.19 ± 0.00 to 0.52 ± 0.00 mg/mL), respectively. Their synergistic effect was better than the effect of individual plant leaf extract. Minimum fungicidal concentration (MFC) values varied across the fungal pathogens when extracts from both plants and their combinations were used. The findings from the current study support the traditional use of P. lanceolata and S. ovata against dermatomycotic fungal infections, which could potentially be exploited for the treatment of superficial infection in humans. Plants have been employed as sources of medicine in Ethiopia for thousands of years to treat different ailments. Studies suggest that about 80% of the Ethiopian population still depend on medicinal herbs for their basic healthcare system –3. Even Because of their capability in providing many benefits to different socio-economic groups worldwide, especially in the line of modern medicine and pharmaceuticals, medicinal plants have gained great attention than ever. A wide range of plants parts such as leaves, roots, stems, bark, flowers, and seeds are used for therapeutic purposes as well as serve as precursors for the synthesis of various useful modern drugs due to their ethnomedical importance in nature. The medicinal potentials of these plants could be traceable to the bioactive phytochemical constituents that are responsible for the physiological action on the human body. Due to their versatility and multipurpose applications, plant-derived substances have gained attention recently. These plant-derived substances which enhance the usefulness of the plant are classified as phytochemicals .P. lanceolata has been given a lot of attention due to its multiple therapeutic applications such as its wound healing remedy [g remedy , 7. Thisg remedy . Previoug remedy .P. lanceolata and S. ovata have been reported to cure numerous diseases from cold to hepatitis, skin diseases, infectious diseases, problems concerning the digestive organs, respiratory organs, reproduction, the circulation, and for reducing fever [Among many popular medicinal plants, ng fever .P. lanceolata and S. ovata have been used either whole or crushed leaves, or juice from leaves of the two plants have been used to treat burns, stop bleeding, and treat all kinds of wounds to enhance the healing process [ process .P. lanceolata and S. ovata in any part of the world including Ethiopia. Therefore, the current study aimed at evaluating the antimicrobial activity of P. lanceolata and S. ovata leaf extracts against dermatomycotic fungi that cause superficial infection in humans. The solvent extracts from the two plants were tested separately and synergistically.As there are highly unexplored natural resources available in Ethiopia, focusing on searching for a novel, highly efficacious, better active, nontoxic, and affordable antifungal agents must be prior activity. In addition, there is scarcity of information about the synergistic antidermatomycotic activity leaf extracts of P. lanceolata and S. ovata plants  and ethyl acetate (9 g) from S. ovata leaf. Then, they were kept at 4°C until the assessments of their antidermatomycotic activities.Ethyl acetate and methanol solvents were used for extraction of P. lanceolata and S. ovata leaves was detected using standard procedures. The presence of flavonoids, alkaloids, saponins tannins, and cardiac glycosides was characterized following the method of Adesegun et al. [The presence of different phytochemical constituents of n et al. . Steroidn et al.  and quinn et al.  were alsn et al. .. albicans, M. furfur, and M. globosa were obtained from microbiology unit, University of Gondar Comprehensive Specialized Hospital. These pathogenic isolates were first grown on sabouraud dextrose broth, incubated for 48 h. About 20 mL of sterilized SDA  medium was poured into Petri dishes. Suspension of the superficial pathogenic fungi was made in sterile normal saline and adjusted to the 0.5 MCFarland's standard. Small volume (20 µL) of fungal suspension was added to each SDA plate and then evenly distributed by means of sterile swap over the agar surface. Agar wells were prepared using a sterilized cork borer with 6 mm diameter, 2 mm deep, and about 2.5 cm apart to minimize over lapping of zone. Dimethyl sulphoxide (DMSO) was used as negative control, while ketoconazole (KC) was used as positive control.The antidermatomycotic effect of the extracts was assessed using agar well diffusion assay . The tesµL of leaf extracts of both plants and ketoconazole or DMSO was placed into wells. Crude extracts and standard antibiotic were allowed to diffuse for about 40 min before incubation, and then the plates were incubated in an upright position at 30°C for 5 days. Results of the qualitative screening were recorded as the average diameter of the inhibition zone surrounding the wells containing the test solution. Results were compared with ketoconazole and DMSO.About 100 mg of dried leaf extract powder was resuspended in 1 mL of 10% DMSO. About 50 P. lanceolata–S. ovata leaf extract mixtures were prepared by mixing 25 µL P. lanceolata leaf extract and 25 µL S. ovata leaf extract that sum up 50 µL, and similar procedures were followed for the remaining activities as it was performed for separate plant leaf extracts.Additionally, μL of sabouraud dextrose broth was added to wells of a microtiter plate for each pathogenic fungal strain. A 100 μL of each dissolved crude plant leaf extract (100 mg/mL) was then added in the first well and serially diluted row by row to give 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.19 mg/mL, and 100 μL of the mixture was discarded from the last row, thus leaving each diluted well with a volume of 100 μL, except for the positive and negative controls. The test strains inocula were prepared from fresh 48 h grown cultures and were adjusted to McFarland standard, and 50 μL was poured to the wells except the last well. Wells containing fungal suspensions and growth media, as well as DMSO, were used as negative control, and wells having fungal suspensions and growth media, as well as KC, were used as positive control. The wells were incubated for 72 h at 37°C temperature. Then, 20 μL of resazurin reagent was added to each and every well to indicate respiratory activity, and a change in color from blue to pink was determined after incubating it for 72 h at 37°C temperature.The MIC is the lowest concentration where no viability was observed after 24 h on the basis of metabolic activity. MIC value of each crude plant extract was estimated using broth microdilution method , 16. 100On the other hand, MFC was determined by first selecting the plate that showed no growth during MIC determination; a loopful from each plate subcultured onto SDA was incubated for further 48 h at 37°C. The lowest concentration at which no colonies were observed was noted as the MFC.t-test after Bonferroni error correction of the predictive value. P ≤ 0.05 was considered statistically significant.The data collected through laboratory experiment were entered to Microsoft Excel 2013 and transferred to SPSS software version 20 for analysis. The results were expressed as mean value ± standard deviation (SD) of three replicates. Where applicable, the data were subjected to ANOVA, and differences between samples were determined by two-tailed P. lanceolata extract; however, steroids and tannins were not detected in S. ovata leaf extract.The different photochemical components of extracts of leaves of both plants were undertaken. All the tested phytochemical components, i.e., steroids, terpenoids, phenols, saponins, tannins, alkaloids, flavonoids, carbohydrates, quinines, and glycosides were found in C. albicans, M. furfur, and M. globosa. Both ethyl acetate and methanol extracts of P. lanceolata were assayed against these pathogens using agar well diffusion assay. The result in the current study revealed that the extracts from both plants, namely, P. lanceolata and S. ovata exhibit stronger inhibitory effect on C. albicans, M. furfur, and M. globosa in comparison to ketokonazle when applied separately and synergistically. Ethyl acetate extract showed higher effects to all tested dermatomycotic fungi. The maximum and minimum average zones of inhibition produced by ethyl acetate extract were 28.67 ± 6.69 mm and 18.17 ± 0.35 mm against M. furfur and C. albicans, respectively  .Test fungal pathogens used in the current study were ectively . Similarectively . Extract ≤ 0.05) . StatistS. ovata leaf extract, M. furfur and M. globosa showed variable susceptibility to ethyl acetate and methanol extracts. There was relatively some degree of similar susceptibility observed by C. albicans to ethyl acetate extract (15.00 ± 0.58 mm) and methanol extract (15.00 ± 0.19 mm). The result obtained revealed that KC had better effect than both extracts. None of the extracts had better effect than KC when applied against C. albicans (21.00 ± 0.00 mm) and M. furfur (19.00 ± 0.00 mm)  (Concerning 0.00 mm) . Statist ≥ 0.05) .M. globosa was highly susceptible to ethyl acetate extract with the average inhibition zone of 32.00 ± 11.64 mm. Statistically, no significance difference was seen with both extracts against all the tested superficial pathogenic fungi (p ≥ 0.05). But their effect was significantly different from Ketoconazole (P ≤ 0.05) (As clearly shown in  ≤ 0.05) .P. lanceolata leaf against tested pathogenic fungi were generally varied from pathogen to pathogen . Concerning the methanol extract, the lowest MIC value (0.19 ± 0.00 mg/mL) was recorded on both M. furfur and M. globosa, while the highest (0.52 ± 0.22 mg/mL) was observed against C. albicans.The MIC values of the tested extracts from the plant pathogen . The ethS. ovata leaf against tested microbes generally varied from pathogen to pathogen. Generally, all tested fungal pathogens were inhibited by the lowest concentration of methanol extract compared to ethyl acetate extract. Therefore, methanol extract was a better choice of extract that can exert effect on tested microbes  MFC was recorded against C. albicans by methanol extract. The MFC values recorded by ethyl acetate and methanol were 1.56 ± 0.00 and 0.39 ± 0.00 mg/mL against M. globosa, respectively.Based on the result obtained, the MFC values of the tested extracts from the plant S. ovata leaves varied across the fungal pathogens. The lowest MFC value of ethyl acetate, i.e., 3.12 ± 0.00 mg/mL, 1.56 ± 0.00 mg/mL, and 1.56 ± 0.00 mg/mL was recorded against C. albicans, M. furfur, and M. globosa, respectively, whereas the lowest MFC value of methanol extract, i.e., 1.56 ± 0.00 mm, 0.19 ± 0.00 mg/mL, and 0.39 ± 0.00 mg/mL was recorded against C. albicans, M. furfur, and M. globosa, respectively  fungi. So, the antifungal activity of ethyl acetate and methanol extracts from the plant a et al. .Previous studies on evaluating antimicrobial activities of the two medicinal plants against dermatomycotic fungi are scanty; the results cannot be compared with any other studies. However, we have tried to compare the results against the works performed on pathogenic bacteria and fungi.P. lanceolata in this study is concurrent with the findings of a study by Eshetie et al. [S. ovata showed different antifungal activities to the three tested dermatomycotic fungal pathogens. The possible explanation for this is that the plants are rich in compounds with potential antifungal activities.The antimicrobial activity of e et al. . LikewisP. lanceolata showed higher effects to all tested fungi compared to methanol extract with the average zones of inhibition 28.67 ± 6.69 mm, 27.00 ± 1.00 mm, and 18.17 ± 0.35 mm against M. furfur, M. globosa, and C. albicans, respectively. The average zones of inhibition formed by ethyl acetate were better than the report from Eshetie et al. [Ethyl acetate extract from S. ovata leaf had good antifungal effect on C. albicans, M. furfur, and M. globosa. Better result was obtained from the study report by Akilandeswariet et al. [S. acuta Burm related species. On the other hand, Abdulrahman et al. [The current study indicated that both ethyl acetate and methanol extracts of the plant t et al.  on antimn et al.  obtainedP. lanceolata and S. ovata leaf extracts had larger inhibition zone against tested dermatomycotic fungal pathogens than that of the leave extract of each plant applied separately. The synergistic effect may be attributed to the possibilities of the extracts affecting the different fungi processes in the cell [According to the findings of this study, the synergistic effect the cell . This miP. lanceolata leaf against M. furfur and C. albicans were 0.19 ± 0.00, while the MIC value of the same extract against M. globosa was 0.65 ± 0.00. The MIC value recorded in this study was much better than the finding report by Eshetie et al. [The present study revealed that the lowest MIC values observed by ethyl acetate extract from the e et al.  who repoS. ovata leaf extracts, all the tested fungal pathogens were inhibited by the lowest concentration of methanol extract compared to ethyl acetate extract. Therefore, methanol extract was a better choice of extract that can exert effect on the tested microbes  when compared to individual application of extracts . The sam39 mg/mL .P. lanceolata and S. ovata leaf were found to contain various phytochemical secondary metabolites. The extracts from both plants showed antimicrobial activity when used against the superficial human fungal pathogens. The combined effect of crude extracts from both plants leaves was found to have higher antifungal activity than the effect of individual plant leaf extracts. The findings from the current study support the traditional use of P. lanceolata and S. ovata against dermatomycotic fungal infections, which could potentially be exploited for the treatment of superficial infection in humans.From the overall study, it can be concluded that extracts from


Acetilactobacillus jinshanensis subsp. aerogenes Z-1. Therefore, it is necessary to characterize the mechanisms underlying their acid tolerance. The results of this study showed a survival rate of 77.2% for Z-1 when exposed to pH 3.0 stress for 1 h. This strain could survive for approximately 15 days in a vinegar solution with 4% or 6% total acid content, and its growth was effectively enhanced by the addition of 10 mM of arginine (Arg). Under acidic stress, the relative content of the unsaturated fatty acid C18:1 (n-11) increased, and eight amino acids accumulated in the cells. Meanwhile, based on a transcriptome analysis, the genes glnA, carA/B, arcA, murE/F/G, fabD/H/G, DnaK, uvrA, opuA/C, fliy, ecfA2, dnaA and LuxS, mainly enriched in amino acid transport and metabolism, protein folding, DNA repair, and cell wall/membrane metabolism processes, were hypothesized to be acid resistance-related genes in Z-1. This work paves the way for further clarifying the acid tolerance mechanism of Z-1 and shares applicable perspectives for vinegar brewing.Several lactic acid bacteria (LAB) are double-edged swords in the production of Sichuan bran vinegar; on the one hand, they are important for the flavour of the vinegar, but on the other hand, they result in vinegar deterioration because of their gas-producing features and their acid resistance. These characteristics intensify the difficulty in managing the safe production of vinegar using strains such as  Bacillus, and lactic acid bacteria (LAB)  . Among t culture .Acetilactobacillus jinshanensis subsp. aerogenes Z-1 demonstrated acid tolerance, and the optimal pH for its growth was pH 3.6 .The inoculum of strain Z-1 was prepared by incubation at 33°C for 3 days in modified MRS medium  . The pH 6° vinegar: 6% total acid content  was provided by a vinegar manufacturer located in Sichuan, southwest China; 1% glucose was added prior to use, and the mixture was subjected to autoclaving in a boiling water bath for 30 min.4° vinegar: 4% total acid content ; 1% glucose was added prior to use and subjected to autoclaving in a boiling water bath for 30 min.600 of approximately 3.5. Then, 10 mL of culture was centrifuged  at 4°C. The obtained biomass was washed twice using sterile saline and subjected to further incubation in 10 mL of modified MRS medium with the pH adjusted to 2.5, 2.8, 3.0, 3.2, and 3.6. Samples were withdrawn at designated times and subjected to serial dilution with sterile saline for viable cell counting. The dilute solution was spread on a modified MRS Petri dish, followed by 7–10 d of anaerobic incubation at 33°C. Each test was performed in triplicate.After transferring 1% (v/v) inoculum into 100 mL fresh modified MRS, the culture was incubated at 33°C for 65 h, achieving an ODTen millilitres of inoculum was centrifuged , and the resultant pellets were resuspended in plastic vials containing 100 mL of 4° and 6° vinegar. Then, the culture was incubated at 33°C. Samples were taken periodically for viable cell counting as described above. Triplicate experiments were carried out in parallel.Batch experiments were performed in 250 mL Erlenmeyer flasks containing 100 mL modified MRS medium, where aspartic acid (Asp), glutamic acid (Glu), arginine (Arg) and lysine (Lys) were fortified individually at 10 mM, 20 mM, 30 mM and 40 mM. After inoculation , the cultures were incubated at 33°C for 108 h. The substrate concentration was selected as an independent variable. Nonspiked medium was used as a control. All treatments were conducted in triplicate. Samples were withdrawn at 12-h intervals to measure the absorbance at 600 nm.Then, the optimal concentration of each amino acid was selected for another batch following the same protocol. However, the medium was adjusted to different pH values . The cell density of each culture was quantified at an interval of 12 h using the OD600.600 of approximately 3.5, and then 50 mL of culture was centrifuged  at 4°C. The harvested cells were washed twice using sterile saline and subsequently resuspended in 50 mL modified MRS medium at pH 3.0 and 3.6 (control) and maintained for 1 h at 33°C. All tests were performed in triplicate. Afterwards, samples were taken for analyses, including scanning electron microscopy (SEM), cellular membrane fatty acid content, H+-ATPase activity, intracellular ATP content, intracellular pH (pHi), and intracellular amino acid content.After transferring 2% (v/v) inoculum into 250 mL Erlenmeyer flasks containing 100 mL fresh modified MRS (pH 3.6), the culture was incubated at 33°C for 65 h, achieving an ODField emission scanning electron microscopy (FESEM) images were collected on an Evo 18 scanning electron microscope. The samples were prepared following the method of The fatty acid content of the cell membrane was determined through gas chromatography–mass spectrometry (GC–MS), and sample pretreatment and measurement were carried out according to +-ATPase activity was determined using a Cell ATP bioluminescence quantitative detection kit , and ATP content was determined using a Bacterial H+-ATPase activity colorimetric assay kit . One U (μmol/min) of H+-ATPase activity is defined as the amount of enzyme that catalyses the conversion of 1 μM of reduced NADH per minute at 37°C. ATP content was defined as nmol/mg intracellular protein. The protein concentration was determined using a protein quantification test kit .Hi was measured by the fluorescence method using 5- (and 6-)-carboxyfluorescein succimidyl ester as the fluorescent probe, that loading of cells with 5- (and 6-)-carboxyfluorescein succimidyl ester, determination of pHi, and calibration of pHi all followed pHAfter acid stress treatment, 30 mL of treated culture was removed and centrifuged  for 10 min to collect the bacteria. The pellets were resuspended in 1 mL of 50 mM PBS buffer (pH 7.0) after washing 3 times with the same buffer. Then, the samples were boiled in water for 15 min and centrifuged  for 10 min to obtain the supernatant for protein content quantification. An equal volume of 20% (m/v) sulfosalicylic acid was added to 0.5 mL of supernatant. The mixture was maintained at 4°C for 1 h and then centrifuged  for 5 min, followed by filtration with a 0.22 μm water phase filter membrane. An automatic Hitachi L-8900 amino acid analyser was employed for analysis.600 of approximately 3.5, and then 50 mL of culture was centrifuged  at 4°C. The harvested cells were washed twice using sterile saline and subsequently resuspended in 50 mL modified MRS medium at pH 3.0 and 3.6 (control) and maintained for 1 h at 33°C. The biomass was harvested through centrifugation  at 4°C and washed twice using sterile saline. Then, the biomass was flash frozen using liquid nitrogen for RNA extraction. Each library was constructed in triplicate.To perform RNA sequencing and transcriptomics analysis, strain Z-1 was first exposed to acid stress. After transferring 2% (v/v) inoculum into fresh modified MRS (pH 3.6), the culture was incubated at 33°C for approximately 65 h, achieving an ODp value ≤0.05 were considered criteria for screening significant differentially expressed genes (DEGs) between different groups. All DEGs were searched against the nonredundant protein data in the NCBI, GO, Swiss-Prot and EggNOG databases, accompanied by cSNP/InDel screening.RNA extraction, transcriptomics sequencing and bioinformatics analysis were performed by Shanghai Majorbio Biopharm Technology Co., Ltd. (China). Total RNA was extracted using TRIzol Reagent , and the total RNA quality was determined using a Nanodrop spectrophotometer . The validated RNA underwent purification, fragmentation, reverse transcription, end repair, amplification, and circularization successively to obtain a library according to the company’s standard guidelines. A bioanalyzer was employed for quality control. Finally, whole-run sequencing was performed on Roche 454 GS FLX Titanium instrument . High-quality reads of the transcriptome in each sample were aligned to the whole genome of strain Z-1 using the Bowtie 2 program. The gene expression level was quantified by RSEM software in transcripts per kilobase per million mapped reads (TPM) values. Next, differential expression analysis between two conditions/groups was carried out using edgeR (v2.12), DESeq2 (v3.11) and DESeq (v3.11) software. Fold change (FC) ≥ 1.8 and adjusted 2O. The PCR protocol included a step of 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, 60°C for 20 s, and 95°C for 15 s. Three biological replicates were performed for each reaction.To confirm the reliability of the RNA-Seq analysis, RT–qPCR was performed for 10 DEGs with the primers listed in Lactobacillus jinshani sp. nov. HSLZ-75  status, it is believed to be a survival strategy of bacteria to avoid adverse effects. This state can be revived under appropriate conditions, for example, strain Z-1 cannot grow with ordinary MRS medium during the early screening and can be isolated after adjusting the composition of the MRS medium. To date, several LAB have been reported to be able to survive under acidic stress and to harbour acid tolerance ability, which is mainly gained through external substances, cell membrane structure and intracellular metabolism  after 1 h, suggesting that Z-1 adapted to an acidic environment by altering the membrane fatty acid content, which enhanced its cell membrane fluidity  at pH 3.6 than at pH 3.0, and the intracellular ATP concentrations were not much different at these two pH levels. This result may be related to the fact that the optimum pH of H+-ATPase activity is slightly acidic (pH 5.0 ~ 5.5), and H+-ATPase activity is gradually eliminated in a very low-pH environment  and Q30 (%) were greater than 97.97 and 93.83%, respectively  greater than 20 and 30 in the total base, respectively), and the rRNA content was far below 15%. Other detailed information is compiled in Transcriptome sequencing was performed to further reveal the acid tolerance characteristics of Z-1. The total number of bases in 6 samples was 2.41 × 10To further analyse these DEGs, GO and KEGG analyses were conducted . AccordiBased on the enrichment analysis described above, acid tolerance was derived from amino acid, energy and lipid metabolism, RNA degradation and nucleotide repair, and signalling pathways.gabD, glnA, carB, carA, arcA, asd, purB and serA  was increased, accompanied by intracellular accumulation of Asp., Ser, Glu, Gly, Ala, Lys, His and Arg. Transcriptional evidence showed that amino acid metabolism, energy and lipid metabolism, RNA degradation and nucleotide repair, and signalling pathways were closely related to acid tolerance. Accordingly, the glnA, carA/B, arcA, murE/F/G, fabD/H/G, DnaK, uvrA, opuA/C, fliy, ecfA2, dnaA and LuxS genes were presumed to be the key genes. This work is helpful for the formation of a comprehensive strategy to solve swelling deterioration based on exploration of the acid tolerance mechanism of Z-1.While LAB play an important role in the formation of flavor in vinegar, several LAB can also cause vinegar deterioration, as their gas-producing properties and acid tolerance exacerbate the difficulties in managing safe vinegar production. Therefore, it is important to grasp the intrinsic factors of acid tolerance in these gas-producing strains. https://www.ncbi.nlm.nih.gov/, PRJNA922687.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: QL: conceptualization, supervision, formal analysis, writing - original draft. KH: investigation, formal analysis, methodology, writing-review and editing. JM: data curation, investigation, and formal analysis. JL, AL, and YY: formal analysis, methodology, writing-review and editing. XA, LH, SC, LZ, and MG: resources and methodology. SL: conceptualization, supervision, project administration, writing-review and editing, and funding acquisition. All authors contributed to the article and approved the submitted version.This work was supported by the financial support from the Science and Technology Department of Sichuan Province (2023ZHCG0081).MG is employed by Sichuan Baoning Vinegar Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The linear range was found to be ~1–20 ng mL−1 for Co, Ge, As, Cd, Sb, and Pb and 5–80 ng mL−1 for Al and Zn elements. The LODs are ~0.10, 0.25, 0.12, 0.13, 0.11, 0.12, 0.61, and 0.85 ng mL−1, and the LOQs are 0.33, 0.83, 0.40, 0.43, 0.36, 0.40, 2.01, and 2.81 ng mL−1 obtained for Co, Ge, As, Cd, Sb, Pb, Al, and Zn, respectively. In addition, the recovery percentages received ranged 85.4%–94.1% for Co, 82.6%–95.1% for Ge, 86.3%–97.9% for As, 87.3%–96.3% for Cd, 88.0%–104.4% for Sb, 96.3%–106.0% for Pb, 88.4%–104.0% for Al, and 95.1%–99.7% for Zn. Finally, the migration of these heavy metals from polypropylene (PP) and polystyrene (PS) into foodstuffs was also simulated according to EU legislation, showing that the most leached element was Zn, followed by Al and Pd, with the migration of ~8.38% and ~0.41%, and ~0.19%, respectively.Plastic food packaging is an essential element for customer convenience and the preservation of food quality. Nonetheless, heavy metals in the packaging materials, either intentionally or nonintentionally added, can be transferred to the food. Therefore, determining heavy metal contents in these packaging materials is essential. In this study, heavy metals, including Co, Ge, As, Cd, Sb, Pb, Al, and Zn from different intrinsic plastic food packaging materials were analyzed using the inductively coupled plasma‐mass spectrometry (ICP‐MS) method. Moreover, the migration of these elements into the environment was also investigated. This method is validated following the new technique's requirements, which include linearity range, accuracy, precision, the limit of detection (LOD), and the limit of quantitation (LOQ). The method has been suitably validated with the regression equation from the standards prepared in HNO Plastic food packaging is an essential element for customer convenience and the preservation of food quality. Nonetheless, heavy metals in the packaging materials, either intentionally or nonintentionally added, can be transferred to the food. Therefore, determining heavy metal contents in these packaging materials is essential. In this study, heavy metals, including Co, Ge, As, Cd, Sb, Pb, Al, and Zn from different intrinsic plastic food packaging materials were analyzed using the inductively coupled plasma‐mass spectrometry (ICP‐MS) method. Moreover, the migration of these elements into the environment was also investigated. This method is validated following the new technique's requirements, which include linearity range, accuracy, precision, the limit of detection (LOD), and the limit of quantitation (LOQ). Polymers are built from small organic molecules, monomers, usually hydrocarbons but often other materials, and are often used in synthesizing plastics. These monomers generally originated from oil or natural gas  of Co, Ge, As, Cd, Sb, Pb, Al, and Zn and internal standard solutions of Sc, Rh, In, Te, and Ir were purchased from Sigma‐Aldrich Co. Ltd. Nitric acid (HNO3 65%), citric acid (C6H8O7 99.5%), hydrochloric acid (HCl 37%), and acetic acid (C2H4O2 99.7%) of ultra‐trace analysis grade were purchased from Merck. Before use, all glassware were cleaned using pure water diluted HNO3 obtained through a Sartorius Pure Water System.Standard solutions , baffled cyclonic spray chamber, PFA nebulizer, and quartz torch with a 2.5 mm i.d. removable quartz injector. The instrument was operated using kinetic energy discrimination (KED) using pure He as the collision gas in the collision/reaction cell (CRC). To automate the sampling process, an Elemental Scientific SC‐4 DX Autosampler was used. Typical ICP‐MS operating parameters are summarized in Table 2.3−1 level for each component . An online internal standard addition system automatically added internal standards to the instrument during analysis. The standard solutions were prepared with concentrations of 1.0, 2.0, 5.0, 10.0, and 20.0 ng mL−1 for Co, Ge, As, Cd, Sb, and Pb; and 5.0, 10.0, 20.0, 60.0, and 80.0 ng mL−1 for Al and Zn. All these solutions were prepared from the stock multi‐standard (1000 ng mL−1 for each) by dilution using ultrapure nitric acid (1% v/v) as a diluent that was used for the linearity range study of the method in Table −1 for Co, Ge, As, Cd, Sb, and Pb; and 15.0, 45.0, and 60.0 ng mL−1 for Al and Zn were prepared, respectively, by spiking of the metals in samples that were immersed in acetic acid (4% v/v) solution to get three spiked concentrations of 0.9, 3.0, and 6.0 ng mL−1 for Co, Ge, As, Cd, Sb, and Pb; and 15, 45, and 60 ng mL−1 for Al, and Zn. Each sample was analyzed three times, and the results were expressed as mean ± SD (SD: standard deviation). The solutions prepared for linearity had concentrations of 0.9, 3.0, and 6.0 ng mL−1 for Co, Ge, As, Cd, Sb, and Pb and 15.0, 45.0, and 60.0 ng mL−1 for Al and Zn used for the precision study. To determine the LOD and LOQ of the method, the solution with a concentration of 1 ng mL−1 for Co, Ge, As, Cd, Sb, and Pb and 5 ng mL−1 for Al and Zn was prepared for linearity study.The internal standard mixture was prepared at 100 ng mL2.4Four different types of plastic food packages, including polypropylene (PP), polyamide (PA), polyvinyl chloride (PVC), and polystyrene (PS) were obtained from local food markets.2.4.1According to the guidelines given by the current regulation  was added to the residue, and the liquid was allowed to evaporate to dryness in a water bath. Then, 10 mL of 1 mol L−1 nitric acid was added, and the mixture was placed on a hot plate until the residue was dissolved. After cooling, the digest solution volume was adjusted to 50 mL.First, 1 g was cut into small pieces and weighed out in a quartz crucible. After adding 5‐mL nitric acid (65%), the crucible was placed on a hot plate and heated until thin white fumes of SO2.4.3−1 intermediate mixed standard solution for Co, Ge, As, Cd, Sb, and Pb and 3.125 mL from the 80 ng mL−1 intermediate mixed standard solution for Al and Zn before extraction solution and digestion, to give final spiking concentration levels of 1 and 5 ng mL−1. The extraction and digest solution of samples were then filtered, diluted, and transferred into a 50‐mL volumetric flask. The blank samples were carried out similarly but without using samples containing the metals of interest.The samples were prepared under the same conditions in the previous experiments and spiked with 2.5 mL from the 20 ng mL33.1The validation parameters included in this study are linearity, accuracy, precision, the limit of detection (LOD), and the limit of quantification (LOQ). The method validation followed the protocol from EURACHEM guidelines  is not <.990 for the least squares method of analysis of the line.The linearity is the ability of a method to elicit test results that are directly proportional to analyte concentration within a given range. Linearity is reported as the variance of the slope of the regression line. The range is the interval between the upper and lower levels of the analyte that has been demonstrated to be determined with precision, accuracy, and linearity using the developed method. The range is usually expressed in the same units as the results obtained by the process. A minimum of five concentration levels and specific minimum specified degrees are required. Acceptance criteria for linearity are that the correlation coefficient  of the metal analyte divided by the response of the internal standards LOQ−1 for Co, Ge, As, Cd, Sb, and Pb and 5 ng mL−1 for Al and Zn were analyzed, and the results are summarized in Table To determine these values, sample blank and spiked samples with an expected concentration of 1.0 ng mL3.1.3Cspk was the analyte concentration in the sample spiked; Cref was the analyte concentration in the unfortified sample; Cadd is the analyte concentration in the added sample. Mathematically, the % Rec is defined as follows:The accuracy of an analytical method, also called recovery, is defined as the closeness between the true value of the analyte in the sample and the value obtained by the analytical procedure. It is a parameter that ensures no loss or contamination occurs during the test procedure, contributing to quantifying the compounds. The samples with low metal contents were spiked with three different analyte levels. The samples were subjected to the highest possible steps of the procedure for at least 3 days with two replicates per day. The mean, standard deviation, relative standard deviation (RSD), and recovery (% Rec) were calculated, where −1 for Co, Ge, As, Cd, Sb, and Pb and 5.0, 15.0, and 30.0 ng mL−1 for Al and Zn of heavy metals into 4% v/v metals free of acetic acid immersed at 60°C for 30 min . The mean value, standard deviation (SD), and relative standard deviation (RSD) are calculated by using the following formulas:−1 for Co, Ge, As, Cd, Sb, and Pb and 5.0, 15.0, and 30.0 ng mL−1 for Al and Zn. All samples were immersed into 4% v/v metals free of acetic acid at 60°C for 30 min ; methodology ; writing – original draft (lead). Truong Thi Truc Linh: Data curation ; resources ; validation . The‐Ky Vo: Conceptualization ; formal analysis ; resources ; writing – review and editing . Quoc Hung Nguyen: Conceptualization ; methodology ; supervision . Thanh‐Khue Van: Project administration ; writing – review and editing .The authors greatly appreciate the financial support of the Faculty of Chemical Engineering, Industrial University of Ho Chi Minh City (IUH), Vietnam.The authors declare that they have no conflicts of interest.


Peripheral nerve injury can cause neuroinflammation and neuromodulation that lead to mitochondrial dysfunction and neuronal apoptosis in the dorsal root ganglion (DRG) and spinal cord, contributing to neuropathic pain and motor dysfunction. Hyperbaric oxygen therapy (HBOT) has been suggested as a potential therapeutic tool for neuropathic pain and nerve injury. However, the specific cellular and molecular mechanism by which HBOT modulates the development of neuropathic pain and motor dysfunction through mitochondrial protection is still unclear.Mechanical and thermal allodynia and motor function were measured in rats following sciatic nerve crush (SNC). The HBO treatment (2.5 ATA) was performed 4 h after SNC and twice daily  for seven consecutive days. To assess mitochondrial function in the spinal cord (L2–L6), high-resolution respirometry was measured on day 7 using the OROBOROS-O2k. In addition, RT-PCR and Immunohistochemistry were performed at the end of the experiment to assess neuroinflammation, neuromodulation, and apoptosis in the DRG (L3–L6) and spinal cord (L2–L6).2+ permeability, was reduced along with the apoptosis marker (cleaved-Caspase3) and mitochondrial stress marker (TSPO) in the DRG and spinal cord of the HBOT group. Additionally, HBOT prevents the reduction in mitochondrial respiration, including non-phosphorylation state, ATP-linked respiration, and maximal mitochondrial respiration in the spinal cord after SNC.HBOT during the early phase of the SNC alleviates mechanical and thermal hypersensitivity and motor dysfunction. Moreover, HBOT modulates neuroinflammation, neuromodulation, mitochondrial stress, and apoptosis in the DRG and spinal cord. Thus, we found a significant reduction in the presence of macrophages/microglia and MMP-9 expression, as well as the transcription of pro-inflammatory cytokines  in the DRG and (IL6) in the spinal cord of the SNC group that was treated with HBOT compared to the untreated group. Notable, the overexpression of the TRPV1 channel, which has a high CaMitochondrial dysfunction in peripheral neuropathic pain was found to be mediated by neuroinflammation and neuromodulation. Strikingly, our findings indicate that HBOT during the critical period of the nerve injury modulates the transition from acute to chronic pain via reducing neuroinflammation and protecting mitochondrial function, consequently preventing neuronal apoptosis in the DRG and spinal cord.The online version contains supplementary material available at 10.1186/s12967-023-04414-x. In 2016, during the Euro final in Paris, Cristiano Ronaldo suffered a devastating knee injury and pain that forced him to withdraw during the game. To speed recovery, and more importantly to relieve pain, Cristiano received intensive Hyperbaric Oxygen Therapy (HBOT), the treatment has shown a positive result in relieving pain and enhancing the recovery process . However2+ due to the injury is a key event in initiating the domino effect of neuronal apoptosis in the DRG neurons and spinal cord [Peripheral neuropathic pain (PNP) remains one of the leading causes of global disability, affecting around 10% of the population . PNP is nal cord , 10.2+ into the injured axon contributes to neuronal apoptosis in the DRG [2+ in neurons, hyperexcitability, secretion of pro-inflammatory cytokines , which eventually induce neuroinflammation, and neuronal apoptosis [2+ influx probably leads to mitochondrial membrane permeability, where calcium accumulates in the mitochondrial matrix, resulting in energy crisis (ATP depletion), cytochrome-c (Cyt-c) release, generation of free radicals and reactive oxygen species (ROS), and consequently, activation of the apoptosis pathway [The massive influx of Ca the DRG . In addipoptosis , 13. The pathway , 15. Pre pathway , 17. The pathway . However pathway , 19. Con2+ permeability, after nerve injury in the DRG neurons and spinal cord, suggests that this channel may be involved in the Ca2+ influx that damages the mitochondria and initiates neuronal apoptosis [2+ influx, prevents the mitochondrial membrane-potential damage and decreased the levels of mitochondrial ROS after nerve injury in an animal model of neuropathic pain [+), bradykinin, and oxidative stress [Interestingly, the elevated expression of Transient Receptor Potential vanilloid 1 (TRPV1), which has a high Capoptosis , 21. Unshic pain . TRPV1 ie stress , 22–25. e stress , 25, 26.HBOT, in which patients are exposed to 100% oxygen under increased atmospheric pressure, has beneficial effects in treating pain disorders, including inflammatory pain, fibromyalgia, and migraine . In addiMale Sprague–Dawley rats  were used in this study. All animal procedures were approved by the University Animal Care Committee and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. During the study, animals were housed in groups of 3–4 rats in a sterilized solid bottom cage with contact bedding under controlled temperature and a 12:12 h light/dark cycle. Animals were maintained on a standard pellet diet, and water was supplied ad libitum. All efforts were made to maintain animals suffering to a minimum.The sciatic nerve crush injury model (SNC) was produced using a modified technique previously described by Savastano et al. . BrieflyHBOT was conducted based on our previous studies . A cylinMechanical sensitivity was assessed using an electronic Von Frey (eVF) device. Rats were transferred to the testing room while in their cages for one hour for habituation. Thirty minutes before the measurement began, rats were placed in the measuring chambers  located 20 cm above the bench for acclimatization. An Electronic von Frey device  was used: the withdrawal threshold was evaluated by applying a force ranging from 0 to 100 g. The punctuate stimulus was delivered to the hind paw, a positive response was defined as an immediate withdrawal of hind paw in response to stimulation, and the withdrawal threshold was automatically displayed on the screen. Five measurements were performed/collected by an observer blinded to the experimental condition for each rat.2 = 1). An observer blinded to the experimental condition documented hind paw lickings, escape behavior (jump), and rearing for each degree interval.Thermal sensitivity was assessed using a computer-controlled hot plate analgesia meter . The animal was placed in a Plexiglas cylinder  with a drilled cover, where the plate can be heated up to 65 °C. For the dynamic hot plate test, first, the animals were transferred to the measurement room while still in their home cage and stayed there for one hour as a habituation to the testing room. The animals were then placed on the plate for 15 min for habituation to the instrument at a temperature of 35 °C. For the dynamic hot plate test (DHP), the plate temperature increased to 45 °C with 1 °C per min speed . The Open Field test consists of large square, . The animals were transferred to the measurement room while in cages and stayed there for one hour to habituate them to the different environments, and then, the animals were placed in the center of the maze. The session lasted 10 min, and measurements of locomotor activity and time spent in the center/peripheral of the arena were quantified. The open field was wiped with 30% alcohol solution between trials.Motor coordination and balance were assessed using the Rotarod . Animals were trained with the Rotarod one week before SNC. In training trials, rats were placed on the rod, which rotated at a constant speed of 10 rpm. The training trial continued until the rat could stay on the rod for 60 consecutive seconds without falling, turning around, or clinging to the rod. If they fell from the rod or turned around, they were correctly placed back on it, and the timer restarted. An accelerating protocol was used in test trials, where the rotation speed increased from 4 to 40 rpm for 300s. Each trial was terminated if an animal fell, clung, rotated for two complete rotations, or remained on for more than 300s. Latency to falls was automatically recorded for each trial. The average of the three tests was calculated and used for analysis. In addition, the delayed gains/motor improvement in performance was computed as the difference in performance between sessions. The traAt the end of the experiment, animals were deeply anesthetized, sacrificed, and transcardially perfused with heparinized saline, 10% sucrose in buffered saline, and 4% buffered formaldehyde. The sciatic nerve, Dorsal root ganglion (L3–L6) and spinal cord (L2–L6) were excised and fixated in 5% paraformaldehyde in phosphate buffer for one hour and then embedded in paraffin. Sections of 5 μm thickness were cut from the SN, DRG and SC with a microtome, and every 5th section was collected for analysis. The sections were then mounted on Matsunami adhesive glass slide .Immunofluorescent staining was performed as described before . BrieflyMicroscopic observation was done using the Eclipse Ci microscope . The images of 3–5 fields were captured by the Nikon DS-Ri1 camera  with the same microscope settings and exposure time. The images were analyzed using NIS Elements analysis software. The density of Microglia/Macrophages, MMP-9, Cas3, TSPO and TRPV1 was determined by the number of positively stained. The co-localization/expression of immunoreactive (IR)-TRPV1 with Caspase3 or TSPO was determined by the percentage of IR of TRPV1 with Caspase3 or TSPO. The images were analyzed by an observer blinded to the condition.At the end of the experiment, animals were deeply anesthetized, sacrificed, and transcardially perfused with heparinized saline, the spinal cord (L2–L6) or the sciatic nerve and DRG (L3-L6) was isolated and kept at – 80 °C until used. The RNA was extracted from the spinal cord and the sciatic nerve-DRG samples, by combining TRI reagent  and Purelink TM kit . cDNA was prepared using High-capacity cDNA Synthesis Kit  and real-time PCR was performed as described before , 34. Bri+-lactobionate, 0.5 mM EGTA, 3 mM MgCl2, 20 mM taurine, 10 mM KH2 PO4, 20 mM HEPES adjusted to pH 7.1 with KOH, and 1 g/L BSA essentially fatty acid-free) for 5 min at 4 °C and then proceeded using a specially designed mitochondria isolation kit  according to the manufacturer instructions. Briefly, tissue samples were cut into small pieces and homogenized using a pestle and glass on ice with 10 volumes of ice-cold extraction buffer  and centrifuged at 600×g for 10 min at 4 °C. The supernatant was then collected and re-centrifuged at 11,000×g for 10 min at 4 °C. Pellets were resuspended with 10 volumes of a second ice-cold extraction buffer  and processed by sequential centrifugation as above. Finally, the pellet was then resuspended in MiR05. The concentration of mitochondrial proteins was determined according to the Bradford assay using the Pierce, Coomassie Plus Protein Assay with BSA as a standard.Mitochondria were isolated from the spinal cord (L2–L6) using a differential centrifugation method . Briefly+-lactobionate, 0.5mM EGTA, 3 mM MgCl2, 20 mM taurine, 10 mM KH2 PO4, 20 mM HEPES adjusted to pH 7.1 with KOH, and 1 g/L BSA essentially fatty acid-free) 30 min at least before the experiment.High-resolution respirometry was performed only on fresh tissue using the OROBOROS Oxygraph-2k . The oxygraph was calibrated at 37 °C according to the manufacturer’s instructions with each chamber filled with 2 mL of MiR05 . Leak-resting State non phosphorylation state after the activation of Succinate-CII, and inhibiting ATP synthase: oligomycin (2.5 µM). Electron transfer (ET) capacity: Uncoupler (0.5 µM). Non-mitochondrial residual oxygen consumption (ROX) after inhibition of complex III: antimycin A (2.5 µM).We used a modified protocol described by Holody et al., (2021) to evaluate mitochondrial respiration in different states . The pro2 per second per milligram wet tissue mass-pmol O2/s/mg) after correction for residual oxygen consumption (ROX), corrected automatically by DatLab® software for instrumental background. The following equations were used for the calculation of the rates of oxygen consumption in a different state:Mitochondrial respiration was expressed in flux per mass . Significant main effects and interactions were further pursued using Post-hoc by Tukey’s test and independent sample t test. The accepted significance value for all tests was set at P < 0.05.Statistical analyses were performed using IBM SPSS statistics version 26 and GraphPad Prism. All data were expressed as Mean ± SEM. Differences between groups were assessed by Student (HBOT) group [t(24) = − 0.24, P = 0.82; t(24) = 0.258, P = 0.79, respectively, Fig.  = 984.10, P < 0.001, η2 = 0.97, Fig.  = 39.92, P < 0.001, η2 = 0.62, Fig. (HBOT) group shows higher mechanical sensitivity threshold  = 3.40, P = 0.002 Fig. Prior the SNC, there was no significant difference in mechanical and thermal sensitivity between the SNC and SNC(HBOT) group (t(24) = − 0.87, P = 0.38, Fig. (HBOT)) show a significant decrease in the latency to fall [F = 392.52, P < 0.001, η2 = 0.94, Fig. (HBOT) group shows a superior motor performance compared to the untreated group (SNC) ) shows a significant improvement in performance between sessions  group shows improvement between session, compared to approximately 50% in the SNC  between the SNC group and SNC = 5.81, P = 0.007; F = 5.81, P = 0.007, respectively, Fig. (HBOT) group  group  = 28.84, P < 0.001; F = 108.87, P < 0.001, DRG, spinal cord, respectively, Fig.  = 84.37, P < 0.001, Fig. (HBOT) group expressed lower level of the pro-inflammatory cytokines  group compared to the SNC group  group compared to the SNC group  in the sciatic nerve/DRG and spinal cord  = 8.78, P = 0.003; F = 6.79, P = 0.009, TRPV1, Caspcase-3, respectively, Fig. P < 0.05, Fig. (HBOT) groups compared to the naïve group [F = 0.37, P = 0.69; F = 0.96, P = 0.40; F = 1.6, P = 0.23, BAX, Bcl2, BAX/Bcl2 ratio, respectively, Fig. The overexpression of TRPV1 channels might be associated with neuronal apoptosis after nerve injury . Here, w = 13.26, P < 0.001; F = 32.32, P < 0.001, TRPV1, Caspcase-3, respectively, Fig.  = 66.46, P < 0.001; Fig. P < 0.05, Fig. (HBOT) group compared to the naïve group  = 7.06, P = 0.008; F = 1.18, P = 0.19; F = 10.12, P = 0.002; F = 4.20, P = 0.03, BAX, Bcl2, BAX/Bcl2 ratio, Caspcase-3, respectively, Fig.  = 15.64, P < 0.001, Fig.  = 53.44, P < 0.001, Fig. P < 0.05, Fig. (HBOT) group expressed reduced level of BAX, and Caspcase-3 compared to the SNC group  group compared to the SNC group , we observed similar results. Thus, after 12 days of the SNC, a significant increase in the transcription of the apoptosis marker [F2,1 = 7.06, 2,1 = 7.06, 2,1 = 7.06, 2,1 = 7.06, 2,1 = 7.06, 2,1 = 7.06,  = 9.00, P = 0.002; F = 15.41, P < 0.001, respectively, Fig.  = 25.48, P < 0.001; Fig. P < 0.05, Fig. (HBOT) group expressed lower level of TSPO at the transcription and protein level in the DRG compared to the SNC group  group expressed a higher level of TSPO, and an elevated level of co-expression of TSPO with TRPV1 in the DRG compared to the naïve group.Here, we found that after SNC there was a significant increase in the expression of the translocator protein (TSPO) at the transcription and the protein level in the DRG [F = 7.27, P = 0.005; F = 75.86, P < 0.001, mRNA, protein, respectively, Fig. (HBOT) group compared to the SNC group  group expressed higher level of TSPO in the spinal cord compared to the naïve group  group [F = 2.64, P = 0.09, Fig.  = 4.43, P = 0.02; F = 6.61, P = 0.006; F = 7.13, P = 0.004, LEAK-1, LEAK-2, Sum LEAK, respectively, Fig. P < 0.05, Fig.  = 3.97, P = 0.03, Fig. (HBOT) group in the maximal mitochondrial respiration. However, on average, the SNC (HBOT) group showed a normal maximal respiration compared to the SNC group  after 7 days of SNC, in order to evaluate the mitochondrial function in different respiration states including: Leak state, OXPOHS state and Electron transfer (ET) capacity  group [F = 2.46, P = 0.11, F = 3.43, P = 0.052, OXPHOS-CI, OXPHOS-CI&CII, respectively, Fig.  = 4.51, P = 0.02, Fig. (HBOT) groups show higher mitochondrial oxygen consumption in oxidative states than in the SNC group  = 6.08, P = 0.008, Fig. P < 0.05, Fig. P < 0.05, Fig. Stimulation of oxidative phosphorylation states  via saturating of ADP and glutamate , and succinate  shows a non-significant reduced level of mitochondrial oxygen consumption in the SNC group compared to the naïve and SNCThe intricacy of the link between mitochondrial dysfunction, neuronal apoptosis, and neuroinflammation in the development of neuropathic pain conditions has been discussed before . HoweverPeripheral nerve damage leads to abnormal changes in the activity and the metabolism of the damaged neurons, resulting in motor dysfunction, allodynia, and hyperalgesia at the injured site . In the (HBOT) group compared to the untreated group. These results align with previous evidence that HBO treatment decreases the expression of pro-inflammatory cytokines and enzymes  and enhances the expression of anti-inflammatory agents, such as IL-10, in nerve injury conditions [The dramatic changes in the immune cell activity and the neuro-immune signature of gene expression in the spinal cord that characterize animals with neuropathic pain might result from the hyperactivation of the somatosensory neurons and the over-nociceptive signal that can stimulate the release of pro-inflammatory cytokines from glial cells , 42 evennditions , 44, 46.(HBOT) group compared to the untreated group. Notable, in the SNC(HBOT) group, we noted a significant enhancement in the mechanical allodynia after 7 days of HBO treatment.As expected, the beneficial effects of reducing neuroinflammation after nerve injury were remarkably observed at the behavioral level. Consequently, the HBO treatment significantly modulates the development of neuropathic pain. Thus, we observed a lower level of mechanical and thermal allodynia/hypersensitivity among the SNC(HBOT)) was hampered after the SNC, the SNC(HBOT) shows a slight reduction in motor performance and superior performance in motor coordination and locomotor activity compared to the untreated group. More critically, the advantage of the HBO treatment was more pronounced between the motor test sessions. Indeed, only the SNC(HBOT) had a significant advantage of improved performance  between the testing days compared to the SNC group. Taken together, the results of the current study underscore the fact that HBO treatment has beneficial effects in preventing the development of neuropathic pain and motor dysfunction through reducing neuroinflammation after peripheral nerve injury.In addition, while the motor performance of both groups  compared to the SNC group. Moreover, we noted a significant reduction in the co-expression of TRPV1 at the protein level in cells that initiate the apoptosis pathway through cleaving Caspase-3 in the DRG.Interestingly, our result has shown that the elevated expression of the apoptosis marker cleaved-Caspase-3 at the protein level in the DRG was strongly associated with the overexpression of the TRPV1 channel in the SNC. Overall, after SNC, we found a significant increase in the transcription of the TRPV1 channel in the DRG and spinal cord. More importantly, in the DRG, we found a substantial increase in the expression of the TRPV1 channel at the protein level in cells that initiate the apoptosis pathway through cleaving Caspase-3. Unsurprisingly, our results are compatible with previous evidence that indicates the neuroprotective effects of HBO treatment in neuropathic pain conditions . Thus, w2+ influx into the cells, which causes hyperexcitability, mitochondrial stress, and ROS production [Previous evidence suggests that the elevated expression of the TRPV1 channel, frequently reported after peripheral nerve injury , plays aoduction . These coduction . Hence iCurrent results support the notion that the overexpression of the TRPV1 channel after nerve injury is associated with an increased level of mitochondrial stress . First, Following current results, we speculate that the overexpression of TRPV1 and TSPO in the spinal cord after nerve injury may promote mitochondrial dysfunction. To examine this hypothesis, we assessed the mitochondrial oxygen consumption in different respiration states after 7 days of nerve injury. The current result supports this hypothesis. Despite the normal mitochondrial respiration at the basal state in the SNC, challenging the mitochondria via stimulation/inhibition complex I, complex II, and ATP-synthase revealed that sciatic nerve injury reduces mitochondrial respiration in a resting state when electron transfer occurs without phosphorylation. In addition, the proportion of oxygen consumption specific to ATP synthase activity and complex II was reduced by nerve injury. Thus, the reduction of mitochondrial respiration was observed in states that include activation of ATP-synthase and complex II. For instance, the maximal mitochondrial respiration state (CI&CII and ATP-synthase) and oxidative phosphorylation state (CII and ATP-synthase). Additionally, reduced mitochondrial respiration was noted in the electron transfer (ET) capacity state. Notable, the HBO treatment enhanced the mitochondrial respiration in each respiration state compared to the SNC group.The reduction in the mitochondrial oxygen consumption in the different respiration states that we observed may result from a decrease in membrane potential that probably occurs under metabolically stressful conditions  that requires high ATP demand and activation of the mitochondrial corrective mechanism to reduce ROS. Previous evidence supports our hypothesis. Indeed, a reduction in membrane potential contributing to cytochrome-c release and a change in mitochondrial respiration following sciatic nerve injury were described before , 58, 59.2+ influx, and accumulation of cellular damage in the spinal cord as a result of the neuroinflammation, hyperactivation, and remodeling of the somatosensory neurons and the over-nociceptive signal from the injured nerve leads to losing the mitochondrial transmembrane potential resulting in energy crisis (ATP depletion), ROS production and cytochrome-c (Cyt-c) release, and TSPO overexpression. However, increasing the cellular oxygen by HBO at the injury site and in the spinal cord probably improves the electron transport chain by providing free electrons available for the electron transport chain (ETC). Additionally, the abundant oxygen supply as the final electron acceptor facilitates the efficient transfer of electrons and ATP production. More critically, as was proposed before by Soustiel, et al., (2008), the protective effect of HBO might be due to the negative regulation of the proapoptotic function of the TSPO [(HBOT) compared to the untreated group.Nevertheless, it’s possible that the increased metabolism level, Cathe TSPO . This miSeveral limitations of our study should be acknowledged. Here, we focused on the effect of the early HBO treatment on the development of neuropathic pain in a sciatic nerve crush model, thus the results obtained in the current study may not provide information to understand the effect of HBO treatment on other types of pain conditions , or the late effect of HBO treatment on mitigating neuropathic pain long after the development phase.Despite these limitations, our findings suggest that allodynia, hyperalgesia, and motor dysfunction induced by sciatic nerve crush is mediated by neuromodulation (TRPV1 upregulation), neuroinflammation, and elevated level of apoptosis in the DRG. To our knowledge, the current result represents the first report demonstrating the association between the TRPV1 channel, apoptosis, and neuroinflammation in neuropathic pain development. Furthermore, the current result indicates that the development of peripheral neuropathic pain is accompanied by mitochondrial dysfunction, neuroinflammation, and apoptosis in the spinal cord. Crucially, HBO treatment during the critical period of the nerve injury modulates the transition from acute to chronic pain and the development of motor dysfunction by reducing neuroinflammation, neuromodulation, and apoptosis in the DRG and the spinal cord. Additionally, the HBO treatment protects mitochondrial function in the spinal cord following sciatic nerve injury, probably via modulation of neuroinflammation and neuromodulation. HBO treatment is a non-invasive therapy and easy procedure to implement. Therefore, HBOT has an advantage over surgical intervention that has lower success ratio, and medication treatment which had a significant challenging problem such as non-specific drug delivery, stability, and the ability to cross the blood–brain barrier (BBB) , 62. TakAdditional file 1: Table S1. The gene primer sequences.


The classical osteoporotic signaling pathways include the four key genes  influencing the regulation of osteogenesis and osteoclastogenesis. This study investigates the expression of these four genes associated with bone remodeling during fracture healing.  Ovariectomized rats as an osteoporotic group were randomly divided into three groups-group A, group B, and group C. Nonosteoporotic rats as the control group were likewise divided into three groups A0, B0, and C0, using the same method. The rats were killed on the third day of fractures in groups A and A0, on the seventh day of fractures in groups B and B0, and on the fourteenth day of fractures in groups C and C0. The bone specimens were taken from the femoral fracture site, and the expression level of each gene in the bone specimens was detected using RT-qPCR, Western blotting, and immunohistochemistry.  LRP5, Runx2, and Osterix expressions were decreased in osteoporotic rat fractures and then increased over time. The expression of RANKL was elevated in osteoporotic rat bone specimens, which decreased after that.  The expressions of the four genes varied with time after fracture, which could be associated with the various stages of bone repair. The four genes can inform practice in ideal interventions in the prevention and management of osteoporosis. Osteoporosis is a metabolic disorder associated with systemic bone aging and degradation. Osteoporosis is characterized by decreased bone mass, structural degradation, increased brittleness, and susceptibility to fractures . Worldwiβ-catenin, BMP-2/Osterix, and RANKL/RANK signaling pathways include the key genes influencing the regulation of osteogenesis and osteoclastogenesis. LRP5, Runx2, and Osterix are the key osteogenic genes associated with the Wnt/β-catenin and BMP-2/Osterix signaling pathway, while RANKL is a key gene associated with the RANKL/RANK signaling pathways [Osteogenesis and osteoclastogenesis have been the subjects of interest in osteoporosis. The classical Wnt/pathways , 9. HoweFollowing the Animal Experimentation Ethics Guidelines, the Ethics Committee of Foshan Sanshui District People's Hospital approved the procedures of the study . A total of 100 Sprague–Dawley female rats  were purchased from the Guangzhou medical university. Then, the rats were housed in a specific pathogen-free (SPF) facility  with a 12/12-h light/dark cycle, and standard laboratory animal food and tap water were available ad libitum. First, the rat model of an osteoporotic model of ovariectomy (OVX)-induced osteoporosis was established by removing the ovaries bilaterally, and then, after 12 weeks, we selected 90 rats weighing 280–300 g. Another 90 rats of the same age and weight were purchased as a control group (without being subjected to sham procedures). A micro-CT  for small animals was used to determine the femur bone mineral density (BMD) in all rats. Ovariectomized rats were sure to be Osteoporotic rats. With the anesthetic, an osteotomy was established using an oscillating sagittal saw at the left proximal femur in all rats, and then the incision was closed. Ninety osteoporotic rats were randomly divided into groups A, B, and C, with 30 cases in each group. Ninety nonosteoporotic rats were randomly divided into groups A0, B0, and C0, with 30 cases in each group. The rats were killed on the third day of fractures in groups A and A0, on the seventh day of fractures in groups B and B0, and on the fourteenth day of fractures in groups C and C0. A bone specimen was taken from the fractured femur when the rats were killed. Specimens  were collected from the fracture sites. Specimens were collected and stored immediately in liquid nitrogen.−ΔΔCT) equation.Briefly, 100 mg of bone tissue was first ground in liquid nitrogen. Total RNA was then extracted from the bone tissues using the RNAiso Plus kit . cDNA was synthesized from the RNA using PrimeScript RT Master Mix . The DNA was amplified using Real-time PCR (RT-qPCR) with the SYBR Premix kit . Primers used in this research were synthesized by Thermo Fisher Scientific company. Conditions of PCR were as follows: initial denaturation at 94°C for 5 minutes, 30 elongation cycles at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 40 seconds, and final extension at 72°C for 10 minutes. GAPDH was used as the internal control. The amplification of the DNA was expressed based on the Ct . The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody  at room temperature for 2 h. The bands were visualized by using enhanced chemiluminescence (ECL) reagent kit  and semiquantified with Image J software.Same with the aforementioned method, 200 mg of bone tissue was ground. Total protein was extracted from bone tissues using RIPA lysis buffer . The protein concentration was determined using a BCA assay kit . A total of 30 μm thick sections and treated with 3% hydrogen peroxide for 10 min. Afterward, the sections were rinsed with phosphate-buffered saline, incubated with primary and secondary antibodies  sequentially, and exposed to DAB . The sections were counterstained with hematoxylin solution . Ten visual fields were randomly selected and observed under a high magnification microscope , and the number of positively stained cells was calculated.Intercepted bone mass was thawed, fixed in 10% neutral formalin for 48 hours, and embedded in paraffin after decalcification in 10% EDTA solution . The specimens were then cut into 5 s). A one-way analysis of variance was used for multigroup comparison, and differences between the two groups were determined by LSD t-test. P < 0.05 was considered statistically significant. Data were analyzed using GraphPad Prisma 5 software and SPSS V. 22.0 .All experiments were performed at least three times. Continuous data were expressed as mean ± standard deviation (±P > 0.05). The weights (g) of the whole osteoporotic and control groups were 286.3 ± 6.4 and 286.0 ± 2.6, without significant differences (P > 0.05).None of the rats died during the experiment. The weights (g) of groups A, B, C, A0, B0, and C0 were: 285.3 ± 2.5, 287.3 ± 3.6, 283.6 ± 7.4, 284.6 ± 6.2, 287.3 ± 8.9, and 286.5 ± 8.0, respectively, and no significant differences existed between any two groups (2) of groups A, B, C, A0, B0, and C0 were 0.42 ± 0.25, 0.44 ± 0.06, 0.43 ± 0.14, 0.21 ± 0.02, 0.23 ± 0.09, and 0.22 ± 0.08, respectively. There were no significant differences between any two groups in groups A, B, and C, and there were no significant differences between any two groups in groups A0, B0, and C0 (P > 0.05). The values of BMD (g/cm2) of the whole osteoporotic group and control group were 0.43 ± 0.18 and 0.22 ± 0.05, and there was a significant difference between them (P > 0.05).The values of BMD (g/cmP < 0.05), while the level of RANKL was higher in the osteoporotic group than in the control group (P < 0.05)  .P < 0.05). However, the differences between groups A0, B0, and C0 were insignificant across the four genes (P > 0.05). The elevated expressions of LRP5 and Runx2 were found lowest in group A (the third day) than in groups B (the seventh day) and C (P < 0.05), and the elevated expressions of Osterix were highest in group C (the fourteenth day) than in groups A and B (P < 0.05), while the depressed expressions of RANKL were highest in group A (the third day) than in groups B and C (P < 0.05). The differences in LRP5 and Runx2 between groups B and C, Osterix between groups A and B, and RANKL between groups B and C were not found to be significant (P > 0.05) , and OPG (a pseudoreceptor). Given the high affinity between OPG and RANKL, OPG can competitively inhibit the interaction of RANKL and RANK, further disrupting the differentiation of osteoclasts . The difβ-catenin, BMP-2/Runx2/Osterix, and RANKL/RANK pathways regulated osteogenesis and osteoclastogenesis in osteoporotic fractures of rats. The expressions of the four genes associated with bone remodeling during fracture healing varied with time after fracture, which could be associated with the various stages of bone repair. The characteristic variations in the expression of the four genes may inform future ideal interventions in preventing PMOP and managing PMOPF.Although we did not investigate the mechanisms underlying the abnormal variations of LRP5, Runx2, Osterix, and RANKL in the bone specimens of osteoporotic rats, our findings provided strong evidence that the Wnt/