Ctxn3+Müller and Ctxn3−Müller cells. We analyzed the characteristics and significant differences between the two groups of Müller cells and emphasized the importance of the Ctxn3+Müller subgroup in diseases. In endothelial cells, we found possible mechanisms of self-protection and adhesion and recruitment to pericytes. In addition, we constructed a communication network between endothelial cells, pericytes, and Müller subsets and clarified the complex regulatory relationship between cells. In summary, we constructed an atlas of the iBRB in the early stage of DR and elucidate the degeneration of its constituent cells and Müller cells and the regulatory relationship between them, providing a series of potential targets for the early treatment of DR.As the basic pathological changes of diabetic retinopathy (DR), the destruction of the blood-retina barrier (BRB) and vascular leakage have attracted extensive attention. Without timely intervention, BRB damage will eventually lead to serious visual impairment. However, due to the delicate structure and complex function of the BRB, the mechanism underlying damage to the BRB in DR has not been fully clarified. Here, we used single-cell RNA sequencing (RNA-seq) technology to analyze 35,910 cells from the retina of healthy and streptozotocin (STZ)-induced diabetic rats, focusing on the degeneration of the main cells constituting the rat BRB in DR and the new definition of two subpopulations of Müller cells at the cell level,  Due to the continuous increase in the global prevalence of diabetes, diabetic retinopathy (DR) is still the main cause of vision loss in many developed countries . Among 2The BRB is composed of the inner BRB (iBRB) and the outer BRB (oBRB), which regulate the movement of liquids and molecules between the ocular vascular bed and retinal tissue and prevent macromolecules and other potentially harmful substances from penetrating the retina . The iBRTo solve the above problems, researchers have used single-cell RNA sequencing (scRNA-seq) technology. We used five rat retinal samples , constructed a single-cell map of a total of 35,910 transcripts, and explored the cellular mechanism of iBRB injury in the early stage of DR. Single-cell sequencing can better reveal the structure of the iBRB and the interaction between iBRB cells through high-resolution detection of each cell in the retina.We explored the characteristics of pericytes, endothelial cells and two Müller cell subtypes, and constructed a regulatory network among them in the early stage of DR. Our research once again emphasizes the important role of the iBRB and provides a new target for protecting the iBRB and inhibiting the further development of DR, which has far-reaching clinical significance and social value.To simulate early DR, we used streptozotocin (STZ) to induce type 2 diabetes in rats. We stripped the retinas of 5 rats  and isolated and diluted these samples for single-cell transcriptome sequencing . After qBased on CellChat analysis, we found that endothelial cells, pericytes, and Müller cells have more complex connections and communication modes than other cells . These cApoe, Glul, and Clu), we identified two groups of Müller cells and named the two groups of cells Ctxn3+Müller and Ctxn3−Müller  and aquaporins (AQP4). GSTs protect retinal neurons from oxidative damage. AQP4 is involved in the drainage process of the neuroretina and avoids retinal tissue edema. Interestingly, glutathione transferase and AQP4 are more highly expressed in Ctxn3+Müller cells. In addition, the overexpression of matrix metalloproteinase (MMP-14) and inhibitor of DNA binding/differentiation (ID) were observed in Ctxn3+Müller cells. The former participates in the degradation of collagen in the extracellular matrix, which has an important protective effect on the fibrosis of retinal tissue after ischemia and hypoxia. Moreover, MMP-14 and Id proteins were related to neocapillaries. In terms of inflammation, Ctxn3+Müller cells specifically overexpressed the vascular adhesion molecule VCAM1, which may suggest its possible role in the pathogenesis of diabetic microangiopathy. Furthermore, Müller cells can release inflammatory factors such as interleukin 33 (Il33), suggesting that Ctxn3+Müller cells can participate in the inflammatory response in the retina. Interestingly, however, many genes related to photosensitivity, such as Pde6b and Cngb1, were observed to be highly expressed in some Ctxn3−Müller cells. Combined with previous studies, Müller cells may exist as a special type of optical fiber while researchers have found that Müller cells also have an inseparable relationship with photoreceptor differentiation. However, our data cannot avoid the possibility of photoreceptor containment and whether Ctxn3−Müller cells have these effects here is our further research content  AP-1 family members, such as Jun, Fos, and Jund; and (2) SOX family members, such as Sox2 and Sox9. We showed the expression of target genes downstream of important transcription factors with UMAP plots  and transforming growth factor Tgf-β  superfamily. We observed that Sox2 and Sox9 jointly regulate many genes related to Müller cell structure and function, such as retinol-binding protein 1 (Rlbp1), receptor Gpr37, and integrated membrane protein. Many studies have shown that Notch signaling is an important regulator of developmental and postdevelopmental processes. Notch2 and the Notch downstream genes Hes1 and Sox9, which are abundant in mature retina, are important factors for retinal Müller cells to maintain structural stability and development and survival  and occludin (Ocln). Claudin-5 is the main structural determinant of the paracellular endothelial barrier, which can promote the closure of tight junctions, thereby reducing vascular permeability and enhancing endothelial barrier function. Occludin is also a transmembrane component of tight junctions between the endothelium, which can regulate the permeability of the BRB. Platelet-derived growth factor receptor beta (pdgfrβ), which plays essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells, was highly expressed in clusters 20 and 27. Therefore, we defined these two cell groups as pericytes, named Pericyte_A and Pericyte_B, respectively  is the main mediator in response to Vegf stimulation  is highly expressed in endothelial cells and is an important factor that aggravates vascular endothelial cell damage and promotes the development of retinopathy (Ccn2), which is related to the pathogenesis of microvascular complications in diabetes  and nuclear factor erythrocyte-related factor 2 (Nfe2l2). The lack of the former is a factor leading to severe endothelial inflammation . In ischemic diseases such as DR, the upregulation of Angpt2 inactivates Tie2, leading to vascular leakage, pericyte loss, and inflammation. The coexpression of Angpt2 and Vegfa will accelerate neovascularization in developing retina and ischemic retina models (Igf1), and the overexpression of Igf1 may lead to the accumulation of Vegf. These changes increase the permeability of blood vessels and are related to the loss of vascular tight junction integrity  showed that Pericyte_A had increased ECM receptor interactions and other pathways, while vascular smooth muscle contraction and actin cytoskeleton regulation and other pathways were significantly enriched in Pericyte_B . CombineFlt1) and Vegfr2 (Kdr) receptors are simultaneously expressed on endothelial cells, and Vegf mainly exerts its effect by binding to Vegfr2. Notably, Müller cells themselves also express Vegfr. We speculate that Vegf produced by Müller cells can also act on themselves, but the effect is not clear. However, studies have shown that the Vegf/Vegfr2 signaling pathway is most likely to affect the biological synthesis, secretion, or degradation of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) in Müller cells  is secreted by endothelial cells and not only acts on Müller cells and pericytes but also affects itself . PreviouThe iBRB is an important structure that ensures the homeostasis of the retina and plays an important role in the pathogenesis of various retinal diseases. It is important to further study and explore the related mechanisms of the steady state and destruction of the blood–retinal barrier to promote the treatment of diabetic retinopathy. Due to the heterogeneity of the retina and the loss of cell type specificity, high-resolution methods such as scRNA-seq with bulk RNA-Seq approaches have inestimable value for the study of the destruction mechanism of the iBRB in DR. Using scRNA-seq, we identified two different Müller cell subsets and constructed a communication network among endothelial cells, pericytes, and Müller cells in the iBRB.Ctxn3+ Müller and Ctxn3− Müller, according to the expression of the Ctxn3 gene. However, our immunofluorescence experiment results are somewhat ambiguous, and since our results are based on the scRNA-seq, they may just reflect a series of Müller cell phenotypes, so more accurate experiments are needed to verify the correctness of clustering. Our work is consistent with the previous observation that Müller cells participate in the formation and regulation of nerves and blood vessels by secreting cytokines to maintain BRB integrity. Interestingly, there were significant functional differences between the two groups of Müller cells. Our analysis data showed that Ctxn3+Müller cells specifically overexpressed many genes related to metabolic waste clearance of the BRB, such as aquaporin AQP4 and GABA transporter. However, Ctxn3− Müller cells showed upregulation of many genes related to photosensitivity, such as Pde6b and Cngb1. In addition, compared with Ctxn3− Müller cells, Ctxn3+ Müller cells upregulated the regulation of differentiation inhibitors (Id1-4) and transcription factors Hes1 and Sox9, suggesting that they may have potential roles in promoting endothelial injury and angiogenesis. The above research shows that there is a special Müller cell subpopulation, Ctxn3+ Müller, which may have important significance in maintaining iBRB and early retinal vascular leakage. It is urgent to reduce the damage of retinal Müller cells to iBRB and thus hinder the progress of DR. Therefore, further research is needed to determine the more specific mechanism of Ctxn3+ Müller cells in the early stage of DR.Müller cells are important cells for maintaining the normal structure and function of the iBRB, especially in cell communication. We defined two new Müller cell subpopulations, Dll4, Bmpr2, and Nrf2 were highly expressed in endothelial cells. Inhibition of the Dll4-Notch1 pathway promotes excessive sprouts and endothelial proliferation in the retina  diluted with sterile fresh citrate buffer  to establish the diabetic rat model Animals believed to have diabetes were maintained on a high sugar diet, and tail vein blood samples were regularly collected and measured to assess blood glucose levels. The control group was fed with D12450B 10 kcal% feed, and the experimental group was fed with D12492 60 kcal% feed. The imported feed is produced by American Research Diets. The rats in the experimental group  and the control group were anesthetized by intraperitoneal injection and fixed in the supine position. First, the eyeball was removed, and then, the corneoscleral margin was cut. The eyeball was radially incised, the anterior segment and vitreous were removed, and the iris structure was slowly separated from the intact retina and placed in a protective solution.The tissues were transported in sterile culture dishes with 10 ml of 1x Dulbecco’s phosphate-buffered saline  on ice. The residual tissue storage solution was removed, and the tissues were then minced on ice. We used 0.25% trypsin  and 10 μg/ml DNase I  dissolved in PBS with 5% fetal bovine serum  to digest the tissues. The tissues were dissociated at 37°C with a shaking speed of 50 RPM for approximately 40 min. We repeatedly collected the dissociated cells at intervals of 20 min to increase cell yield and viability. The cell suspensions were filtered using a 40 μm nylon cell strainer, and red blood cells were removed by 1X Red Blood Cell Lysis Solution . The dissociated cells were washed with 1x DPBS containing 2% FBS. The cells were stained with 0.4% Trypan Blue , and viability was assessed on a Countess® II Automated Cell Counter (Thermo Fisher).Beads with unique molecular identifiers (UMIs) and cell barcodes were loaded close to saturation so that each cell was paired with a bead in gel beads-in-emulsion (GEMs). After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. The beads were retrieved into a single tube for reverse transcription. In cDNA synthesis, each cDNA molecule was tagged on the 5′ end  with the UMI and a cell label indicating its cell of origin. Briefly, 10× beads were subjected to second-strand cDNA synthesis, adaptor ligation, and universal amplification. Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3′ ends of the transcripts linked with the cell barcodes and UMIs. All the remaining procedures, including library construction, were performed according to the standard manufacturer’s protocol (CG000206 Rev. D). The sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2,100 and with a Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). The libraries were sequenced on a NovaSeq6000 instrument (Illumina) in 2 × 150 bp mode.The Read10X function in the Seurat package (3.2.2) was used to merge the data from all samples into R (4.0.2) and generate an aggregated Seurat object . Low-quap value <0.01 and fold change [FC] >1.3). The R package clusterProfiler  for 3 h at room temperature. After immersion and fixation, the eyes were washed with phosphate-buffered saline (PBS), and the lenses were carefully removed. The eyes were immersed in 10% sucrose for 30 min, 20% sucrose for 2 h, and 30% sucrose overnight at 4°C for cryoprotection before being embedded in Tissue-Tek optimal cutting temperature compound (OCT) and frozen on dry ice. Eighteen micron sections were cut on a cryostat. The sections were blocked for 1 h with 10% normal goat or donkey serum in PBS with 0.5% Triton X-100, incubated overnight at 4°C with primary antibodies, and then incubated with secondary antibodies.SCENIC is a computational method for simultaneous gene regulatory network reconstruction and cell-state identification from single-cell RNA-seq data .1 We firAccording to the expression of TFs and target genes and their importance, we visualized the results using the Cytoscape tool . The interactions of each TF and target were merged manually to analyze the overall interactions. We also visualized the target genes of several important TFs with a UMAP plot.The datasets presented in this study can be found in the National Center for Biotechnology Information Gene Expression Omnibus database under accession number GSE209872.The animal study was reviewed and approved by the Science and Technology Commission of Shanghai Municipality.YW designed the experiments. YZ performed the data analysis and constructed the datasets. XY drew the figures. SY collected the biopsies and performed the immunofluorescence experiments. XY, QL, and YZ wrote the manuscript. YW, MF, GY, and MC supervised this project. All authors reviewed and edited the manuscript, contributed to the article, and approved the submitted version.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.https://www.frontiersin.org/articles/10.3389/fnmol.2022.1048634/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


Dry eye disease (DED), a multifactorial ocular surface disease, is estimated to affect up to 34% of individuals over 50 years old. Although numerous animal models, including rodents and rabbits, have been developed to mimic the pathophysiologic mechanisms involved in dry eye, there is a lack of non-human primate (NHP) models, critical for translational drug studies. Here, we developed a novel desiccating stress-induced dry eye disease model using Rhesus macaque monkeys. The monkeys were housed in a controlled environment room for 21 to 36 days under humidity, temperature, and airflow regulation. Following desiccating stress, NHPs demonstrated clinical symptoms similar to those of humans, as shown by increased corneal fluorescein staining (CFS) and decreased tear-film breakup time (TFBUT). Moreover, corticosteroid treatment significantly reduced CFS scoring, restored TFBUT, and prevented upregulation of tear proinflammatory cytokines as observed in dry eye patients following steroid treatment. The close resemblance of clinical symptoms and treatment responses to those of human DED patients provides great translational value to the NHP model, which could serve as a clinically relevant animal model to study the efficacy of new potential treatments for DED. Beyond hindering visual acuity, DED poses a great burden on the patient as it can result in continuous ocular discomfort, impacting daily activities and hampering one’s quality of life8. Depending on its etiology, DED is primarily classified into aqueous deficient dry eye disease (ADDE), resulting from reduced tear secretion, and evaporative dry eye disease (EDE)11. Both etiologies result in the disruption of the homeostasis of the tear film, which triggers a cascade of ocular inflammation and subsequent damage11. Tear film breakup time (TFBUT) and corneal fluorescent staining (CFS), measuring tear film instability and corneal epitheliopathy, respectively, are used to measure the extent of dry eye disease2.Dry eye disease (DED), a highly prevalent multifactorial inflammatory disorder, results in symptoms of ocular discomfort, tear film instability, and visual disturbance12; thus, treatment has been focused on regulating the immune pathway to suppress corneal inflammation13. Despite the significant advancement in our understanding of DED and its extensive patient population, only few non-specific anti-inflammatory drugs with moderate efficacy and low tolerability are available for the treatment of DED14. The colossal discrepancy between the demand for DED-specific treatment and the development of such drugs is partly due to the limited informative animal models to study the translational drug efficacy16. A variety of experimental animal models have been developed to study the pathogenesis of DED; however, the species of choice have largely been limited to rodents and rabbits18. Although murine and rabbit models provide the benefit of wide availability and ease of manipulation, their anatomical, physiological, and immunological characteristics of the ocular surface differ from those of humans19. As the eyes of non-human primates (NHP) most closely resemble the human eye, Qin et al.4 have developed a severe dry eye disease model in rhesus monkeys by excising the lacrimal gland; however, this is an irreversible aqueous-deficient dry eye disease model, and currently, there is no NHP model of evaporative dry eye disease. Moreover, current animal models of EDE utilize both the dry eye chamber and atropine or scopolamine injection16, which concurrently suppresses immune cell infiltration20, hindering an accurate assessment of the pharmacodynamics in potential drug studies.Since being recognized as a disease in the 1990s, our understanding of the underlying pathophysiology of DED has greatly expanded. Ocular surface inflammation, especially perpetuated by T cells, damages the corneal barrier in DED11, and the need for an animal model that closely resembles humans, the purpose of our study was to develop a novel NHP model of EDE for the use of pharmacokinetic and pharmacodynamics studies. Here, we induced DED by exposing the NHPs only to the controlled environment room without the utilization of secondary pharmacology intervention. The monkeys developed DED, similar to those observed in human patients, as demonstrated by increased corneal fluorescein staining score, upregulation of inflammatory cytokines, and decreased TFBUT and tear volume.Given the greater prevalence of dry eye of EDE etiologyDuring the induction of dry eye disease and treatment, the monkeys were housed in a controlled environment room with a relative humidity maintained at 6.4% ± 1.4% , which remained high through day 21 , and day 36 , compared to the baseline , corneas showed minimal or no punctate staining (3.5 ± 1.5) Fig. A. A statp = 0.007) and day 21  following dry eye induction  and 5 min  compared to their naïve baseline control  , tear film break-up time decreased significantly on day 14  and IL-2 (p < 0.05), and a 1.5-fold increase in IFN-γ and TNF-α  and day 14     Fig. A. Simila01) Fig. A. Intere01) Fig. B. In con24, affecting millions of patients in the US alone25. Here, we develop for the first time a non-human primate model of evaporative dry eye. Our findings demonstrate that by housing the monkeys in a controlled environment room of desiccating stress, the monkeys develop experimental DED, as evidenced by increased corneal epitheliopathy, reduced tear film breakup time, and ocular surface inflammation. This new model greatly advances the experimental tool of studying dry eye and its therapeutics as the non-human primates closely resemble the anatomical, physiological and immunological systems of humans27.Dry eye disease is a significant public health issue4. Although murine and rabbit models have been beneficial in studying the underlying pathophysiology of DED, distinctions in anatomical and physiological differences between mice and humans have limited translational research27. Thus, we developed a new experimental animal model utilizing non-human primates and a well-established dry eye model of desiccating stress. Since its first introduction in the early 2000s, the dry eye model of desiccating stress using a controlled environment chamber has been widely used to study evaporative dry eye disease28. Similarly, rhesus monkeys were housed in a controlled environment room with relative humidity below 15% and a constant airflow of 12 L/min. The monkeys did not exhibit any stress behaviors, and their body weight remained relatively constant throughout the entire study, demonstrating that the experimental conditions did not induce excessive stress. The animals developed clinical symptoms of DED by day 14 following desiccating stress induction, as shown by increased corneal epitheliopathy, decreased tear volume and disruption to tear film stability. As indicated by an increase in CFS score, significant corneal epitheliopathy was observed by day 14 remained high throughout the experiment until day 36. Moreover, to measure tear film instability, a hallmark of dry eye disease29, tear film breakup time was measured in the monkeys throughout the experimental conditions. Significant shortening in tear film breakup time was observed following desiccating stress exposure. Similar to the aqueous-deficient dry eye model in rhesus monkeys4, our EDE non-human primate model showed significant reduction in tear volume, as indicated by a two to threefold shorter Schirmer test readings following DED induction. In conjunction, a significant upregulation of proinflammatory cytokines was observed in tears. Our data clearly demonstrate that the controlled environment room effectively induces EDE in non-human primates.Due to its multifactorial etiologies and symptoms, numerous experimental dry eye animal models have been developed30. Given their high degree of resemblance in physiological responses and anatomy, non-human primate model is considered the gold standard of animal models for drug development and approval by the FDA26. In particular, the single main lacrimal gland and larger eye size4, similar to those of humans, make NHP an ideal model to study not only the therapeutic efficacy but the pharmacokinetics and effective drug delivery to the eye15. Despite its many benefits, only one NHP model of DED has been characterized due to the costs and need of a special facility to house non-human primates. Qin et al.4 have demonstrated that complete excision of the principal lacrimal gland results in severe dry eye disease. However, the lacrimal gland excision model is an effective model to study aqueous deficient dry eye disease and not EDE, which affects a larger population of dry eye patients11. Moreover, complete excision induces irreversible damage and is not an ideal model to study the progression and underlying mechanism of therapeutic improvement. In contrast, the new NHP model of desiccating stress-induced DED allows observation of pathophysiological reversal and progression of disease in response to treatment. Topical corticosteroid, a potent anti-inflammatory drug, reduces signs and symptoms of DED by suppressing inflammation9. To test the comparability of our NHP model to DED observed in humans, including therapeutic response, monkeys were treated with a known DED treatment of 1% prednisone. Following prednisone treatment, a significant improvement in CFS scoring was observed relative to the placebo treatment. Moreover, the treatment restored tear homeostasis and prevented the exacerbation of ocular inflammation, as exemplified by the normalization of TFBUT and the maintenance of tear inflammatory cytokine levels. Despite being exposed to continuous desiccating stress, we observed a trend of Pred Forte treatment controlling the upregulation of tear cytokine levels, suggesting that following more prolonged treatment and without continuous desiccating stress, our primates will have a similar therapeutic response to those seen in the clinic. Taken together, these data demonstrate that the NHP EDE model resembles not only the clinical symptoms and signs of EDE but also the therapeutic response, highlighting it as an effective model to study the therapeutic efficacy of new dry eye disease drugs in the preclinical setting.The disparity between the growing DED patient population and the lack of effective targeted treatment is primarily due to the limited successful translation of preclinical studies to the clinic4. Our model does not utilize any invasive manipulation of the monkeys, and the controlled environment room does not induce excessive stress, as exemplified by their stable weight and lack of abnormal behavior. In addition, the NHP model of the controlled environment room is superior to the murine model utilizing a controlled environment chamber not only in the anatomical similarity between the human and monkeys but in that the environment more closely resembles those of humans. When housed in their cages, mice are constantly exposed to the dust from the bedding and bodily fluid buildups in the cages. Such irritants can confound the pathogenesis and therapeutic efficacy but are absent in the controlled environment room NHP model.One of the biggest limitations of using NHP experimental models is the ethical concerns15. We have successfully developed a new NHP model of evaporative dry eye disease, which shows comparable clinical symptoms and therapeutic response to patients with dry eye disease. As an effective preclinical validation model, the NHP model will provide an invaluable tool for novel drug development and vastly improve the efficacy of translational research.Murine and rabbit models have enormously expanded our understanding of the pathophysiology of dry eye disease; however, due to cross-species differences, the existing models have limited efficacy in developing dry eye therapiesM. mulatta) between the age of 4 to 5 years were used in these experiments. Monkeys had free access to drinking water and were fed with monkey chow . In addition, daily allotment of fruits, vegetables, or additional supplements and various toys were also provided. All experimental protocols (AW2038) were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sichuan Primed Shines Bio-tech Co., Ltd.. No animals were sacrificed for the purposes of this work. This study adhered to the tenets of the Declaration of Helsinki and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Association for Research of Vision and Ophthalmology guidelines.Twelve female Rhesus macaque monkeys (The monkeys were housed in a controlled environment room with relative humidity (RH) below 15%, an airflow of 12 L/min, and at a temperature of 21 °C to 26 °C, for 36 consecutive days. Dry eye was clinically evaluated using corneal fluorescein staining on days 0, 14, 21, and 36 post-dry eye induction.Twenty-one days after the induction of dry eye, the monkeys in the controlled environment room were randomly divided into two treatment groups (n = 6 monkeys/12 eyes in each group): (1) a group receiving topical normal saline as placebo, (2) a group receiving topical Pred Forte 1% . Two drops of normal saline and Pred Forte 1% were topically applied to both eyes of unanesthetized monkeys three times a day for 14 days . Dry eye was clinically evaluated using fluorescein staining on treatment days 0, 7, and 14 (treatment day 0 equates to post-induction day 21).17.Corneal fluorescein staining was performed on days 0 (baseline), 14, 21, and 36 after induction of dry eye and on days 0, 7, and 14 of the treatment regimen. Ten microliters of liquid 10% fluorescein  were applied to the inferior-lateral conjunctival sac of the monkey, and after 10 min, corneal fluorescein staining was examined under the cobalt blue light using a slit lamp biomicroscope . Punctate staining was evaluated in a masked fashion using the National Eye Institute grading system, giving a score from 0 to 3 for each of the five areas of the cornea11. Ten microliters of preservative-free solution of 2% fluorescein was applied to the conjunctival sac with a micropipette. The monkeys were made to blink three times by an ophthalmologist to ensure adequate mixing of the dye. The time interval between the last complete blink and the appearance of the first corneal black spot, indicating disruption to the tear film, was measured using a stopwatch. The background illumination intensity was kept constant , and an integrated yellow filter was used to enhance the visibility of the tear film over the entire cornea. TFBUT was measured three times per eye, and the mean value of the measurements was calculated.Tear film breakup time was measured following the guidelines outlined by the Report of the International Dry Eye Workshop (DEWS) 20174. In brief, a 35 mm Schirmer test strip  was inserted into the lower conjunctival fornix at the junction of the middle and lateral third of the lower eyelid margin. Eye lids were gently closed, and the extent of wetting was measured after 1 and 5 min, respectively.Schirmer I test following local anesthetic application was performed on days 0 (baseline), and 21 after induction of dry eye to measure the basic tear secretion in the monkeys as described previouslyMonkeys were laid on a table in a supine position after being anesthetized with intramuscular injection of 10 mg/kg ketamine hydrochloride . Thirty microliters of phosphate-buffered saline were instilled into the inferior fornix, and monkeys were manually made to blink eight times. A total of 25 μL of tear wash was collected with a micropipette from the lateral canthus. To minimize ocular surface irritation, we collected the tear wash immediately after the application. The tear wash was placed into a 1.5 mL Eppendorf tube and stored at − 80 °C until further examination.LEGENDplex™ bead-based immunoassays  were used to measure the levels of IL-17, IL-2, TNF-α, IFN-γ in tears according to the manufacturer’s instructions. Briefly, 25 μL of the solution composed of the tear, standard, mixed beads, and buffer solutions were added to each tube. Next, the tube was spun at 800 RPM and incubated for 2 h at room temperature. After three washes to remove the unbound proteins, 25 μL of corresponding detection antibodies were added to each tube. The tube was then spun at 800 RPM and incubated for another hour at room temperature. After three washes to remove the unbound detection antibodies, the samples were read on a flow cytometer  and then analyzed using the LEGENDplex™ V8.0 software .The groups were compared by paired or unpaired two-sample t-tests. All values are expressed as mean ± standard error of the mean (SEM). A p value < 0.05 was considered statistically significant. All statistical analysis was performed using Prism 9 software.


Datura innoxia, was monitored by some physio-biochemical indices.A potential solution for recycling and reusing the massively produced sewage water (SW) is to irrigate certain plants instead of highly cost recycling treatment. Although the extensive and irrational application of SW may cause environmental pollution thus, continual monitoring of the redox status of the receiver plant and the feedback on its growth under application becomes an emergent instance. The impact of SW, along with well water (WW) irrigation of medicinal plant, D. innoxia plants compared to the WW irrigated plants. The total chlorophyll, carotenoid, non-enzymatic antioxidants, viz. anthocyanin, flavonoids, phenolic compounds, and total alkaloids increased by 85, 38, 81, 50, 19, and 37%, respectively, above WW irrigated plants. The experiment terminated in enhanced leaf content of N, P, and K by 43, 118, and 48%, respectively. Moreover, stimulation of carbon and nitrogen metabolites in terms of proteins, soluble sugars, nitrate reductase (NR) activity, and nitric oxide (NO) content showed significant earliness in flowering time. The SW application improved not only Datura plants’ quality but also soil quality. After four weeks of irrigation, the WW irrigated plants encountered nutrient deficiency-induced stress evidenced by the high level of proline, H2O2, and MDA as well as high enzyme capabilities. Application of SW for irrigation of D. innoxia plant showed the improvement of secondary metabolites regulating enzyme phenylalanine ammonia-lyase (PAL), restored proline content, and cell redox status reflecting high optimal condition for efficient cellular metabolism and performance along the experiment duration.The SW application amplified the growth, yield, minerals uptake, and quality of D. innoxia and the feasibility of generalization on multipurpose plants grown in poor soil.These evidences approved the benefits of practicing SW to improve the yield and quality of  Vast ef by 2025 , 8. Howe by 2025 .The Egyptian government is rising significant concerns about human health from SW-irrigated crops; thus, inedible plants (grasses and forest trees) have been replaced compared to crops grown in control, where SW was not previously applied . Using SDatura received high awareness nationally [Datura were cultivated for secondary metabolites production. Datura innoxia, a perennial plant with inedible parts, is a commercially important plant for having a broad range of heavy market demand for bioactive metabolites due to their extensive use in medicine, where its leaves contain tropane alkaloids with pharmacological activities and significant medicinal properties such as atropine, scopolamine, and hyoscyamine that are used as parasympathicolitics as having the ability to suppress the parasympathetic nerve activity [Datura via following up some growth parameters in terms of plant dry weight (DW), branches, and a number of flowers, besides pigments content, secondary metabolites content, nutritional profile, and redox status in terms of stress markers and membrane damage traits, as well as malondialdehyde (MDA) as a lipid peroxidation marker, H2O2, and its quenching enzymes. Moreover, the study of antioxidant response in addition to metabolites in the inedible commercially valuable plant, Datura innoxia, may collectively help donating new reference information for underlining the possibility of practicing SW and the feasibility of generalization on large-scale plants. Thus, plant growth assessments and the characterization of enzymatic as well as non-enzymatic antioxidant responses were parallel conducted. This research also investigated the changes in soil physicochemical parameters as a secondary objective.Recently, the growth of medicinal plants such as tionally . Many spactivity , 14. Plaactivity . Some practivity , 16. Thi− 1, respectively) than that of WW . Moreover, almost all measured nutrients determined in the SW samples collected from the Arab Elmadabegh sewage line were higher than the corresponding criteria of the WW (tap water). However, the micronutrients and HMs in the SW herein are relatively low and within the limits of standard concentrations.Data shown in Table Physico-chemical characteristics of the soil were interpreted in Table Data depicted in Table p < 0.0001) increased plant biomass in terms of DW with a percent increase amounted to 163%, while plants irrigated with WW increased by only 26% when compared to corresponding starter values .The obtained results in Table Datura innoxia’s morphological feature in terms of branching and flowering criterion. Early branching and flowering resulted within the 2nd and 4th week, respectively, submitted the highest number of branches (4 branches/plant) and flowers (7 flowers/plant) by the end of the experiment. In contrast, the WW irrigated plants exhibited branch initiation at 5th week and floral initiation at 7th week with final relatively low branch and flower number (2 branches and two flowers/plant).Visibly, the application of SW irrigation had a potential impact on SW application positively influenced the photosynthetic pigment synthesis in terms of the total chlorophyll and carotenoids over the corresponding values recorded in WW irrigated plants Table  on the 1Datura plants (P < 0.0001).Unlike the chlorophyll fluctuation, proline showed heterogeneous activities, and it was kept the baseline of control and restored by SW application along the study duration while it was promoted considerably by the end of the experiment with WW irrigation with a percent increase of 433% over the corresponding starter value (1.5 mg/g DW). The displayed data in Table Datura plants. Amino acids, protein, and soluble sugars content were accumulated by the irrigation of SW and reached the maximum values in plant leaves after the 5th week of irrigation with percent increases amounting to 32, 105, and 59%, respectively, over the corresponding values recorded for WW irrigated plants and maintained the highest values up to the end of the experiment; thereby, improving final plant growth.The data presented in Figs. a, b, c d2 g/h) while their content kept low fluctuation and diminished at the end of the experiment under WW application with percent increase of only 54 and 120%, respectively.NO content of the leaves of SW-irrigated plants was elevated across the time of application with a maximum percent increase of 166% over the corresponding starter value (50 nmol/g FW) Fig. a. LikewiP < 0.0001) increased over time. Data depicted in Table P < 0.0001) on secondary metabolites content.SW irrigation noticeably increased secondary metabolites in terms of anthocyanins, flavonoids, phenolic compounds, and alkaloids recording final values with a percent increase of 81, 50, 19, and 37%, respectively, when compared to the corresponding values of WW irrigated plants Table . This auThe secondary metabolites-mediating enzyme, PAL, reached the maximal (122 μmol/mg protein/min) on the 10th week with a percent increase of 110% over the starter value Fig. d, wherea2O2, a stress marker) and membrane damage were evaluated as the levels of lipid peroxidation , as well as variations in enzymatic antioxidant capacities of Datura innoxia leaves, were monitored over a period of ten weeks for insight evaluation of the performance of Datura plants under different irrigations. Data in Fig. (a-b) revealed that irrigation of SW did not affect hydrogen peroxide (H2O2.). Conversely, MDA concentration was reduced considerably submitted the lowest values (7.04 μmol/g FW) at the 6th and the 7th week.Some more indicative physio-biochemical parameters, such as hydrogen peroxide  and micronutrients  in es Table , with a 3  caused soil alkalinity in many regions of Wadi El- Assiuty and the OM, total nitrogen and total phosphorus content ranged between 0.019–0.73%, 30–370 mg/kg and 2.2–10.7 mg/kg, respectively. This finding may be because of ammonium nitrification as well as organic compound oxidation [Water resource scarcity is a vital problem in several localities in Egypt; thus, recycling and the reuse of alternative resources is a potential solution for getting rid of that massively produced SW. Furthermore, irrigating of certain plants is a good substitution for highly cost recycling treatment; however, the over-use on land for irrigation practices imposes considerable hazards causing chemical pollution problems. However, the plant growth and physio-biochemical parameters evaluated in the current study for along ten weeks showed an enrichment response rather than a toxic one, which can be due to the SW proprieties normally varying according to its locality and the source from which it is produced . Additioxidation . The curxidation , who repxidation . The curxidation  who statxidation . Furtherxidation . Similarxidation  indicatexidation  and Mojixidation  revealedxidation , the curxidation , who men2+ enhances the rate of total electron transport from water to NADP. Mn2+ is essential for PSII (O2 evolving system). In addition, there is also a direct interaction between ferredoxin and copper in the reducing site of PSI [Datura plant biomass. Teixeira et al. [Datura plant leaves had the highest concentration of photo-assimilates, accelerating the transition from the vegetative to the flowering stage; consequently, flowering jointed with the triggering of photo-assimilate production in the green area, which was the primary source until reaching the reproductive. Chen and Chu [Ottelia acuminata flowering, and the total number of flowers increased as nitrogen content in SW increased. The early flowering was supported by highly upregulated nitrogenous metabolism of the leaves, where proteins and amino acids accumulation as a result of SW irrigation suggest that the leaves are well-constructed and have an elevated metabolic efficiency [Ten-week period was the limit of our study as the further application of WW completely stressed the plants grown in poor desert soil in terms of high values of ROS, alarming antioxidant capacity and decreasing primary metabolites manufacturing. Interestingly, SW irrigation exhibited substantial plant liveliness than WW irrigated plants, in addition to safely promoting characteristics of plants evaluated and continued for the 10 weeks. In this respect, the most substantial dry matter production and chlorophyll accumulation were recorded by SW application. This growth enhancement impact is compatible with Urbano et al. . Some mie of PSI . Consequa et al.  indicatea et al. , who foua et al. . Soluble and Chu  demonstrficiency . Moreoveficiency . In the ficiency  reportedficiency . In the Hence, the exacerbation of proline under WW irrigation across time was combined with the decrease in soluble protein generation. This apparent proline production was not always advantageous; rather, it may have been a negative consequence of extended WW irrigation of nutrient-poor soil. Göring and Thien  indicateNumerous studies indicated that NR substantially contributes to plants’ NO biosynthesis . TherefoDatura was the enhancement of secondary metabolisms and augmented production of highly valuable secondary metabolites that imparted the valuable medicinal properties and quality of Datura plant, viz. anthocyanins, flavonoids, phenolic compounds, and alkaloids. All of them meaningfully increased across the duration of cultivation owing to SW. It was suggested that SW irrigation improved de novo nitrogenous components synthesis, thus increasing the production of secondary metabolites compared to control [Another healthy effect stimulated in SW irrigated  control .Datura innoxia since it is a factory of interest gained alkaloids, it is noteworthy that the increase of alkaloids due to SW irrigation was concomitant with the low proline level along the study duration. This finding could be since ornithine, the precursor of tropane alkaloid, and the proline have the same precursor, namely: glutamic acid [Datura plants irrigated with SW [Regarding mic acid  that hea with SW . Further with SW . In cont with SW . Contrar2O2 that were concomitant with decreasing membrane integrity and stability evidenced by high MDA concentration, which may be another main reason for suppressing the growth rate of Datura plants under WW irrigation compared to SW irrigated plants. As stress indicators, the enhanced levels of these toxic molecules (H2O2 and MDA) indicated that plants under prolonged WW irrigation might encounter nutrient deficiency after the fourth week and thus undergo nutrient deficiency-induced stress. Tewari et al. [Whereas plants with WW irrigation exhibited elevated levels of Hi et al.  reportedi et al. .2O2 and MDA content maintained near the control values reflects the high optimal condition and maintained membrane function for effective cellular performance as well as metabolism that can be attributed to the stabilized cell redox status [Datura. Moreover, activating GST as a defensive mechanism alarming the cell may be due to some xenobiotic agrochemical loaded in the delivered SW, which may trigger wide disciplines of antioxidants and metabolic pathways that cumulatively improve leaf physiological status in Datura plants. Hence, SW irrigation positively influenced the growth and physiological parameter as well as valuable secondary metabolites production of Datura.The constant Hx status , leadingDatura leaves plants compared to WW irrigated ones. The contents were still within the critical limits recorded for the metals’ phytotoxicity [D. innoxia plants [Datura plant in the presence of SW indicated suspected resistance against HMs accumulated in the soil.However, SW irrigation manifested a noticeable accumulation of macro-  and micronutrients  in toxicity . Nitrogetoxicity  and are toxicity . In thistoxicity  stated ttoxicity  and Herntoxicity  stated ta plants  as well.a plants  suggestea plants . TherefoDatura innoxia, jointed with a well-furnished metabolic profile and positive antioxidative changes in response to SW irrigation, it can be deduced that SW could be safely reused for Datura cultivation while at the same time to provide poor soil with adequate amendments. Hence, the soil was fertilized by the nutrient favorable to plant performance and development rather than be diseased. Nevertheless, the existence of high concentrations of HMs and traces of some toxic compounds lead to the instance for continual monitoring of the redox status of the receiver plant and the feedback on its growth. In that case, it will have priority for irrigation purposes. Thus, experiencing the crises of water scarcity and costive fertilizers, sewage might be effectively generalized for irrigation multipurpose plants.The ten-week monitoring period was the timeframe/limit of our study as the further application of WW completely stressed the plants grown in poor desert soil in terms of high values of ROS and alarming antioxidant molecules and the dramatically diminished primary metabolites’ content. Optimistically, according to the revealed results regarding healthy growth and improved medicinal property of The experiment was conducted over ten weeks, from March to mid-May 2019. Two water samples were collected, one from Arab Elmadabegh, Assiut, Egypt (27°12 N and 31°09 E), where the most extensive sewage line in Assiut governorate  (source 3) [3) was estimated by calcimeter method [For both water and soil, soluble Ca and Mg concentrations were measured using the EDTA titration method, and Na and K were estimated using a flame photometer. OC for soil was evaluated by adopting the method of Jackson . Soil to3) . Cation 3) . Free car method . Availabr method . The pH r method . Electrir method . Water aThe experiment was performed during spring 2019. Plants were gathered from the botanical garden of the Faculty of Agriculture, cut into 160 uniform/same-sized plantlets of approx. 25 g. Plantlets were randomly divided into two equal groups; WW and SW irrigations. Afterward, plantlets were washed with fresh water and weighed before being transplanted into the pots. The pots  were organized in a completely random arrangement with four replicates for each group, and plantlets were transplanted at the rate of 20 plantlets/pot. Immediately, they were transported to a greenhouse at the Department of Botany and Microbiology, Faculty of Science, Assiut University, receiving natural light (transmitted through glass panels) under ambient sunlight, the temperature ranging between 27 and 38 and 12–15 °C at day and night, respectively; relative humidity (34–42%) and reference evaporation (4.65–5.48 mm).At the beginning of the experiment, using watering can, the dry soil received the WW and SW in field capacity at the rate of 180 ml/kg, soil with polyethylene bags to avoid soil treatment leaching. During cultivation, the frequency of SW was maintained once a week as per the plant water requirement (compensating the lost water via evaporation and maintaining the moisture level at field capacity). Along with experiment duration, soil water content was sustained in field capacity by weight method via the addition of WW day by day if required.The harvest of all treatments was scheduled at 7-day intervals and performed by picking up the whole plant, root and shoot, from the soil. At the end of each week and for ten weeks, the observations were recorded on 2 randomly selected plants per replication per treatment for all indices. For the chemical analysis, 2 randomly selected plants were handled by blending as one sample.Fresh weight (FW) of harvested plants was determined immediately and then cleaned via thoroughly rinsed with distilled water to be oven dried at 60 °C to constant weight for two days to evaluate DW. The number of branches and flowers per plant was also calculated.Chlorophyll was extracted from 0.5 g of fresh leaves suspended in 5 ml of 95% ethyl alcohol at 60–70°C in a water bath. Absorbance readings were taken with a spectrophotometer (Unico UV-2100 spectrophotometer). Chlorophyll was estimated as mg/g FW at 663 and 644 nm using equations of Lichtenthaler .NO content was quantified according to Ding et al.  and Hu e2 g/hr. was estimated by adopting the described method of Downs et al. [3. The resultant Nitrite was detected by adding naphthyl-ethylenediamine dihydrochloride and sulfanilamide. Absorbance readings at 540 nm were taken with a spectrophotometer (Unico UV-2100 spectrophotometer).NR activity expressed as micromoles of NOs et al. , in whic2O, which was blended with 4.5 ml anthrone reagent and boiled in a water bath for 5 min before cooling down on an ice bath. The absorbance of the developed blue-green color was determined at 620 nm using a Unico UV-2100 spectrophotometer. Proline was determined in dry leaves. Leaves tissue was ground in 6 ml sulfosalicylic acid (3%) before the centrifugation of the mixture. Then, the outcome supernatant was mixed with 2 ml of glacial acetic acid as well as 2 ml of ninhydrin. The reaction mixture was extracted with 4 ml toluene to quantify at 520 nm [First, 1 ml of Stannus Chloride reagent was combined with 0.5 ml of the water extract, and then the tubes were heated in a water bath for 20 minutes and then cooled. The plant water extract was made by steeping 0.5 g of dry leaves in 10 ml of distilled water for 1 hour at 95 °C. The extinction of violet color was measured at 570 nm using the aforementioned reagents and distilled water instead of the extract of the plant sample . Solublet 520 nm .2CO3 (20%). The absorbance of the developed color was measured at 725 nm with a Unico UV-2100 spectrophotometer. The methanolic extract of fresh leaves was utilized to analyze flavonoids by the method by Harborne and Williams [3 (1:10) were added. After 5 min, 2 ml 1 M CH3- COOK was added, the total volume was made up to 10 ml, and absorbance was measured at 415 nm. A routine quantification method for analysis of the total alkaloidal content spectrophotometrically. The yellow-colored complex formed followed at 435 nm based on Dragendorff’s reagent (DR) described by Sreevidya and Mehrotra [Determination of anthocyanin pigments was done according to the method described by Dawood and Abeed  on acidiWilliams . Five mlMehrotra .2O2; μmol/g FW) was measured spectrophotometrically in the leaves. Fresh leaves were ground in cold acetone (5 ml). Afterward, 3 ml of the acetone extract was added to 1 ml of titanium dioxide (0.1%) in H2SO4 (20%) before centrifuging the mixture at 6000 rpm for 15 min. The yellow color developed was measured at 415 nm [The content of hydrogen peroxide  and ascorbate peroxidase , glutathione peroxidase (GPX/EC.1.11.1.9), glutathione-S-transferase , and  were assayed following the method of Abeed et al. [Plant samples (four replicates from each treatment) were extracted via homogenizing leaf samples in 0.1 M phosphate buffer (pH 7.4) containing 10 mM β-mercaptoethanol, 1 mM EDTA, and 1% polyvinylpyrrolidone. The homogenates were centrifuged at 10,000 d et al. , Flohé ad et al. , Habig ed et al. , and Sykd et al. , respect3:HClO4 mixture), as described by Eissa and Abeed [Potassium and sodium concentrations were measured utilizing the flame emission technique (Carl-Zeiss DR LANGE M7D flame photometer) according to Abeed and Dawood . Nitratend Abeed . SummariThe analysis of variance for completely randomized design (CRD) was carried out using Costat  with two main treatments, WW and SW. The observations were recorded on plot mean basis analysis . Means w


Gardnerella, Prevotella, and Atopobium), as well as the abnormalities of the vaginal secretion. Overall, our results showed that applying the postbiotic gel ameliorated BV, and the symptom improvement was accompanied by significant changes in the bacterial vaginal microbiota. Our study provides valuable clinical data in managing BV.Vaginitis is the most common disease in gynecology. Vaginal dysbiosis is a main reason of bacteria vaginitis (BV), as the disrupted microecological environment facilitates the growth of various vaginal pathogens. The most dominant bacteria in the vaginal microbiota are lactic acid bacteria, which are important for maintaining vaginal health. At present, antibiotics and other drugs are often used in clinical treatment, but there are many adverse reactions and easy to relapse, and the intervention of probiotics can help restore vaginal microbiota and alleviate BV. This study is a human clinical trial of 50 patients with bacterial vaginitis (BV). The alleviation effect of applying a postbiotic gel for one week in BV was evaluated. Changes in patients’ clinical indicators of BV  and the vaginal microbiota after using the postbiotic gel were monitored. Our results showed that apply the postbiotic gel improved the symptoms of BV, indicated by improvement in the abnormalities of patients’ vaginal secretions. After applying the gel, the relative abundance of vaginal lactobacilli increased compared to baseline. Significant negative correlations were found between lactobacilli and potential vaginal pathogens (including  Lactic Lactobacillus crispatus CTV-05 (Lactin-V) significantly reduced the recurrence rate of BV  and after (day 7) intervention. All (n = 50) patients diagnosed with BV at this hospital were considered for participation in the study and were screened by hospital professionals for eligibility. The inclusion criteria were: (1) women clinically diagnosed with diagnostic criteria for BV, aged 18-55 years; (2) body mass index between 19-24; (3) no history of heart, liver, lung, kidney, digestive tract, nervous system, and metabolic abnormalities; (4) not taken other vaginal preparation drugs 30 days before this trial; (5) voluntarily signed the informed consent. Subjects that were allergic to the postbiotics gel were excluded from this study Figure 12.2The postbiotic gel used in this work was produced by Qingyitang Industrial Co., Ltd. . Probiotic strains were provided by the Lactic Acid Bacteria Collection Center of Inner Mongolia Agricultural University . The gel was prepared by mixing the raw materials Table 1 2.3Participants was registered to participate in this trial by providing basic demographic information (age: 35.8 ± 8.96) before the start of the trial. The gel was applied every night and avoided the menstrual period. To apply the gel, subjects first cleaned their hands and vulva with warm water, took a product, slightly lifted the hips in a supine position, slowly inserted the gel catheter containing the gel into the deep part of the vagina, pushed it into the pubic area with a pusher, and maintained the posture for about 15 minutes. The product was applied every night for seven days.Sample collection is carried out by professional doctors. Fresh vaginal secretions were collected with sterile cottons before the start of medication and the next day of the last gel application. In order to obtain fresh samples, we collect the secretions of volunteers according to strict methods and submit them for examination as soon as possible. First of all, sterile cotton swabs were gently rotated in the subjects’ posterior vaginal fornix to collect fresh vaginal secretions. After sampling, cottons with the samples were placed in test tubes containing physiological saline and stored at -80°C until further processing. At the same time, symptoms of subjects, including severity of vulvar itching and leucorrhea, color and odor of vaginal secretions were recorded. The biochemical and microbiological properties of vaginal secretion were determined in the laboratory within 30 minutes of sample collection by a medical professional. Hydrogen peroxide test was performed using a combined aerobic vaginitis/bacterial vaginosis five-item qualitative test kit . The presence of lactobacilli, fungi, and the cleanliness of collected samples were observed using a microscope (Olympus Corporation of Japan). The pH of the vaginal secretion was determined using pH test strips. All assessments were operated by professional doctors or personnel, and the data were entered and confirmed by more than two medical staff to ensure data consistency and accuracy. A total of 50 subjects completed the clinical assessment in the trial, and 47 subjects provided a complete sample .2.4The metagenomic DNA in the collected vaginal secretions was extracted by QIAamp kit , and the purity and concentration of the extracted DNA were detected by Nanodrop  and agarose gel electrophoresis. Qualified DNA samples were amplified, targeting to the 16S rRNA V4 region using barcoded region-specific primers, 515F (GTGYCAGCMGCCGCGGTA) and 806R (GGACTACHVGGGTWTCTAAT). The amplification conditions were pre-denaturation at 95°C for 1 min; denaturation at 95°C for 30 s; annealing at 60°C for 40 s; extension at 72°C for 1 min, for a total of 30 cycles; terminal extension at 72°C for 7 min and termination at 4°C. Amplified samples were checked by agarose gel electrophoresis for the amplicon product size and purity. Follow-up analysis was only performed on amplicon products appearing as a single and bright band.The samples that met the quality requirement were used for DNA library construction and sequenced using the Illumina novaseq PE250 platform. The original sequences were quality controlled and grouped by the sample nucleotide barcode. Microbial diversity analysis was performed using the QIIME platform  as follo2.5P < 0.05; Wilcoxon test). PICRUST2  was used to calculate the Shannon index, chao1 index, and Simpson index. The Shannon curve and the observed species number curve were plotted using R (version 4.2.1), which were used to assess the bacterial diversity of each sequenced sample and the sequencing depth. Wilcoxon tests were used to evaluate the α diversity between samples collected at the two time points. β diversity (weighted and unweighted Unifrac) was calculated by R packages (vegan), and principal coordinate analysis (PCoA) was used for descending presentation. Association between β diversity and study groups was assessed using a non-parametric analysis of similarities  with 999 permutations. Followed by comparative analysis to identify significant differential marker bacteria of treatment  and positive reaction in hydrogen peroxide test indicate poor BV or at least poor vaginal health. Initially, 47 patients showed a positive result for hydrogen peroxide, and 18 (remission rate = 38.3%) of them showed a negative hydrogen peroxide result after using the gel. Moreover, 29 people had a cleanliness grade of III or IV, and most (18) of them showed significant improvement after using the postbiotic gel. These data suggested that applying the postbiotic gel for a week could improve BV and vaginal health.3.2A total of 5,479,608 high-quality sequences were obtained from 87  sequencing samples. The rarefaction curves of the Shannon diversity , Gardnerella (6.02%), Prevotella (3.30%), Streptococcus (1.62%), and Atopobium (2.89%). After using the postbiotic gels, the relative abundances of some pathogenic bacteria, including Gardnerella  , which revealed no significant differences before/after the gel intervention , the content of Lactobacillus in vaginal secretions of 24 patients increased  pathogens, i.e., Gardnerella, Streptococcus, Prevotella, Atopobium . Streptococcus showed a weak positive correlation with Prevotella , but negative correlations with the amount and characteristics of vaginal secretion . For evaluating the amount of secretion: a small amount was scored as 2, a medium amount was scored as 1, and a large amount was scored as 0. For evaluating the nature of secretion: a thin amount was scored as 1, and a thick amount was scored as 0. For evaluating the odor of secretion: an odorless amount was scored as 1, and an odorous amount was scored as 0. For evaluating the color of secretion: a clear color was scored as 3, a light yellow color was scored as 2, a yellow color was scored as 1, and a green color was scored as 0. Then we used Spearman’s correlation analysis to analyze the association between the dominant bacterial genera and clinical features of the collected vaginal samples Table S34Gardnerella vaginalis, Prevotella spp., and Atopobium vaginae  for 11 weeks was effective in reducing the recurrence rate of BV in 228 patients  after using the postbiotic gel. Gardnerella can adhere to vaginal epithelial cells, forming a dense biofilm, which can enhance the bacterial tolerance to high concentrations of antibacterial molecules like hydrogen peroxide and lactic acid  for the publication of any potentially identifiable images or data included in this article.XS: Formal analysis, data curation, visualization, writing of the original draft. LX: Clinical trial implementation, specimen collection. ZQZ: Conceptualization, design of methodology. YTY and PXL: Formal analysis, software testing and verification. TM and SG: Supervision of clinical trial, manuscript revision. L-YK: Writing, critical evaluation and revision of the original draft, resource provision. ZHS: Conceived and designed the experiments. All authors contributed to the article and approved the submitted version.


Our data support previous regional archaeobotanical evidence and establish a more precise chronology of the dispersal of millets into northern Iberia during the Bronze Age, becoming an essential crop until the arrival of maize from America after AD 1492. This study emphasizes the importance of multidisciplinary methods to ascertain the origin and development of agricultural practices during recent prehistory.Despite being one of the most important crops in the recent prehistory of Eurasia, the arrival and exploitation of millets in the westernmost part of Europe are still largely underexplored. Here and for the first time, we report multipronged biomolecular evidence of millet consumption along the Atlantic façade of northern Iberia through a combination of radiocarbon dating, stable isotopes, and dental calculus analyses on the human individuals found in the burial site of El Espinoso cave . The high-resolution chronological framework established for individuals placed the burials between 1235 and 1099 cal. BC. The discovery of high δ Two millet species have been traditionally cultivated in Europe during recent prehistory: Setaria italica (foxtail millet) and Panicum miliaceum (broomcorn millet)2. These plants have a short lifecycle3, allowing for annual crop rotation with other cereals, like wheat or barley4. Millets are crops with great ecological adaptability to different altitudes, soils, and climates5 and a rich nutritional value6. They are used to feed both people and animals7, and as ingredient in diverse alcohol fermentation techniques8. Unfortunately, despite their relevance among farming societies during the Iron Age, little is known about when and how millets were introduced into western Europe, specifically in northern Atlantic Iberia9.Millets are small-seed spring/summer domestic grasses that played a key role in human diet and culture across Europe by the first-millennium cal. BC12, although broomcorn millet has slightly earlier chronologies than foxtail millet in these areas. From East and Central Asia, the spread of millets would have occurred in the late third millennium cal. BC across the Inner Asian Mountain Corridor13 and the Eurasian Steppes14. In West Asia and East Europe, the spread of millet cultivation took place during the second millennium cal. BC, producing the first episode of food globalization which laid the foundations of an interconnected Eurasian continent15. In these areas, mobile pastoralists might have constituted key agents in the diffusion of these crops16 based on the coexistence of livestock and plant cultivation practices across Europe17. Direct radiocarbon dates obtained from several broomcorn millet grains, recovered at different Central and Eastern European sites, confirm that millet cultivation arrived in these regions during the second half of the second millennium cal. BC20, a considerably younger chronology than what was previously thought21. Instead, for western regions such as Britain, the latest direct radiocarbon dates point to a slightly later arrival around the first half of the first millennium cal. BC22.The earliest evidence for domesticated broomcorn and foxtail millets comes from north-eastern China and eastern Inner Mongolia, dating to around 6000–5500 cal. BC23. Their small-seed morphology made them hard to find in old excavations, but the implementation of intensive collection programs and the systematic use of flotation systems during recent decades24 have favored the recovery of millet seeds, significantly enriching the archaeobotanical record worldwide. Ethnographic and experimental studies have also improved knowledge about millet cultivation, including harvesting, threshing, sieving, milling, storage, and cooking activities26. Furthermore, comparisons of nutritional components of millets show a similar, and even higher, dietary intake in terms of proteins, minerals, and vitamins than other cereals crops27. Recently, ancient starches and phytoliths retrieved in dental calculus and ground stone tool surfaces have provided new direct and indirect evidence for the prehistoric consumption of millets, given their potential to be differentiated at the genus or species level30 and between wild or domestic species32. Simultaneously, isotopic evidence for C4 plant consumption has been reported in different European regions, questioning the relevance of millets in their diets during the Bronze Age33. Thus, the current evidence about millet consumption is continuously growing, and millets might have been more relevant than previously thought in the recent prehistory.Millets have been traditionally seen as minor crops, with a secondary role in past human economies, used as food for people of low social status and/or as fodder for animals34. Despite the richness of these assemblages, biomolecular approaches have rarely been applied to explore evidence related to the origin and development of its regional agriculture, such as the timing of the arrival of millets and how the human populations exploited them. Until the 1990s, millet cultivation was considered to be an Iron Age innovation associated with the hillfort culture35. The currently available carpological data support the presence of broomcorn and foxtail millets in this area, at least, since the Late Bronze Age, with some limited evidence from the Middle Bronze Age9. However, no direct dates on those seeds confirm their exact chronology. Similarly, stable isotope analysis of bone collagen from Bronze Age individuals failed to detect isotopic signals related to C4 plant consumption. Moreover, dental calculus analysis has never been applied to this spatio-temporal range that would allow identifying millet species' starch grains.In northern Iberia, the Cantabrian region has yielded an extraordinary Bronze Age funerary record where caves were selected as burial locations by early farmers13C, δ15N and δ34S) on human and animal bone collagen, and the study of starch grains entrapped in the dental calculus of humans found at El Espinoso burial cave.With the aim to identify the millet consumption in the Atlantic coast of northern Iberia and to ascertain when and how these crops were systematically exploited during the Bronze Age of this region, this paper combines direct radiocarbon dating on human remains, stable isotope analysis , in the easternmost sector of Asturias province Fig. , which i38. The El Espinoso sequence showed a convergence greater than 99%, and the model and overall agreement indices were 107.9% and 100.2%, respectively. Only OxA-38660 date had a poor agreement of 52.9%. A model of the burial phase at the site was built with start and end boundaries  had δ13C values ranging between − 17.8 and − 14‰ (m = − 15.6‰), δ15N values between 9.1 and 10.4‰ (m = 9.5‰) and δ34S values between 13.5 and 16.9‰ (m = 14.9‰).The two faunal specimens available were analyzed as a local baseline from the site: a cow specimen reported a δ13C signature of − 22.1‰, a δ15N value of 4.1‰ and a δ34S signature of 16.2‰, and a sheep had a δ13C value of − 20.6‰, a δ15N signature of 3.7‰ and a δ34S value of 13.5‰. Humans from La Llana, Los Cinchos, and La Fragua sites reported δ13C values of − 20.9‰, − 21.1‰ and − 20.4‰ and δ15N values of 8.6‰, 9.2‰ and 9.2‰ and δ34S values of 14.9‰, 14.5‰ and 14.8‰, respectively.From El Espinoso cave, the humans analyzed  during the regional Bronze Age, neither in the cave deposits nor in the few documented open-air sites52. However, future archaeomalacological and ichthyofaunal studies will help to verify this.The El Espinoso animal samples had δ13C values, sulfur isotope ratios were also analyzed. The freshwater and marine resources consumption produce elevated δ34S values53. At El Espinoso, humans but also faunal specimens, who follow different diets, showed the dispersion of the same values ranging between 13.5 and 16.9‰. On the other hand, La Llana, La Fragua and Los Cinchos individuals reflected similar sulfur values, but they did not provide enrichment of carbon values. Therefore, the δ34S values recorded were more likely to be associated with the environment where they were living rather than with the influence of fish in the diet. Indeed, humans and animals of El Espinoso were predominantly living near the coast, where the sea spray effect can reach more than 30 km inland, affecting the sulfur signatures of coastal environments54. In this sense, the sulfur isotope values from humans and animals of Los Avellanos I and II, dated in the Late Neolithic/Chalcolithic and without typical carbon isotope values of marine diets, showed the same higher sulfur isotope signals55. Consequently, the most likely hypothesis to explain the δ13C enrichment in El Espinoso individuals could be a significant consumption of C4 plants, likely millets. Isotopic measurements of modern millets have provided δ13C values from − 10 to − 12‰ and δ15N values from 3 to 4‰56, as well as isotopic measurements of Iron Age millets have reported δ13C and δ15N values around − 10‰ and 4‰57. Millet has a protein content of approximately 10%; thus, a significant intake of these crops is required to detect a substantial change in collagen isotope ratios58.In addition, to reject the possible influence of fish on the El Espinoso human diet due to the higher δ13C values, showing that the diet was predominantly terrestrial, based on C3 plants and animal protein. This dietary pattern was also observed in other human bone collagen isotopic studies from the Bronze Age sites, such as Cova do Santo47, La Castañera48, La Garma A59, Santimamiñe60 and Ondarre61 , dated in the Late Bronze Age, showed isotopic signals of C4 plant consumption during the childhood, while two more teeth dated in the Early Bronze Age indicated a C3 exclusive diet62. With the current isotopic evidence, if C4 plant resources were consumed before the Late Bronze Age, the quantities were not high enough to be registered in the long-term bone collagen record. This would suggest infrequent or occasional cultivation of millets during the Middle Bronze Age, becoming a staple crop only in later periods, probably from Late Bronze Age, as the isotopic results from El Espinoso cave demonstrate. Until now, the earliest bone collagen isotopic evidence of C4 plant consumption in northern Iberia is documented at La Hoya village, an inland site 150 km from the current Atlantic sea in Alava and dated in the Iron Age37. El Espinoso individuals are more than 700 years older than La Hoya, setting back the isotopic evidence of intensive millet consumption in northern Iberia to the Late Bronze Age.In contrast to El Espinoso, the isotopic data from the humans of La Llana, Los Cinchos, and La Fragua sites reported lower δ63. A longitudinal gradient for the dispersal from Eastern to Western Europe during recent prehistory is proposed, with the Atlantic coast being one of the last regions to adopt millets. However, the radiocarbon evidence indicates a relatively quick spread of these crops through Europe20. Concerning the published stable isotope analyses from different European regions, some studies have found isotopic evidence of millet consumption around 5500 cal. BC in East Asia65, between 2500 and 2000 cal. BC in Central Asia66, between 1600 and 1400 cal. BC in the Caucasus region67, between 1600 and 1200 cal. BC in northern and central Italy69, between 1400 and 1200 cal. BC in France70 and between 1300 and 1000 cal. BC in northern Spain.According to the evidence, millets were introduced into Iberia across the Pyrenees from Central Europe during the Middle/Late Bronze Age transition, reaching earlier and playing a more important role in the northern regions of Iberia than the southern ones4 plant consumption by age. These minor differences can be explained by the higher importance of millet in the children's diet. This pattern was previously observed at La Hoya and Los Canes71. Here millets were introduced to infants and young children during the weaning process, probably in the form of porridge57. Considering the stable isotope data from different millet consumers of Europe, in some cases, all the individuals of the same group ate large amounts of millet, while in others, the millet consumers lived in a community where most people did not consume this crop. This fact suggests dietary differences related to social, cultural, or economic status33. However, the current isotopic evidence indicates that intensive consumption of millets outside China was sporadic during recent prehistory until the first millennium cal. BC, when the results tend to be more homogeneous from the Eurasian steppes to the Atlantic fringe.Regarding the carbon values across the humans’ age distribution of El Espinoso, three out of four subadults showed the higher carbon values of the sample, suggesting the existence of inequalities in C76. Based on the phytolith and starch grain morphology, these crops show great potential for identifying different genera or species, opening new possibilities for their detection in archaeological contexts31. There is little evidence of preserved millet grains in dental calculus from Europe, with even fewer examples in Iberia. This lack of knowledge can be explained by the absence of studies on the recent prehistory of this geographical area. Among a great diversity of taxa represented, some starches of the Paniceae tribe were recovered within the dental calculus of the Chalcolithic individuals from El Mirador cave , based on their average size and morphological features77. In Italy, 79 starch grains were identified among individuals from the Chalcolithic/Bronze Age site of Grotta dello Scoglietto (Tuscany)28.The richest archaeological evidence of millet grains within dental calculus comes from Asia, where these crops were primarily domesticated. Different studies have revealed starch grains and phytoliths from other millet species4 plants. Experimental starch grains from Panicum miliaceum  found in the El Espinoso individuals may be attributed to different Cgenus) show a larger size distribution than domestic ones (Triticum and Hordeum genus)79. Additionally, B-type grains are more abundant than A-type in wheat and barley species. Our experimental measurements have shown the great variety of starch grain sizes for the different Triticeae domestic species , typical of grass grains, as those of the Triticeae tribe , also known as “forgotten millets”, has been found during the recent prehistory of northern Iberia81. Today they are widely distributed across central and south Europe82 but almost unknown in Atlantic Europe83. The only C4 plants found in Iberia during recent prehistory are domestic millets, particularly broomcorn and foxtail millets . They are the C4 crops that are frequently identified across Europe33, constituting the primary candidates cultivated in large proportions by northern Iberian farmers. In the Vizcaya province, macroremains of foxtail millet were discovered in Kobaederra (Level 1) and Arenaza (Layer 9), chronologically assigned to the Chalcolithic and Early Bronze Age, respectively84. However, those seeds have not been directly dated; thus, we cannot exclude the possibility that they might be intrusive. Alternatively, there is an absence of Panicum milliaceum in the prehistoric carpological records of the Cantabrian Region. Also, in northern Portugal, several millet grains were found in the Chalcolithic sequence of Crasto de Palheiros85, but stratigraphic issues were reported in those layers. In the Middle Bronze Age in northern Portugal, the carpological remains of broomcorn millet were identified in the sites of Sola IIb86 and Terraço das Laranjeiras87. Later, during the Late Bronze Age, a higher presence of millet (exclusively P. miliaceum) is recorded across the region, in the neighboring areas of Galicia province  and northern Portugal 9. Likewise, seeds from both millet species have been identified in different sites in the Ebro basin (northern Mediterranean Iberia) during the Middle and Late Bronze Age, such as at Punta Farisa, Masada de Ratón, El Vilot and Vincament89.Millets are poorly represented in the Bronze Age archaeological record. The first carpological evidence of millet exploitation in Iberia dates to the Middle Bronze Age. However, they were not systematically exploited until the Late Bronze Age and Iron Age90. To better understand the origin and development of millet cultivation, direct dating of millet grains will be needed20 as the morphology and size of these tiny seeds facilitate their vertical movements downwards through the stratigraphical sequences and must be called into question for future research23.Consequently, the available evidence confirms that millet cultivation in northern Iberia seems to have started during the Middle/Late Bronze Age transition. However, it was not until the Late Bronze Age that millets became a significant and regular crop. The transition from the Middle to Late Bronze Age marks the shift of millets from a minor to a major role in the agriculture of the European farming societiesSetaria italica (foxtail millet) and Panicum miliaceum (broomcorn millet) were traditionally cultivated. Ethnobotanical data have shown a higher presence of S. italica or both species sown together in the Asturias province. Such place-names as "Panizales, Panicera, Paniceres," etc., are familiar in the Asturias and refer to the Spanish name of foxtail millet91. At the same time, the most common crop found in Galicia and northern Portugal is P. miliaceum7. This coincides with the archaeobotanical remains recovered from prehistoric sites, with foxtail millet found in the Cantabrian Region and the broomcorn millet in the northwestern area of Iberia. Ethnographic approaches in Galicia during the twentieth century have provided insights into the utilization of millet grains for human consumption. Simultaneously, both grains and plants are used as feed or fodder for animals35. In recent times in the wider northwestern regions of Iberia, millet has been used to make porridge, stews, puddings, or bread for human consumption7. However, there are some differences observed among regions. In Asturias, S. italica is used exclusively for foddering, while in Galicia and northern Portugal, besides animal consumption, P. miliaceum, is also used as human food. Both crops have been cultivated until the present, and their importance was exceptionally high during the Roman and Medieval periods, as testified by ancient sources92 and stable isotope analyses93. However, the arrival of maize (Zea mays) from America in the seventeenth century displaced their cultivation to a marginal role. Also, ethnographic studies in other European regions reported the use of millets to produce an alcoholic drink94.Ethnographic studies can provide valuable information that can be used as a complement to the archaeological evidence about the cultivation and consumption of millets in the past. This is the case in northern Spain, where 13C, δ15N and δ34S); and (3) dental calculus analysis. El Espinoso cave was used as a burial location during the last third of the second millennium cal. BC. The Bayesian model places the span of the burial phase between 0 and 115 years, supporting a practically synchronous use of the cave for a few decades. δ13C and δ15N isotope values show a diet with high consumption of C4 plants, likely millets, and contrast with the values obtained in different Early and Middle Bronze Age funerary sites for the same region, suggesting that the systematic consumption of millets in this area started from the Late Bronze Age. The study of the microresidues entrapped within the dental calculus of El Espinoso individuals confirms the presence of starch grains with a polyhedral morphology typical of the Paniceae tribe. Besides, other round to oval starch grains characteristic of the Triticeae tribe have also been identified, demonstrating a mixed agricultural economy for the Late Bronze Age of northern Iberia. The multidisciplinary research undertaken on the human bone assemblage of El Espinoso cave has generated a corpus of high-quality biomolecular data about millet consumption and constitutes one of the oldest and most direct evidence of the exploitation of millets in northern Atlantic Iberia.In this study, we infer direct and precise data about the systematic use of millets during the Late Bronze Age in northern Iberia by combining different methodologies approaches on human remains including: (1) radiocarbon dating; (2) stable isotope  of humans and animals were performed to ascertain these individuals' carbon, nitrogen, and sulfur signatures. Bone collagen extraction was conducted in the EvoAdapta Group (University of Cantabria) according to the procedures proposed by Richards and Hedges97. Carbon, nitrogen and sulfur isotope analysis of the collagen samples was undertaken by Elemental Analysis—Isotope Ratio Mass Spectrometry (EA-IRMS) in Iso-analytical laboratory . Quality indicators widely used were employed42. Dental calculus analysis was achieved to discern what types of plants were involved in the diet of this human group. The dental calculus matrix was removed from the teeth following the protocol by Sabin and Fellow-Yates98. Decontamination and the extraction procedures for micro-debris were carried out following standard protocols as described by Cristiani et al.99 and Fiorin et al.100, and were conducted in dedicated clean spaces under strict environmental monitoring of the DANTE—Diet and ANcient TEchnology laboratory of Sapienza University of Rome . The analysis of the microfossils was carried out using a Zeiss Imager2 polarised microscope (100×–630×) at the DANTE laboratory and a Leica DVM6 M digital microscope at the EvoAdapta Group. Morphological and size comparisons were carried out with the modern plant reference collections housed at these institutions (See Supplementary Text In this research, a multidisciplinary approach was conducted to reassess the relevance of millets in the prehistoric human diet. Radiocarbon dating was carried out to establish a precise chronology for the humans analyzed here. The dates were calibrated in OxCal v4.4Supplementary Information.