The treatment of hyperuricemia and gout is mostly based on lowering serum uric acid levels using drugs, such as allopurinol, or increasing urinary excretion of uric acid. However, some patients still experience adverse reactions to allopurinol and turn to Chinese medicine as an alternative. Therefore, it is crucial to design a preclinical study to obtain more convincing data on the treatment of hyperuricemia and gout with Chinese medicine. This study aimed to explore the therapeutic effect of emodin, a Chinese herbal extract, in a rat model of hyperuricemia and gout. In this study, we used 36 Sprague–Dawley rats, which were randomly divided into six groups for experimentation. Hyperuricemia was induced in rats by intraperitoneal injections of potassium oxonate. The efficacy of emodin in reducing serum uric acid levels was demonstrated by comparing the positive control group with groups treated with three different concentrations of emodin. The inflammatory profiles, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α levels, were unaffected by emodin treatment. In the experimental results, it was observed that the serum uric acid concentration in the vehicle control group was 1.80 ± 1.14, while the concentrations in the moderate and high concentration emodin groups were 1.18 ± 0.23 and 1.12 ± 0.57, resulting in no significant difference in uric acid concentration between these treatment groups and the control group, indicating that emodin has a therapeutic effect on hyperuricemia. The increase in the fractional excretion of uric acid (FEUA) demonstrated that emodin promoted urinary uric acid excretion without significantly affecting the inflammatory profile. Thus, emodin reduced the serum uric acid concentration to achieve effective treatment of hyperuricemia and gout by increasing urinary excretion. These results were supported by the measured serum uric acid and FEUA levels. Our data have potential implications for the treatment of gout and other types of hyperuricemia in clinical practice. Gout is a joint disorder caused by monosodium urate sediments ,2. It haGout is characterized by increased serum uric acid levels and decreased urinary uric acid excretion. Hyperuricemia is an important risk factor for gout. The incidence of gout episodes increases when serum uric acid levels increase . PrimaryRheum palmatum, Polygonum cuspidatum, Polygonum multiflorum, Aloe vera, and Cassia obtusifolia. Emodin has been used for the treatment of arthritis due to its anti-inflammatory effects, detoxification, and promotion of bowel movements. Emodin has also been reported to inhibit xanthine oxidase  and serum and urinary urea nitrogen levels 24 days after PO injection in the study groups compared with the control group  may occur. The deposited crystals then induce an acute inflammatory response known as a gout attack. More severe cases can result in tophi, chronic gouty arthritis , and structural joint damage. Gout attacks can occur in the joints or tissues surrounding the joints . Gout most commonly occurs in lower-extremity joints. Other locations, such as the distal knuckles, ears, olecranon sac, finger pads, and tendons (such as the Achilles tendon), may also be affected.Monosodium urate, a component of tophi, is phagocytized by macrophages and other monocytes and induces an inflammatory response by activating the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. Activation of the NLRP3 inflammasome leads to the cleavage of pro-IL-1β and the secretion of IL-1β. IL-1β then binds to the IL-1β receptor, inducing the downstream signaling of pro-inflammatory cytokines and chemokines. Activation of the NALP-3 inflammasome causes monocytes and macrophages to release chemokines , which promote neutrophil chemotaxis and proliferation. An increase in the number of macrophages and monocytes leads to the secretion of TNF-α and IL-1β, which cause a strong inflammatory response. The NLRP3 inflammasome does not directly induce an inflammatory response. Therefore, the deposition of monosodium urate crystals in the joints may not necessarily be accompanied by obvious inflammatory symptoms ,18,19,20Allopurinol is the first-line drug for the treatment of gout. Allopurinol is widely used clinically because of its availability, low cost, and therapeutic efficacy. It is a xanthine oxidase inhibitor that inhibits uric acid production. Allopurinol is well tolerated by most people; however, approximately 1–2% of users experience allergic reactions, such as a maculopapular rash. Although rare, patients treated with allopurinol may experience severe or even fatal skin reactions, such as Stevens–Johnson syndrome or toxic epidermal necrolysis . AlthougTherefore, other compounds or natural Chinese herbal medicines can be used to replace or reduce the dosage of allopurinol to achieve the same or even greater therapeutic effects. Thus, the adverse reactions to allopurinol can be reduced with a reduction in its dosage. Chinese medicine has recorded hyperuricemia and gout for more than 2000 years. There is more than 1700 years of history of treating hyperuricemia and gout with Chinese herbal medicine, dating back to the synopsis of prescriptions in the Golden Chamber (Jingui Yaolue). Several Chinese herbal medicines have been used to treat gout and hyperuricemia in clinical practice ,24,25. DOur study was a pilot study of the effect of emodin on lowering serum uric acid concentrations by increasing urinary excretion of uric acid. Emodin is a chemical compound  that is abundant in the TCM herb rhubarb. Recently, several studies have shown that emodin has anti-cancer, anti-bacterial, antiviral, antidiabetic, anti-inflammatory, anti-allergy, neuroprotective, liver-protective, and anti-osteoporosis activities . HoweverThis study had several limitations. We used rats as the animal model of hyperuricemia. Previously, a PO-induced hyperuricemia mouse model was constructed ,31. Due In addition, the animal sample size used in our study may seem insufficient to yield conclusive findings. Nonetheless, we are committed to upholding ethical standards, and, as such, we cannot increase the number of animals at this time in line with the principles of replacement, reduction, and refinement. Despite the limited number of animals used, we were able to achieve statistically significant positive results in our study. In addition, a specified score for assessment of the histopathological changes in the renal tissues with immunohistochemical and electron microscopic examination of the renal tissues will add value to this manuscript. We unfortunately cannot perform this analysis due to various constraints such as time, budget, and technical expertise. However, we will explore the possibility of utilizing a specified scoring system to assess the histopathological changes in the renal tissues. The interaction with the microbiota is an important factor to consider in the study of emodin. It should be considered and explored for the potential impact of emodin on the microbiota fingerprint ,36. AddiPotassium oxonate , carboxymethylcellulose , Tween 80 (Product No.: P4780), uric acid , dimethyl sulfoxide , and allopurinol  were purchased from Sigma–Aldrich . The uric acid inhibitory agent, emodin , was purchased from Combi-Blocks, Inc. . All test agents were of analytical grade. Emodin (10 mg/kg body weight) was prepared by dissolving 10 mg in 0.05 mL of DMSO (≥99.9%) and adding water to a final volume of 10 mL . Other doses of emodin were prepared as required. Serum and urinary biochemical profiles were assessed using commercial kits .Uric acid can be metabolized to allantois by uricase in animals. However, there is an evolutionary defect in uricase in humans. Animal models of gout are generated by intraperitoneal injection of PO, a uricase inhibitor. Gouty arthritis in rats was induced by an intra-articular injection of monosodium urate.n = 36) were purchased from BioLASCO Taiwan Co., Ltd. . The rats were acclimated to the animal room for 1 week with a standard diet and ad libitum access to water. All protocols were approved  by the Institutional Animal Care and Use Committee of the China Medical University . Efforts were made to reduce the number of animals and their suffering. The rats were housed in pathogen-free conditions in an animal room with a humidity of 50 ± 10%, a 12 h/12 h light/dark cycle, and a temperature of 25 °C [Six-week-old male Sprague–Dawley rats (of 25 °C . Before The rats were divided into the following groups (6 per group): (a) normal control, (b) hyperuricemia and gout (VEH), (c) allopurinol treatment , (d) low-dose emodin 10 mg/kg (EMO10), (e) moderate-dose emodin 30 mg/kg (EMO30), and (f) high-dose emodin 50 mg/kg (EMO50) groups. The body weight of the rats was measured, and the rats were intraperitoneally (i.p.) injected every day from the start of the experiment until the end of the study (24 days). Phosphate-buffered saline  was injected in rats in the control group, and PO  was injected as the induction agent in rats in the other groups. At day 21, the rats, except those in the control group, were anesthetized with 2.5% isoflurane and monosodium urate  into the right fourth intermetatarsal space. From the 21st day to the 24th day, oral gavage of 10 mL/kg bodyweight PBS was performed for the control and VEH groups. The ALL group was orally administered allopurinol after intraperitoneal administration. The EMO10, EMO30, and EMO50 groups were administered emodin at the dosage described above . g for 5 min, and sent for analysis. On the 25th day, the rats underwent abdominal surgery under anesthesia. Blood samples were collected from the vena cava. The kidneys were excised during the operation. The right paw was cut for pathological examination. The rats were killed after the experiment was completed.The perimeter of the paw was measured using silk and then transferred to a centimeter scale daily after injection. Urine samples (24 h) were collected from the metabolic cage the day before study completion, centrifuged at 2000× g, 5 min). GOT, GPT, creatinine, and uric acid levels were measured using DiaSys GOT (ASAT) FS, DiaSys GPT (ALAT), DiaSys Creatinine FS, and DiaSys Urea FS , respectively. TNF-α, IL-1β, and IL-6 levels were measured using enzyme-linked immunosorbent assay kits . Paw specimens were examined using polarized microscopy to determine the presence of uric acid crystals. The minor lobe of the liver was homogenized in cold saline and centrifuged at 10,000× g for 10 min (4 °C). The supernatant was used to measure xanthine oxidase activity. Uric acid excretion was calculated as the fractional excretion of uric acid , according to the following equation:We measured uric acid, creatinine, and nitrogen levels in urine and blood samples. Blood samples were stored at room temperature for 1 h to allow coagulation. Serum was obtained after centrifugation of the blood samples  staining, a widely employed staining technique that imparts color to the various components of the cells, thus facilitating their visualization under a microscope.The stained tissue samples were then meticulously examined using TissueFAXS software, a powerful tool that allows for the acquisition of high-resolution images of biological samples. The use of this software was particularly advantageous as it enabled us to capture detailed images of the kidney tissues down to the subcellular level. By analyzing these images, we were able to discern subtle differences in the microstructure and cellular composition of the tissues, providing us with valuable insights into the biological processes occurring in these organs. Overall, this approach allowed us to obtain comprehensive and detailed microscopic results, which were critical to our research findings .The primary outcome was the serum uric acid concentration in rats. Swelling of the rat paw was evaluated on experimental days 21 and 25. The secondary outcome measures were the urinary uric acid concentration, serum and urinary creatinine concentrations, and serum biochemistry profile. The enzymatic activities of FEUA and xanthine oxidase were also evaluated. The urinary concentrations of the cytokines IL-1β, IL-6, TNF-α, and OSM were evaluated at the end of the experiment . The hisp < 0.05.The data in this study were presented as the mean ± S.D. To perform the statistical analysis, the researchers utilized the Statistical Package for Social Sciences . To compare the differences between groups, a one-way analysis of variance was performed. The level of statistical significance was set at In this study, we successfully established a rat model of hyperuricemia. Emodin significantly lowered serum uric acid levels in rats with PO-induced hyperuricemia. Emodin also had renoprotective and hepatoprotective effects in rats with PO-induced hyperuricemia. This effect was attributed to increased uric acid excretion from the urine. Emodin had no anti-inflammatory effect on gouty arthritis. This requires further investigation to determine the pathway of renal excretion of uric acid. Overall, our findings indicate that emodin has clinical potential for the treatment of patients with hyperuricemia. In clinical use, emodin may be considered for independent use as a pure substance or in existing traditional Chinese herbs containing emodin to lower serum uric acid levels. This may help prevent or avoid the occurrence of gout, uric acid-induced kidney damage, and uric acid-related cardiovascular diseases.


Isobolographic analysis showed a superadditive interaction for rosuvastatin and duloxetine, as both the ED30 and ED50 for the rosuvastatin/duloxetine combination contained only a quarter of each drug compared to the individual drugs. The rosuvastatin/duloxetine combination reversed paclitaxel-induced GFAP overexpression, indicating that such effects might depend in part on astrocyte inactivation. Results suggest that statins could be useful in synergistically enhancing the efficacy of duloxetine in some chemotherapy-induced neuropathic conditions.Paclitaxel, a widely used cancer chemotherapeutic agent, has high incidence of neurotoxicity associated with the production of neuropathic pain, for which only duloxetine has shown significant but moderate analgesic effect. Since statins, classically used to reduce hypercholesterolemia, have shown antinociceptive effect in preclinical studies on neuropathic pain, we studied whether the antinociceptive efficacy of duloxetine could be synergistically potentiated by rosuvastatin in a model of paclitaxel-induced neuropathy in mice. The astrocytic and microglial responses in the spinal cord of paclitaxel-treated mice were also assessed by measuring GFAP and CD11b proteins, respectively. Paclitaxel treatment did not impair motor coordination and balance in rotarod testing. Rosuvastatin, duloxetine, and the rosuvastatin/duloxetine combination (combined at equieffective doses) dose-dependently decreased mechanical allodynia (ED Cancer is the second leading cause of death globally and is responsible for nearly 10 million deaths in 2020 . CIPN isThe mechanisms by which antineoplastic drugs cause neuropathic pain are complex, with numerous modifications found in primary sensory neurons and in dorsal horn cells in rodent models of CIPN, such as changes in ion channels  and in transient receptor potential channels (increase in TRPV1 channel), together with deficits in mitochondrial function and changes in immune cell interactions , with concurrent high levels of pro-inflammatory cytokines (for recent reviews see ).The current treatment of CIPN relies on two main approaches: prevention , and drug-based symptomatic treatment . Among tIt has recently been reported that rosuvastatin, a synthetically made hydrophilic statin that undergoes low first-pass extraction in the liver , produceTherefore, in the present preclinical study, we set out to compare the antinociceptive therapeutic effects of rosuvastatin and duloxetine on mechanical allodynia and thermal hyperalgesia in a mouse model of paclitaxel-induced neuropathy, and to examine by isobolographic analysis whether the antinociceptive effects of these two drugs can interact synergistically when combined in equieffective doses. Intraperitoneal paclitaxel administration under different dosages and protocols has shown to induce sensory neuropathy in rats and mice, with hyperalgesic/allodynic effects that are largely dose-independent ,25,26,27Paclitaxel-induced neuropathic pain was assessed through the production of mechanical allodynia and heat hyperalgesia using the electronic von Frey and the hot plate algesimetric tests, respectively. In the electronic von Frey test, mice receiving 2 mg/kg paclitaxel developed significant mechanical allodynia starting from day 7 after the first administration of paclitaxel, reaching quite stable allodynic scores from day 10 with respect to both the baseline pretreatment measurement and the sham group. Allodynia remained significant until day 17, where von Frey testing was stopped A. In theIn addition, in order to rule out motor effects possibly induced by paclitaxel, which could alter the nociceptive response of mice to algesimetric testing, the rotarod test was performed. The 2 mg/kg paclitaxel group did not differ statistically from the vehicle group with respect to motor coordination and balance, and both groups of mice similarly improved motor performance scores over time as compared with pre-paclitaxel scores, indicating a similar learning ability of motor skills .30 value for rosuvastatin was 12.98 mg/kg, with a 95% confidence interval (95% CI) of 10.36 mg/kg to 16.26 mg/kg , i.p. administration of rosuvastatin during 7 consecutive days induced a dose-dependent increase in the withdrawal threshold in the right hindlimb, expressed as % change of the basal score before rosuvastatin A. The % 26 mg/kg C.30 value for duloxetine was 7.10 mg/kg with a 95% CI of 5.06 mg/kg to 9.97 mg/kg . The administration of the combination of rosuvastatin and duloxetine, in equieffective proportions of their respective ED30, induced a dose-dependent increase in the withdrawal threshold in the right hindlimb , expressed as % change of the basal score before rosuvastatin A. The % 91 mg/kg C.50 value for duloxetine was 15.53 mg/kg with a 95% CI of 6.72 mg/kg to 35.87 mg/kg . The administration of the combination of rosuvastatin and duloxetine, in equieffective proportions of their respective ED50, induced a dose-dependent increase in the hot plate latency .Expression levels of GFAP and CD11b proteins, as markers for astrocytic and microglial activation, respectively, was quantified by Western blot in dorsal horn tissue of mice with paclitaxel-induced neuropathy, following a similar antinociceptive drug treatment protocol to that used for algesimetric evaluation. Mice were daily injected with 2 mg/kg i.p. paclitaxel for 5 consecutive days, and 10 days after the first injection of paclitaxel, a 7-day antinociceptive treatment with rosuvastatin/duloxetine combination started at day 0. Mice with paclitaxel-induced neuropathy that did not receive the rosuvastatin/duloxetine combination served as controls for the effects of the rosuvastatin/duloxetine combination. The antinociceptive treatment consisted of daily i.p. injection of the EDWe successfully replicated the model of peripheral neuropathy induced in mice by 5 consecutive daily injections of 2 mg/kg paclitaxel, as proposed by Nieto et al. , a model30 of rosuvastatin or duloxetine alone upon von Frey testing were 12.98 and 7.10 mg/kg, respectively, while the equieffective dose of the rosuvastatin/duloxetine combination, i.e., the ED30 of the combination, only contained 3.02 mg/kg rosuvastatin and 1.65 mg/kg duloxetine. This means that the association of the drugs made it possible to reduce the dose of both drugs to less than a quarter when used to treat mechanical allodynia, at least in mice. Upon hot plate testing, the ED50 of rosuvastatin or duloxetine alone was 32.50 and 15.53 mg/kg, respectively, whereas the equieffective dose of the rosuvastatin/duloxetine combination contained 8.46 mg/kg rosuvastatin and 4.04 mg/kg duloxetine, meaning that the combination of the drugs again led to a reduction in the dose of each drug to near to a quarter, but this time in face to thermal hyperalgesia of the mice. If extrapolated to humans, this might be an important finding because the most common side-effects of duloxetine in clinical settings, such as fatigue, insomnia, anxiety, nausea, constipation, eye distention, and sexual dysfunction [The main and most important result of the present study was that rosuvastatin and duloxetine, which individually are effective in some neuropathic pain models ,28,29,30function ,32—and pfunction ,34—are sfunction ). In addfunction .It seems worth remarking that the superadditive interaction between rosuvastatin and duloxetine, detected by isobolographic analysis upon von Frey and hot plate testing, originated from quite parallel regression lines obtained in the dose–response plots of the individual drugs, implicating that the potency ratio for these two drugs remained constant during testing of mechanical allodynia and heat hyperalgesia in neuropathic rat ,37,38. TAs it is known, the von Frey test is the most widely used to determine tactile allodynia related to neuropathic pain in both rats and mice and is highly sensitive to peripheral changes in nociceptive processing such as those produced during characterization of non-steroidal anti-inflammatory drugs . InsteaOn the other hand, the fact that GFAP protein was overexpressed in the lumbar dorsal horn of mice with paclitaxel-induced neuropathy supports the involvement of astrocytes in allodynia and hyperalgesia observed in those animals. In contrast, a small but non-significant increase in the microglia marker CD11b was found in dorsal horn tissue. Although some studies have shown increased levels of microglial proinflammatory cytokines and chemokines in the spinal cord of paclitaxel-treated animals ,50,51,52Results also showed that the rosuvastatin/duloxetine combination was able to reverse the GFAP protein overexpression in the lumbar dorsal horn of mice treated with paclitaxel. This is a rather expected result, because previous works have found that duloxetine alone downregulates increased levels of GFAP found in the spinal cord of mice with diabetic neuropathy . SimilarIn contrast, the increase in CD11b protein expression levels induced by the rosuvastatin/duloxetine treatment in the dorsal horn of paclitaxel-treated mice was unexpected. Indeed, duloxetine administration has been found to downregulate the spinal expression of CD11b in a mouse model of diabetic neuropathy . In addiAlthough the mechanism underlying the ability of rosuvastatin to exert a synergistic action upon the duloxetine antinociceptive effect remains yet uncertain, this issue could constitute a potential basis for future clinical applications addressed to lower duloxetine and/or statin dosing together with a lowering of their side-effects. As mentioned elsewhere , due to 30 (von Frey testing) and the ED50 (hot plate testing) for the rosuvastatin/duloxetine combination contained about one-fourth of each drug than the ED30/50 of each drug individually. The 2-fold increase in spinal GFAP with no apparent change in the level of CD11b protein points to a role of astrocytes in paclitaxel-induced neuropathy. The reversal of overexpressed GFAP by the rosuvastatin/duloxetine combination suggests the antiallodynic and antihyperalgesic effects exerted by the drugs combination in some extent could depend on astrocyte inactivation. Extrapolated to clinical settings, the results suggest that statins could be useful in synergistically enhancing the efficacy of duloxetine in CIPN, potentially allowing for a reduction in the dose of duloxetine and its associated side effects, while achieving better analgesia.In conclusion, the present results showed that in mice with paclitaxel-induced neuropathy, the rosuvastatin/duloxetine combination was more effective than the individual drugs in both algesimetric tests, giving rise to a superadditive antinociceptive effect against both mechanical allodynia and thermal hyperalgesia. Of note, the EDAdult male CF-1 mice weighing 25–35 g, from the rearing facility of the Faculty of Medicine of the University of Chile, were used. They were maintained at room temperature (22 ± 2 °C) under a light–dark cycle of 12:12 h, and with free access to food and tap water. Before each experiment, the mice were acclimatized to the laboratory environment for at least 2 h. The experimental protocols were performed according to the Guide for the Care and Use of Laboratory Animals of NIH  and apprThe drugs used were paclitaxel , rosuvastatin , and duloxetine . Paclitaxel, dissolved in 50% Cremophor and 50% absolute ethanol , was administered once a day for five consecutive days at a daily dose of 2 mg/kg i.p. Rosuvastatin was dissolved in 4% DMSO and 30% PEG-300 (both from Sigma-Aldrich) using double-distilled water, and administered once a day for seven consecutive days, at doses of 3, 10, 30, and 100 mg/kg i.p. Duloxetine was dissolved in 2% DMSO, 30% PEG-300, and double-distilled water, and administered once a day for seven consecutive days, at doses of 1, 3, 10, and 30 mg/kg i.p. All drugs were administered in a volume of 10 mL/kg, while controls groups received 10 mL/kg of the vehicle used to dissolve the respective drug.The primary antibodies employed in the study were polyclonal anti-GFAP , polyclonal anti-CD11b , and monoclonal anti-β-actin  that was employed to normalize the levels of protein detected. As secondary antibody, the goat polyclonal 115-035-174 antibody  was used.Mechanical allodynia: Mechanical allodynia was evaluated by means of the electronic von Frey test. After the animals were acclimatized for 30 min in a box of plexiglas with a wire-grid floor, a stimulus of increasing pressure was applied to the center of the plantar area of the right hind paw by means of an electronic von Frey filament  until the animal withdrew the stimulated limb. The force (in grams) required to achieve the withdrawal response was recorded, and an average of 3 consecutive measurements was defined as the mechanical threshold of the animal [e animal .Heat hyperalgesia: Heat hyperalgesia was evaluated using the hot plate test. Each mouse was placed on a hot plate  at a temperature of 55 ± 0.5 °C, without movement restrictions, and the time (in s) in which the mouse presented a behavioral reaction to heat  was measured, with a cut-off time of 20 s to prevent tissue damage [e damage . The resTo rule out a posible effect of paclitaxel in motor performance that may affect measurement of mice nociceptive behavior, the rotarod test was used according to the protocol of Dunham and Miya . AnimalsPaclitaxel was administered at a concentration of 2 mg/kg i.p. in a volume of 10 mL/kg, for 5 consecutive days, as described by Nieto et al. , doses gX  by linear regression analysis, the maximal antiallodynic or antihyperalgesic effect being the 100% Antiallodynia/Antihyperalgesia level  shown by the respective control group of sham mice. In the present study, the ED50 was used to assess the antihyperalgesic effect of rosuvastatin, duloxetine, or their combination on the hot plate test, while the ED30 was used to assess the effect of rosuvastatin, duloxetine, or their combination on mechanical allodynia, since the low sensitivity of the von Frey test against duloxetine and its combination with rosuvastatin did not allow reaching 50% antiallodynia, even with the highest doses used with these drugs. As reported elsewhere by Tallarida [50), although any other level of effect reached by the drugs to be combined can be used. For example, Pelissier et al. [25 when the algesimetric test was not very sensitive to a particular drug, either alone or in combination.The antinociceptive effects of rosuvastatin and duloxetine (as well as the effects of the respective vehicles) in mice with paclitaxel-induced neuropathy were evaluated using the hot plate and the electronic von Frey tests. All algesimetric evaluations took place at noon, immediately before rosuvastatin, duloxetine, or vehicle administration. Dose-response curves were carried out using daily administration of rosuvastatin  or duloxetine  during 7 consecutive days, starting 10 days after the first administration of paclitaxel once stable levels of allodynia and hyperalgesia were observed. Dose ranges for rosuvastatin and duloxetine were taken from Miranda et al.  and fromallarida , concernr et al.  used ED230 (for von Frey testing) or ED50 (for hot plate testing), mixing equieffective submultiples of them . This procedure allowed to build a dose-response curve for the combination, from which the experimental ED30 or ED50 of the mixture can be calculated. The relation between the experimental value of the combination  and the theoretical value  in the isobologram determines the type of interaction, which can be expressed by the interaction index  between the drugs tested.The antinociceptive interaction of rosuvastatin with duloxetine in mice with paclitaxel-induced neuropathy was performed by isobolographic analysis, as described elsewhere ,77,78. T2 integrin family of adhesion molecules. The expression levels of these proteins, measured by the Western blot technique, were used to detect activation or inactivation of astrocytes and/or microglia in the dorsal horn of mice after induction of neuropathy with paclitaxel, and after treatment with the rosuvastatin/duloxetine combination. Animals were sacrificed by sodium pentobarbital overdose on days 0, 10, or 17 as related to the first injection of paclitaxel. The spinal cord was extracted into lumbosacral segments, washed 3 times in PBS, and immediately stored at a temperature of −80 °C. The tissues were homogenized in RIPA lysis buffer , and a cocktail of protease and phosphatase inhibitors . The homogenate was centrifuged at 15,000 rpm for 20 min at 4 °C and the supernatant was collected for protein concentration determination using the bicincoinic acid technique .Astrocytes were identified by the expression of GFAP that forms the intermediate filaments of the cytoskeleton, while the cells of the microglia were identified by the presence of the CD11b protein, a member of the βFor SDS/PAGE, 20 µg of total proteins was taken and mixed with loading buffer with blue bromophenol, heated at 95 °C for 5 min and loaded into 12% polyacrylamide gels . Electrophoresis was carried out at room temperature for 2 h at 80 V. The bands were electrotransferred to nitrocellulose membranes (trans-blot turbo mini-nitrocellulose transfer packs) using a Transwell equipment  for 7 min. After incubating the membranes overnight at 4 °C with the appropriate antibodies (see above), they were rinsed with Tris buffer and incubated with the peroxidase-conjugated secondary antibody (see above). Bands were developed with luminol and revealed in a Chemiscope equipment  adjusted to the best exposure time.t-test. Comparisons involving three or more datasets  were performed by one-way ANOVA, two-way ANOVA, or repeated measures two-way ANOVA (where appropriate), with Dunnett  or Bonferroni (intergroup comparisons against corresponding scores in sham mice) post hoc test. Significance was accepted at a probability level of 0.05 or less.Data are presented as means ± standard error of the mean (S.E.M.) and all statistical analyses, including calculation of area under curves and linear regression analyses, were performed with GraphPad Prism 9.5.1 software . Comparisons of data between two groups was performed by two-tailed unpaired Student’s


This 3.6-year prospective study examined combined impacts of physical activity, dietary variety, and social interaction on incident disability and estimated population-attributable fraction for disability reduction in older adults.Participants were 7,822 initially non-disabled residents  aged 65–84 years of Ota City, Tokyo, Japan. Sufficiency of moderate-to-vigorous-intensity physical activity (MVPA) ≥150 min/week, dietary variety score (DVS) ≥3 (median), and social interaction (face-to-face and/or non-face-to-face) ≥1 time/week was assessed using self-administered questionnaires. Disability incidence was prospectively identified using the long-term care insurance system’s nationally unified database.P < 0.001 for trend). Population-attributable fraction for disability reduction in satisfying any one, any two, and all three behaviors were 4.0% , 9.6% , and 16.0% , respectively.During a follow-up of 3.6 years, 1,046 (13.4%) individuals had disabilities. Independent multivariate-hazard ratios (HRs) and 95% confidence intervals (CIs) of MVPA, DVS, and social interaction sufficiency for incident disability were 0.68 , 0.87 , and 0.90 , respectively. Incident disability HRs gradually reduced with increased frequency of satisfying these behaviors , in an inverse dose-response manner (Combining physical activity, dietary variety, and social interaction substantially enhances the impacts on preventing disability among older adults, with evidence of an inverse dose-response manner. Improving insufficient behavior elements through individual habits and preexisting social group activities may be effective in preventing disability in the community. It is also essential to create a social environment in which older adults can improve their physical, mental, and social health through social participation.Maintaining functional ability and preventing disability are important challenges in an aging society. Functional ability is determined by the person’s intrinsic capacity , relevant environmental factors, and the interaction between the two.2–4 However, the health behaviors examined previously were limited to behaviors related to the prevention of non-communicable diseases, such as PA , diet , smoking, and alcohol consumption.2,3 In order to prevent frailty and sarcopenia, which are major causes of disability, it is important to also include moderate-to-vigorous-intensity PA  and the consumption of a varied diet containing protein and vitamin D.5 There is also growing recognition of the importance of the social aspect of frailty,6,7 with studies reporting that social relationships greatly impact health outcomes, such as disability8 and mortality.9 Thus, it is necessary to focus on MVPA, dietary variety, and social relationships from the perspective of disability prevention in the community.There is increasing evidence that combining healthy lifestyle behaviors effectively reduces disability risk.10 examined the synergistic impacts of the combinations of any two of PA, protein intake, and social network on the development of frailty, they did not investigate the combined impact of all three variables. Pérez-Tasigchana et al4 examined the association of six healthy behaviors  with incident frailty and disability. However, they failed to include daily social interaction in their analyses of the association between the number of healthy behaviors an individual performs and the incidence of frailty and disability.4 Moreover, they did not focus on the combination patterns of the health behaviors. Thus, the combined and public health impacts of MVPA, dietary variety, and social relationships on incident disability are still unclear.To our knowledge, only two observational studies have examined the combined impacts of these behaviors on adverse health outcomes. Although Haider et alWe examined the combined impacts of MVPA, dietary variety, and social interaction on incident disability and estimated the population-attributable fraction (PAF) for disability reduction in community-dwelling older adults.11,12 On August 1, 2016, Ota City had a population of 716,645, among whom 162,443 (22.7%) were aged 65 years or older. Of the population aged 65 and older, 15,500 residents  aged 65–84 years, stratified by sex and age group (65–74 and 75–84 years), were selected using random sampling strategies in all 18 districts of the city.11 All participants were physically and cognitively independent, which was defined as the absence of long-term care insurance (LTCI) certification.13We used data from a community-wide intervention study, launched in 2016, on preventing frailty in Ota City, Tokyo, Japan.Of the 15,500 self-administered questionnaires distributed in July 2016, 11,925 were returned (response rate: 76.9%). The mean district response rate was 77.1% (range: 71.8–80.8%). As Figure The study protocol was developed in accordance with the guidelines proposed in the Declaration of Helsinki and was approved by the Ethical Committee of the Tokyo Metropolitan Institute of Gerontology. All participants gave their informed consent before participation.14,15 Its external reliability and validity have been previously reported.14,15 Participants were assessed based on their time spent performing vigorous and moderate PA, as well as their walking time during a typical week. Using these values, we defined total MVPA as 7 days × .16 A total MVPA time ≥150 minutes/week was defined as sufficient PA.17We evaluated individuals’ levels of MVPA using the Japanese version of the International Physical Activity Questionnaire-Short Form (IPAQ-SF).18,19 Although various DVS cut-off scores  were used in previous studies, these DVS scores came from health check-up data from which DVS scores are typically reported as higher (mean scores ranging from 5 to 7)18,19 than those from self-administered mail survey data (mean scores ranging from 3 to 4).11,20 Therefore, based on the distribution of the study population, the median DVS (score of 3) was used as the cut-off value, and DVS ≥3 was defined as sufficient dietary variety in this study.Participants were assessed based on their weekly frequency of consumption of 10 main Japanese food groups . Participants were able to choose from four responses in relation to their consumption of each food group . A Dietary Variety Score (DVS) was calculated for each participant, using the sum of the number of times each participant answered “almost daily” for each food group (range: 0–10).21,22 Thus, sufficient social interaction was defined as contact with others ≥1 time/week.The level of social interaction was assessed based on the frequency of face-to-face and non-face-to-face  interactions they had with relatives, friends, neighbors, and family who did not live with them. Frequency of contact with others (apart from cohabiting family members) of less than 1 time/week was defined as social isolation.13 LTCI certification is based on a nationally standardized multistep assessment.13 Ultimately, the Municipal Certification Committee of Needed Long-Term Care  decides whether an older adult should be certified as requiring long-term care and classifies care needs under one of seven levels .13Disability was identified in the participants using the nationally unified database of the LTCI system, enrolment in which is mandatory for all Japanese adults aged 40 years and older. The system provides formal care and support to Japanese adults aged 65 and older with physical and mental disabilities.23 using the date of the LTCI application as the date of disability incident. Because new LTCI applications were affected by the novel coronavirus disease pandemic,24 we set January 31, 2020 as the endpoint.Disability was defined as the onset of long-term care needs at the support level 1 or above,2), alcohol drinking and tobacco smoking statuses , hypertension, hyperlipidemia, heart disease, stroke, diabetes mellitus, cancer, depressive mood, lower back pain, knee pain, sitting time , and instrumental activities of daily living (IADL) dependence (presence or absence). Equivalent income was calculated by dividing household income by the square root of the number of household members.25 BMI was defined as self-rated body weight (kg) divided by self-rated height squared (m2). Depressive mood was defined as a score of ≥2 on the 5-item Geriatric Depression Scale.26,27 Sitting time was evaluated using the Japanese version of the IPAQ-SF.14,15 IADL was assessed using the instrumental self-maintenance subscale of the Tokyo Metropolitan Institute of Gerontology Index of Competence,28 the total score of which ranges from 0 to 5. Participants whose total score was 5 were classified as independent; those whose total score was ≤4 were classified as dependent.29The covariates included age, sex, living situation , marital status , educational attainment , equivalent income , body mass index . An α of 0.05 indicated statistical significance. To examine whether minutes/week of MVPA, DVS, and other continuous variables were systematically associated with the number of satisfying health behaviors, we used a trend test (“nptrend” command). Chi-square test compared social interaction and other categorical variables related to the number of satisfying health behaviors.First, we examined the impact of individual health behaviors on incident disability risk. Because our data had a multilevel structure comprised of individuals (at level 1) nested within 18 districts (at level 2), we used multilevel survival analyses with fixed slopes and random intercept models, calculating adjusted hazard ratios (HR) and 95% confidence intervals (CIs) for incident disability, the dependent variable. Individual health behaviors were included simultaneously as fixed factors. The district was a random factor. We constructed four analytic models. Model 1 was adjusted for age and sex. Model 2 additionally adjusted for living situation, marital status, educational attainment, equivalent income, BMI, hypertension, hyperlipidemia, heart disease, stroke, diabetes mellitus, cancer, alcohol drinking status, and smoking status. Model 3 additionally adjusted for depressive mood, lower back pain, knee pain, and sitting time. Model 4 additionally adjusted for IADL dependency.Second, the Kaplan-Meier method was used to produce the cumulative probability of remaining disability-free according to the number of satisfying health behaviors. To examine whether the number of satisfying health behaviors was systematically positively associated with the cumulative probability, we used a trend test .Third, we performed two analyses: with the number of satisfying health behaviors as the fixed factor and with the combination pattern of health behaviors as the fixed factor. They were included in a dummy form (not satisfying any behaviors was the reference) as fixed factors in the same statistical approach. We tested the statistical interaction between the number of satisfying health behaviors or combination patterns of health behaviors and sex or age group (65–74 or 75–84 years).30 In the estimation of the PAF of individual health behaviors, we calculated how much the disability rate might have decreased, if participants with insufficient levels of MVPA (<150 minutes/week), DVS (<3), and social interactions (<1 time/week) would have reached the previously defined sufficient level for each behavior. In the estimation of the PAF for the number of satisfying health behaviors, we calculated how much the disability rate might have decreased, if participants with 0 to 2 satisfying health behaviors would have reached sufficient levels for all three health behaviors. The estimations of the PAF when any one or two health behaviors were satisfied were performed in the same manner.Finally, PAFs and their 95% CIs were estimated for individual health behaviors and number of satisfying health behaviors in model 4, using the “punafcc” command.In secondary analyses of the possible influence of reverse causation, we performed sensitivity analyses using the same statistical approach, after excluding individuals who developed disability during the first year of follow-up.We excluded participants with missing information regarding their disability, IPAQ-SF, DVS, and social interaction from the analysis. The missing covariates were assigned to the “missing” category and included in the analysis to reduce the selection bias.During a median follow-up of 3.6  years, 1,046 participants  presented with disabilities, with rates of 46.3 per 1,000 person-years, and 291 participants  presented with disability during the first year.P < 0.001). There were significant differences between groups in sex, marital status, education, equivalent income, BMI, sitting time, and prevalences of hypertension, hyperlipidemia, heart disease, stroke, diabetes mellitus, smoking, depressive mood, lower back pain, and IADL dependency.Table Table P < 0.001 for trend).Figure P < 0.001 for trend). PAF for disability reduction in satisfying any one, any two, and all three behaviors were 4.0% , 9.6% , and 16.0% , respectively. In the combination pattern, disability risk was consistently and significantly lower in the satisfying only MVPA , MVPA + dietary variety , MVPA + social interaction , and all three behaviors,  even in model 4, as compared with people who did not satisfy any behaviors. The significance of dietary variety + social interaction disappeared in model 4 . These results did not vary by age group  or sex .Table P = 0.052). Other results were not substantially different from those of the primary analyses , 3.6-year disability would be reduced by 16.0%. Finally, these results were not substantially different in the sensitivity analysis, suggesting the importance of combining MVPA, dietary variety, and social interaction in disability prevention efforts in the community.4 and disability31 among traditional and nontraditional health behaviors. There is growing evidence that the Japanese dietary pattern is associated with lower risk of disability,32 and dietary variety especially reduces all-cause and cancer mortality.33 Although our results on the association of social interaction alone with incident disability were not robust, they were consistent with a recent study.4Our results for each health behavior were consistent with previous studies. Recent studies showed that PA was most protective against frailty34–37 eating,38–40 and doing social activities35 with others brought various health benefits compared to doing these activities alone. Evidence of the relationship between this kind of interactive behavior and positive psychological outcomes39 is especially robust, and the mechanism can be explained by the psychosocial factors  that mediate the positive effects on mental health.41 Moreover, interaction with others not only promotes PA42 and dietary variety43 but also affects the risk reduction of health outcomes.9 By adding a social interaction element, the preventive impact on disability was considered to be reinforced from a psychosocial aspect.Interestingly, despite the non-robust associations of DVS and social interaction with incident disability, additional risk reduction was observed when combined with MVPA. Previous studies consistently reported that exercising,44 and frailty.45 In particular, for reducing frailty, a combination of exercise and nutritional supplementation/education—not nutrition only—was found to be effective.45 The additive effects on muscle mass, strength, and physical performance may play an important role in preventing frailty and disability. Our results provided further evidence that combining PA and dietary variety with social interaction reduced disability risk and enhanced the public health impact. The clear inverse dose-response association of the number of satisfying MVPA, DVS, and social interaction with disability risk is a noteworthy and important finding of this study.On the contrary, MVPA and dietary variety would enhance the preventive impact on disability from medical and physiological aspects. Previous systematic reviews and meta-analyses of randomized controlled trials showed some positive effects of exercise and nutritional interventions for reducing sarcopenia46,47 Increasing population coverage rates of participation in such groups is an important challenge for achieving population-level disability prevention.From our findings and from a public health perspective, we suggest effective countermeasures against disability in the community setting. To combine MVPA, dietary variety, and social interaction, promoting the establishment and continuation of social group activities in the community is a main strategy. Several quasi-experimental studies reported that participation in social activities, such as community salons and self-managed exercise groups, reduced the risk of disability.12 This could include adding a 10-minute dietary habit check and/or exchange program to a self-managed exercise group, or a simple muscle strengthening activity and/or the dietary habit check to a cultural group. This may lead to increased diversity of activities and strengthen the preventive functions against disability. Promoting such efforts in collaboration with multiple occupations, institutions, and organizations may be also an effective strategy for preventing disability in the community.Another important point is adding the insufficient element among MVPA and dietary variety to preexisting social group activities to make it a habit.15 Moreover, DVS from self-administered questionnaires may be underestimated compared to face-to-face interview surveys.18 Although our results were based on the median DVS (of 3) of the study population, desirable DVS is considered to be a higher value.18,19 Thus, our results should be generalized with caution. Second, selection bias is a concern, as people who did not satisfy any behaviors constituted only 5.8% in this study. The exclusion of 3,649 (30.6%) respondents who had missing IPAQ-SF, DVS, or social interaction data may have led to the biased inclusion of a health-conscious population in the analysis. Third, the follow-up period was relatively short. Although our results have been confirmed by a sensitivity analysis, the possibility that undiagnosed medical conditions may have affected health behaviors and/or incident disability cannot be completely excluded. Fourth, an older adult must contact the municipal government to officially certify his or her care needs.13 Therefore, some disabled individuals might have failed to report themselves, which may have resulted in the underestimation of disability incidence (detection bias). Finally, it should be noted that this study used data sources from a community-wide intervention study. Although the intervention did not affect population-level frailty at 2 years, the walking time and DVS in the intervention subgroup improved at the population-level.12 The present study only assessed baseline health behaviors and was not able to take into account the improvements at 2 years and subsequent changes in these health behaviors during the follow-up. If health behaviors improved further during the subsequent follow-up period after 2 years, the relationships between health behaviors and disability risk shown in this study may have been underestimated.Our study has several limitations. First, the self-administered questionnaire used for measurement might involve recall bias. Although the IPAQ-SF is widely used for the assessment of PA, it tends to overestimate PA, as compared with objective measurement tools.Despite these limitations, to our knowledge, this study is the first to report the combined impact of MVPA, DVS, and social interaction and its PAF for disability risk. These results are strengthened by the large sample of randomly recruited participants and the high response of 76.9% and follow-up rate of 99.84% .The combination of regular PA, dietary variety, and social interaction substantially enhances the impacts on preventing disability of older adults in an inverse dose-response manner. An approach that adds the insufficient behavior element to individual habits and preexisting social group activities may be effective in preventing disability in a community. A further study will be necessary to confirm the hypothesis in real-world settings.


To facilitate continuation of school feeding during COVID-19 school lockdowns, U.S. Congress authorized waivers to allow for school meals to be picked up by parents/guardians in non-school settings. We summarized school meals distribution and characterized reach in socially vulnerable neighborhoods in New Orleans, a city prone to environmental disasters, with a city-wide charter school system, and historically high levels of child poverty and food insecurity.School meals operations data were obtained from New Orleans, Louisiana (NOLA) Public Schools for 3/16/2020–5/31/2020. For each pick-up location, we estimated: average meals available (weekly), average meals served (weekly), number of weeks of operation, and rate of meal pick-up . These characteristics were mapped in QGIS v3.28.3, along with neighborhoods’ Social Vulnerability Index (SVI). Pearson correlation and ANOVA were run to assess differences between operations characteristics and neighborhood SVI.From 38 meal sites, 884,929 meals were available for pick-up; 74% of pick-up sites were in moderately/highly socially vulnerable areas. Correlations between average meals available and served, weeks of operation, rate of meal pick-up, and SVI were weak and not statistically significant. SVI was associated with average rate of meal pick-up but not other operations characteristics.Despite the disaggregated nature of the charter school system, NOLA Public Schools successfully pivoted to providing children with pick-up meals due to COVID-19 lockdowns, with 74% of sites located in socially vulnerable neighborhoods. Future studies should describe the types of meals provided to students during COVID-19, in terms of diet quality and nutrient adequacy. With reported increases in food insecurity nationwide , the UniThis Brief Research Report summarizes the school meal distribution program that operated in New Orleans, Louisiana in response to the COVID-19 pandemic, including an evaluation of the geographic coverage of the program with respect to the social characteristics of the neighborhoods where meal distribution sites were placed. Unlike most U.S. cities with traditional schools with centralized governance through districts, New Orleans has a decentralized city-wide charter school system that serves 99% of the city’s public-school students . CharterNew Orleans has among the highest rates of child poverty  and chil2.Data on meal distribution operations related to the COVID-19 pandemic between March 16 and May 31, 2020 were obtained from NOLA Public Schools, the Orleans Parish’s school board. NOLA Public Schools oversees operations for 76 public schools in Greater New Orleans, including 6 kindergarten through 12th grade (K-12) schools, 48 elementary schools, and 22 high schools, having served 45,022 students in the 2020–2021 academic year, 82% of whom are Black  and Wednesdays . Thus, school meal data were summarized based on weekly numbers to account for differences in daily vs. twice-weekly distributions.socioeconomic status, including poverty, unemployment, income, and education, 2) household composition/disability, comprising of age, single parenting, and disability, 3) minority status/language, comprising of race, ethnicity, and English-language proficiency, and 4) housing/transportation, comprising of housing structure, crowding, and vehicle access  number of individuals 25 years or older without a high school diploma, 5) number of individuals 65 years or older, 6) number of individuals 17 years or younger, 7) number of individuals with a disability, 8) number of households led by a single parent, 9) number of individuals who are not non-Hispanic White, 10) number of individuals who speak English “less than well,” 11) number of individuals who live in a multi-unit structure, 12) number of mobile homes, 13) number of crowded households (defined as having more people than rooms), 14) number of households with no vehicles, and 15) number of individuals living in group quarters.Geocoded data from the American Community Survey (ACS) 2015–2019  obtainede access . Variablper capita income, which was ranked from highest to lowest as a higher income is indicative of lower vulnerability. After, a percentile rank was calculated for each of the 15 variables for each census tract, estimated as follows: Percentile rank = (rank – 1)/(N-1). Next, each of the 15 variable’s percentile rank were summed to create an overall score. To rank census tracts based on their overall scores, an overall percentile rank was calculated following the same formula, with the final SVI ranking score ranging from 0.0 (less vulnerable) to 1.0 (most vulnerable).Following SVI construction procedures , Orleans2.1.Descriptive statistics were used to characterize the school meal distribution operations. For each pick-up location, the following characteristics were summarized: average meals available (weekly), average meals served (weekly), number of weeks of operation, and rate of meal pick-up .In order to visualize the distribution of the meal sites in relation to New Orleans’ socioeconomic characteristics, we created three maps using the software QGIS version 3.28.3, overlaying the SVI ranking score categorized in quartiles and the 1) average meals available, 2) average meals served, and 3) rate of meal pick-up at each meal pick-up location; all three maps provided a similar visual story, so only the map presenting weekly average meals served is presented in this Brief Research Report (the other two maps are available upon request).p <0.05 denoting statistical significance.Further, we ran Pearson correlations between the SVI ranking score (continuous) and each of the meal distribution characteristics . Lastly, using the SVI ranking score categorized in quartiles , we ran an Analysis of Variance (ANOVA) analysis to examine the mean differences in average meals available, average meals served, number of weeks of operation, and rate of meal pick-up by SVI ranking score categories, using Tukey’s test as a post-hoc analysis where needed. All statistical analyses were conducted in SAS v9.4, with a value of 3.Based on data from NOLA Public Schools, 45 school meal pick-up sites were set up throughout the city in the immediate aftermath of the COVID-19 lockdowns . However, seven sites in the dataset had rates of meal pick-up that were greater than 100%, suggesting that more meals were served than were available; since we were unable to trace the source of the error in this accounting, these 7 pick-up sites were excluded from the sample for all analyses. From the remaining 38 sites, a total of 884,929 meals were available for pick-up during the 11-week observation period. On average, 2,405 meals were available weekly per site during the study period , with an average of 2,183 meals picked up weekly , yielding an average meal pick-up rate  of 90.1% (range 59–100%). On average, pick-up sites were open for meal distribution for 9.5 weeks (range 0–11 weeks).n = 14) or highly  socially vulnerable areas (SVI > 0.492), with the exception of the Algiers/Old Aurora region, in the southern tip of the map, which appears to have only one pick-up site. It is important to note, however, that three of the seven pick-up sites excluded from the sample were located in this area (data not shown), suggesting adequate coverage.There were weak and not statistically-significant correlations between SVI ranking (continuous) and average meals available, average meals served, number of weeks of operation, and rate of meal pick-up . We also4.This Brief Research Report summarizes the school meal distribution operations carried out in New Orleans in the immediate aftermath of the COVID-19 triggered lockdowns. We found that NOLA Public Schools operated 45 meal pick-up sites throughout the city between March 16 and May 31, 2020, with reliable data on 38 of these 45 pick-up sites. Average number of meals available and served weekly were 2,405 and 2,183, respectively, with an average pick-up rate of 90%. Almost 37% of the pick-up sites (14 out of 38) were located in the most socially vulnerable neighborhoods, with an additional 37% located in moderately socially vulnerable neighborhoods. Neighborhood social vulnerability was not significantly associated with average meals available, average meals served, nor number of weeks of operation, though our small sample size may have limited statistical power to detect any differences. Neighborhood social vulnerability was associated with rate of meal pick-up, with statistically significant differences among census tracts ranked 2 (~80% pick-up rate) and 3 (~93% pick-up rate) in a 4-category vulnerability ranking, with 1 being the least vulnerable and 4 the most vulnerable.Since New Orleans has among the highest rates of child poverty  and chilOther research has also described COVID-19 related school meal distributions, particularly immediately after the lockdown declarations in March 2020 . In a stThis study has strengths and limitations. Strengths include the detailed operations data available, including the addresses of the meal distribution sites, allowing us to map the sites and evaluate geographic reach. Furthermore, we used a social vulnerability index – a more comprehensive way of assessing neighborhood demographic and socioeconomic characteristics than, for example, just looking at poverty rates or racial/ethnic minority make-up – to assess meal coverage from an equity perspective. As for limitations, there is no available information on who picked up the meals and who consumed the meals. It is possible that some community members without children picked up meals, and that meals picked up by parents or guardians of children were consumed by family members other than the target children. Further, the operations data did not include information on the nutrition composition of meals, so diet quality and nutrient adequacy could not be assessed. Moreover, we only had reliable operations data on 38 pick-up sites, which limited our statistical power for detecting associations between meal operations data and neighborhood characteristics. Finally, while parents/caregivers could pick up meals for their children in the pick-up site closest to their home, this may have been on a different census tract than their residence; thus, our results linking the operations’ data with neighborhoods’ social vulnerability should be interpreted with this caveat in mind.In conclusion, NOLA Public Schools successfully pivoted to providing children with take-home meals due to COVID-19 lockdowns as of March 16, 2020, setting up 45 meal pick-up sites throughout the city, with three quarters of the sites assessed located in socially vulnerable neighborhoods. Future studies should focus on describing the types of meals provided to students during the COVID-19 lockdowns – in terms of quantity, quality, and nutrient adequacy – as well as on compiling “lessons learned” of what worked and did not work for future emergency preparedness. This is particularly important in a city like New Orleans, which is frequently affected by environmental disasters  that affect school operations.https://nolapublicschools.com/contact.The data analyzed in this study is subject to the following licenses/restrictions: data are not publicly available but were shared by NOLA Public Schools upon request by the authors. Requests to access these datasets should be directed to MC, KC, and MK conceptualized the manuscript idea. MC obtained the data. JF, NE, and TN analyzed the data. MC and MK supervised the data analysis. MC wrote the first draft with contributions from JF. All authors contributed to the article and approved the submitted version.This research was supported in part by the Kellogg Foundation (Grant number P-6005931-2021) and by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number TL4GM118968. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.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.


Postpartum depression (PPD) has been associated with adverse health outcomes, including maternal suicide. Mode of delivery has been suggested to be a risk factor for PPD, but no large cohort study has examined the association between mode of delivery and PPD. We aimed to examine the association between mode of delivery and risks of PPD at 1 and 6 months after childbirth.In a nationwide study of 89,954 mothers with a live singleton birth, we examined the association between mode of delivery and risks of PPD. PPD was evaluated using the Edinburgh Postnatal Depression Scale (≥13) at 1 and 6 months after childbirth. Odds ratios (ORs) with 95% confidence intervals (CIs) of PPD were calculated using multivariable logistic regression analyses after adjustment of antenatal physical, socioeconomic, and mental factors.Among 89,954 women, 3.7% and 2.8% had PPD at 1 and 6 months after childbirth, respectively. Compared with unassisted vaginal delivery, cesarean section (CS) was marginally associated with PPD at 1 month but not at 6 months; adjusted ORs were 1.10  and 1.01 , respectively. The association with PPD at 1 month was evident in women with antenatal psychological distress . The observed associations were attenuated after adjusting for infant feeding method.Women who had antenatal psychological distress and underwent CS delivery may be regarded as a target for monitoring PPD. In Japan, maternal and late-maternal deaths by suicide were reported to be 8.7 per 100,000 births, with 63 cases (23 during pregnancy and 40 within a year after delivery) from 2005 to 2014 in Tokyo, Japan; this is in contrast to the nationwide low maternal mortality rate , which is thought to be due to maternal physical complications during pregnancy.9 Globally, postpartum suicide has been listed as a possible cause of pregnancy-related death in the 10th revision of the International Classification of Diseases and Related Health Problems (ICD-10).10 Therefore, the prevention of PPD is important in terms of preventing avoidable maternal deaths. Reported antenatal risk factors for PPD range from physical, socioeconomic, and mental factors.1,3,10–13Postpartum depression (PPD) is a major concern in reproductive health.12,14 In a meta-analysis of 28 studies, cesarean section (CS) was associated with increased risk of PPD.15 This meta-analysis included five Asian studies; three of them had small cohort sample sizes of less than 600, and two were cross-sectional studies. A Canadian cohort study of 2,560 women showed no association between CS and PPD among the overall sample of women; however, in interaction models, CS was associated with an increased risk of PPD in Canadian-born women and a decreased risk in non-Canadian-born women, suggesting a difference in the subjective “desired” mode of delivery according to culture and ethnicity.16 Because the rate of CS in Japan is on the rise,17 it is important to examine the potential adverse outcomes of CS. A previous study of 479 Japanese women showed that emergency CS was associated with an increased risk of PPD, but the number of CS cases was only 109 and the study subjects were limited to primiparous women aged 35 years and older.18 Other than CS, a previous study involving 707,701 Swedish women showed that instrumental vaginal delivery was associated with PPD within a year after childbirth.19 Because vaginal delivery comprises 80% of all deliveries in Japan,17 it is important to examine the effect of assisted and instrumental vaginal deliveries in reference to unassisted  vaginal delivery on PPD among Japanese women if the data allows. One previous study examined the association of induced vaginal delivery and CS with neonatal outcomes compared with unassisted vaginal delivery,20 but there has been no study that examined the association between these modes of delivery and PPD.Mode of delivery has been also suggested to be a risk factor for PPD.The Japan Environment and Children’s Study (JECS) collected information about the mode of delivery and PPD among large nationwide population samples. Using these data, we assessed the association between mode of delivery  and risk of PPD at 1 and 6 months after childbirth.21–23 The present study is based on the jecs-ag-20160424 dataset, which was released in June 2016 and revised in October 2016.The JECS is a nationwide government-funded birth cohort study that began recruiting expectant mothers in January 2011 and finished recruiting in March 2014. Fifteen Regional Centres  were selected. Each Centre recruited women during early pregnancy at obstetrics clinics/hospitals, and local municipal offices issuing the Maternal and Child Health Handbook. The JECS Study Protocol and details have been published elsewhere.n = 92,790). Complete information on mode of delivery was available for 92,356 mothers. In addition to mothers with complete information on their Edinburgh Postnatal Depression Scale (EPDS) scores, mothers with total EPDS scores greater than the cut-off score, even with missing values, were included and regarded as positive for PPD. In contrast, mothers with total EPDS scores less than the cut-off score, when estimated scores for missing items did not exceed 3 points, were included and regarded as negative for PPD. Thus, PPD status at 1 month and 6 months postpartum was available for 89,954 and 84,858 mothers out of the 92,790 mothers (96.9% and 91.5%), respectively.Among 104,065 fetal records, we only included mothers with a live singleton infant .25 The cut-off score to indicate PPD in the EPDS varied in previous literature. According to a systematic review of 37 studies , the cut-off score of 9/10 was suggested to indicate possible PPD and 12/13 was suggested to indicate probable PPD.26 We used the cut-off score of 12/13 to signify PPD in this study, which could allow international comparisons with previous studies.15,26 The covariates based on prior studies included women’s age, parity, pre-pregnancy body mass index (BMI), weight gain during pregnancy, gestational week at birth, and newborn complications at birth as physical factors; marital status, education, annual family income, and residential area as socioeconomic factors; antenatal mental status and history of mental diseases as mental factors; and infant feeding method.We collected Japanese-language EPDS scores at 1 and 6 months after childbirth. The total score of the EPDS, which comprises 10 items listed on a four-point Likert scale from 0 to 3 according to the increasing severity of symptoms, ranges from 0 to 30.27 as <18.50, 18.50–24.99, and ≥25.00 kg/m2, respectively. Weight gain during pregnancy was calculated by subtracting weight just before delivery from pregestational weight and re-categorized into quantiles. Gestational week at birth was dichotomously categorized into term  or preterm . Newborn complications at birth were dichotomously categorized into yes or no. Marital status was categorized as married, never married, divorced, or widowed. Education was categorized as junior high school, high school, vocational school, junior college, or university and higher. Equivalent family income was calculated by dividing the average amount in a six-scale family income category by the square root of the number of family members and re-categorized into quantiles. Women’s antenatal mental status was assessed using the Japanese version of the Kessler 6 (K6) scale. The K6 score was originally used for screening “serious mental illness”28 or mood and anxiety disorder 29 in the general population. The optimal cut-off score for screening mood and anxiety disorders, or possible psychological distress in the general population was 4/5.30,31 Therefore, we dichotomously categorized women into those with (≥5) or those without (0–4) antenatal psychological distress. History of mental diseases was determined using a question regarding history of diagnosed mental diseases  in the past. Infant feeding method at 1 month was categorized as exclusive breastfeeding, bottle feeding, or mixed. Residential areas were represented by the locations of the 15 Regional Centres where the data were collected.Women’s age was categorized as 5-year age groups, and parity as primiparous or multiparous. Pre-pregnancy BMI was calculated as the weight in kilograms divided by the square of the height in meters and re-categorized into underweight, normal, and overweight and obese according to the World Health Organization classificationn = 89,669) using the method of infant feeding to check for any effect modification in the associations between mode of delivery and risk of PPD by infant feeding method. In addition, a stratified analysis by antenatal mental status was conducted for those whose antenatal mental status was obtained . All analyses were performed using Statistical Analysis Software version 9.4 .The categories’ proportions of each variable in the total sample were calculated and tested using a chi-square test across each mode of delivery. Multivariable logistic regression analyses were used to estimate the associations between mode of delivery and PPD. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated after adjusting for potential confounders. Using unassisted vaginal delivery as the reference group, we estimated the risk of PPD (EPDS score ≥13 at 1 and 6 months after childbirth) among women whose delivery was assisted, instrumental, or CS. In model 1, we adjusted for the women’s age. In model 2, we further adjusted for parity, pre-pregnancy BMI, weight gain during pregnancy, gestational week at birth, and newborn complications at birth. In model 3, we further adjusted for marital status, education, annual family income, mental status, history of antidepressant use, and residential area. We further adjusted for infant feeding method in an additional analysis, and conducted a stratified analysis (Table P = 0.20).Compared with women who gave birth by unassisted vaginal delivery, women who gave birth by assisted vaginal delivery, instrumental delivery, or CS had an increased risk of PPD at 1 month in the age-adjusted analysis: ORs were 1.12 , 1.20 , and 1.30 , respectively (Table P = 0.12) in model 3. Women who had assisted vaginal delivery or CS showed no increased risk of PPD in multivariable logistic regression models.At 6 months from childbirth, women with instrumental delivery showed an increased, though insignificant, risk of PPD: OR was 1.15 (0.96–1.37) (P = 0.07) in the additional analysis that was further adjusted for infant feeding method. Among women with no antenatal psychological distress, there was no association between mode of delivery and PPD at 1 month. At 6 months, there was no association between mode of delivery and PPD in either strata of the antenatal mental status.Table P for interactions between mode of delivery and infant feeding method towards the risk of PPD at both 1 and 6 months were >0.1. At 1 month from childbirth among those who used a bottle or mixed feeding, CS tended to be associated with increased risk of PPD compared with unassisted vaginal delivery, but the association was attenuated: adjusted OR was 1.10  (P = 0.09). At 6 months from childbirth among those who used a bottle or mixed feeding, the increased risk of PPD with instrumental delivery was augmented and reached the level of significance: adjusted OR was 1.25 .Table In this nationwide cohort study, we found that CS was marginally associated with an increased risk of PPD at 1 month, but not at 6 months after childbirth after adjustment for antenatal physical, socioeconomic, and mental factors. This tendency was attenuated after adjusting for infant feeding method. In the stratified analyses, we found that an association between CS and risk of PPD at 1 month was evident among women with psychological distress.32 and among 17,648 American women .33 Yet, a Chinese study of 1,823 women and a Japanese study of 99,202 women showed no association between mode of delivery and risk of PPD at 4 weeks, with respective ORs of 1.13 ,34 and 1.02 ,35 partly due to different outcome settings. On the other hand, the null association between CS and risk of PPD at 6 months from childbirth found in our study was similar to the findings from previous studies with 55,814 Norwegian women,36 1,379 Qatari women,37 and 99,202 Japanese women.35Our result indicating a marginal association between CS and PPD at 1 month was consistent with the findings from previous studies that did not adjust for infant feeding method; among 511,422 Finnish women ,1,3 Biological mechanisms, such as hormonal changes, may play a role in the development of PPD.1 The levels of steroid hormones, including estradiol, progesterone, and cortisol, are known to dramatically decrease after childbirth regardless of mode of delivery.1 However, there has been debate concerning the role of steroid hormones in depression in general and in PPD.38The mechanism by which CS may influence risk of PPD is not clear.39 The oxytocin level is known to sharply increase during vaginal delivery,40 and the mean plasma oxytocin level was significantly higher in unassisted vaginal labor  compared with that in elective CS .41 However, a recent systematic review indicated that the infusion of synthetic oxytocin for elective CS raised oxytocin to levels equivalent to those during unassisted vaginal labor.40 Further, the oxytocin level after childbirth is substantially affected by lactation, and lactation increased the oxytocin level and the number of oxytocin pulses by lactation was higher after unassisted vaginal labor than that after CS.42 Thus, the mechanism by which CS biologically affects risk of PPD remains largely unexplained. Unfortunately, we did not collect information about hormone levels in this study. Further studies are necessary to examine the role of these hormones in the association between mode of delivery and PPD.Also, maternal oxytocin concentrations were associated with the risk of PPD.1,3 With CS, the inflammation caused by the surgical operation and trauma,43,44 possible local temporary infection,43 and chronic pelvic pain45 may increase mental and physical stresses.11,15,46 In addition, having CS may be associated with feelings of failure,46 lack of control,46 fear of birth,32 reduced self-esteem,46 and perception of negative delivery experience,47 all of which could contribute to the higher risk of PPD. Moreover, societal and women’s longstanding belief that “normal birth” is a “vaginal birth”16 may affect the mental status of women who undergo CS . This belief does not seem to be prevalent in Canada,16 where the CS rate has been more than 40%; the CS rate was comparatively lower (19%) in our study population.There are possible psychological mechanisms which include various kinds of stresses derived from CS that could induce PPD. Generally, stressful life events are known to lead to PPD.In our study, the association between CS and PPD at 1 month after childbirth was confined to women with antenatal psychological distress. Women with antenatal psychological distress may be more vulnerable to the stresses and fears discussed above. Therefore, more antenatal psychological support should be offered to pregnant women with antenatal psychological distress to reduce the possibility of CS.48 In our study, the method of infant feeding seems to be a strong confounding factor because the association between CS and PPD in the main analysis was greatly attenuated by adjusting for the infant feeding method in the additional analysis. However, we obtained an insignificant interaction by infant feeding method in the stratified analysis (P = 0.60). The association between instrumental delivery PPD was unexpectedly evident at 6 months among women who did not use exclusive breastfeeding. Further research is necessary to confirm or reject the causal effect of breastfeeding on PPD, as well as its moderator effect on the association between different modes of delivery and PPD.Evidence for the association between breastfeeding and PPD is inconclusive so far. A bidirectional association was recently suggested, meaning that failure of breastfeeding, or choosing not to engage in breastfeeding, may increase the risk of PPD, while PPD itself may reduce the ability and motivation to breastfeed the newborn.The major strengths of this study include the use of data from a nationwide cohort study with a large sample size and ample information. We adjusted for potential confounding variables, such as maternal physical, socioeconomic, and antenatal mental factors, and also conducted stratified analyses. We set two time points for the outcomes, at 1 and 6 months after childbirth, which enabled us to clarify how long the effect of delivery mode on risk of PPD lasted.17 Women undergoing emergency CS have been reported to have a higher risk of PPD than those with elective CS , so the investigation of overall CS and PPD could blur the real associations with emergency CS. Second, our outcome, PPD, was measured using self-reported EPDS scores and was not diagnosed by psychiatrists. However, the EPDS is commonly used in research and is a well-validated reliable measurement; therefore, the impact of misclassification is expected to be small. Third, residual confounding could have occurred from unmeasured confounding variables, such as the mother’s anxiety or fear of delivery and general fatigue.However, several limitations should also be addressed. First, there was no information to distinguish between elective and emergency CS. According to the national database of health insurance claims, in 2013, emergency CS consisted of approximately one-third of all CSs in Japan.In conclusion, women who underwent CS had a marginal association with PPD at 1 month after childbirth, but that association did not persist at 6 months. Women who had antenatal psychological distress and underwent CS may be regarded as targets for monitoring of PPD in the early stages after childbirth.


K-mean clustering, correlation analysis, and principal component analysis (PCA) were used to determine the relationship between irrigation hazard indices and the degree of SWI. The tested GW samples were grouped into four clusters , with average SWI degrees of 15%, 8%, 5%, and 2%, respectively. The results showed that the tested GW was unsuitable for irrigation due to salinity hazards. However, a noticeable increase in sodium and magnesium hazards was also observed. Moreover, increasing the degree of SWI (seaf) was associated with increasing salinity, sodium, and magnesium, with higher values observed in the GW samples in cluster C1, followed by clusters C2, C3, and C4. The correlation analysis and PCA results illustrated that the irrigation indices, including EC, PS, SAR, and MAR, were grouped with the SWI index (seaf), indicating the possibility of using them as primary irrigation indices to reflect the impact of SWI on GW quality in coastal regions. The results of this study will help guide decision-makers toward proper management practices for SWI mitigation and enhancing GW quality for irrigation.The degradation of groundwater (GW) quality due to seawater intrusion (SWI) is a major water security issue in water-scarce regions. This study aims to delineate the impact of SWI on the GW quality of a multilayered aquifer system in the eastern coastal region of Saudi Arabia. The physical and chemical properties of the GW were determined via field investigations and laboratory analyses. Irrigation indices , potential salinity (PS), sodium adsorption ratio (SAR), Na%, Kelly’s ratio (KR), magnesium adsorption ratio (MAR), and permeability index (PI)) and a SWI index ( Groundwater (GW) is an important source of freshwater for plants and plays a significant role in meeting increased water demands for urban, industrial, and irrigation purposes in arid regions with low precipitation, high evaporation, and scarce surface water ,2,3. Owi+, and Cl− up to 15 km inland. They found that an increase in the mixing ratio of seawater was the main indicator of seaward SWI, with a maximum value at 5 km from the sea. They recorded a degradation in water quality due to SWI up to 15 km landward. However, the relationship between SWI and irrigation hazards  was not addressed in their study. Sarkar et al. (2021) [2+ concentration in the solution caused by the ion exchange process. Notably, GW salinization poses a major danger to GW resources in coastal regions, reducing the quantity and quality of the water available for human and agricultural consumption. In coastal regions worldwide, GW resources are at risk of salinization owing to global warming and seawater intrusion (SWI) ,8. Rapid. (2021)  investig. (2021)  assessedseaf). We believe that this is the first time that a combination of the saltwater intrusion index, cluster analysis, and irrigation risk indices has been used to study the effect of SWI on GW quality for irrigation in a multi-layer aquifer system. The results of this study may help decision-makers to sustainably manage GW resources and build an effective plan for managing SWI.In this study, we developed a new approach to assess the impact of SWI on the quality of irrigation water using cluster analysis, irrigation hazard analysis, and SWI quantification. All irrigation hazards, including salinity, sodium, magnesium, and carbonate, were addressed and compared with SWI quantification indices to explore their impacts at different degrees of SWI. The aims of this research were to: (1) delineate the impact of SWI on the GW quality of a multi-layered aquifer system in the eastern coastal region of Saudi Arabia using K-mean cluster analysis, irrigation hazard indices, and a SWI quantification index , the transmissivity of the Alat aquifer was approximately 12.96 m2/h. The Khobar aquifer developed within the karstified and fissured dolomitic limestone of the Khobar member of the Dammam Formation. Both the upper and lower limits were bounded by the Alat shale aquitard and shale units  of the Dammam Formation. In both cities, Hassa and Qatif provided most of the water used for drinking and agricultural purposes. The hydraulic conditions of the Khobar aquifers in eastern Saudi Arabia range from 0.02–324 m2/h for transmissivity and 0.001–0.0001 for storativity. In the research area, the Khobar aquifer has a transmissivity of approximately 312 m2/h.The hydrogeological framework of the study area is a multi-layered aquifer system consisting of three principal aquifers separated by two aquitards. The three aquifers are the Neogene, Alat, and Al-Khobar. The Neogene aquifer is under unconfined conditions in the research area and is formed within the clastic unit of the Hadrukh Formation, with a transmissivity of 25.5–144 m of 0.01 ,34. The fsea)), and irrigation hazard analysis was utilized to determine the impact of the degree of SWI on the quality of the tested GW and its suitability for irrigation. The relationship between the SWI indicator (fsea) and the irrigation hazard indices—EC, PS, SAR, Na%, KR, MAR, and PI—was also evaluated using correlation and dimensional reduction statistical techniques  to determine which group of indices would be most suitable for assessing GW quality in SWI-prone regions.Forty-one wells in the eastern coastal region of Saudi Arabia that provide water for irrigation and human consumption were sampled in March 2021. Water samples were collected from observation and production wells. The observation wells tapped the shallow aquifer, which ranged in depth between 5–10 m. Production wells ranged in depth between 70–130 m, producing water from the middle and deep aquifers ,26. Wate3 was used to acidify the samples for major cation and trace-element analyses.We utilized a portable Hanna GPS Multiparameter Meter (HI9829) to perform in situ measurements of pH, temperature, salinity, oxidation-reduction potential (ORP), EC, and dissolved oxygen (DO). Water samples were stored in polyethylene bottles before being filtered in real-time through membranes with a pore size of 0.45 μm. Two separate 250 mL sampling kits were prepared for anion, major cation, and trace element analyses using the filtered samples. Ultrapure HNO+, K+, Ca2+, and Mg2+) and major anions  were analyzed using high-performance ion chromatography (IC) at the King Fahd University of Petroleum and Minerals. Bicarbonate was measured using acid titration in the laboratory, and the level of total dissolved solids (TDS) was determined using gravimetric analysis. For quality control, blank, duplicate, and blind water samples were analyzed to verify the precision and calibration of the equipment. The ionic charge balance for all the water samples was cross-checked as follows:Major cations , using the approach proposed by Appelo and Postma (2005) [fsea). The fraction of the seawater index (fsea), which represents the degree of salinization and SWI into the GW system, was calculated as follows:Cl,sam, CCl,sw, and CCl,f stand for chloride concentrations in the GW samples, seawater, and freshwater, respectively. The values of the fraction of seawater index (fsea) range from 0 to 1, where zero reflects freshwater and one represents seawater.The degree of SWI into the tested GW was quantified through the fraction of the seawater index (a (2005) . The conSeveral water quality indices, such as EC, PS, SAR, Na%, KR, MAR, and PI, have been widely used to analyze the water quality and its degree of suitability for irrigation purposes in various situations ,47,48,49+, Ca2+, and Mg2+ ions. Water quality can be classified into four categories based on the SAR values: excellent (<10), good (10–20), permissible (20–40), and unsuitable (>40) [One of the most crucial indices for evaluating the sodium hazard of irrigated water is the SAR. The SAR indices reflect the relative concentrations of Nale (>40) ,49. The Na% is another index used for the classification of irrigated water and for reporting Na hazards . AccordiThe KR serves as another criterion for evaluating the risk of sodium contamination in irrigation water. It is used to classify water used in irrigation as suitable (<1) or unsuitable (>1) ,46,47. K− and SO42−, which can increase the osmotic potential of a soil solution. Irrigated water was classified as suitable (<3 meq/L), moderate (3–5 meq/L), or unsuitable (>5 meq/L). PS was computed as follows:The PS serves as a criterion for evaluating the effect of salinity in irrigation water on soil salinity and fertility . AccordiEC serves as the main index for evaluating the effect of irrigation water salinity on soil salinity and fertility ,43. The The MAR index is typically used to evaluate the effects of irrigation water on soil quality and crop productivity ,50. ThisThe PI was used to evaluate the carbonate and alkalinity hazards of irrigation water to soil and crops . This re4.The minimum, maximum, and median values of major ions and physicochemical parameters in the GW of the multi-aquifer system in the eastern coastal region of Saudi Arabia are illustrated in K-mean clustering has been utilized by researchers in GW quality studies ,54. It iseaf) [seaf) for the GW samples tested in each cluster are illustrated in seaf for each cluster  were calculated, and are presented in seaf) ranging from 14.4% to 15.4%, with an average value of 14.5%. These samples were obtained from wells that only penetrated a shallow aquifer and were located within 2–3 km of the Arabian Gulf region. Cluster C2 comprises GW samples with values of seaf ranging from 7.2% to 10.1% and an average of 7.9%, indicating an intermediate degree of SWI. These samples were collected from a shallow aquifer and wells located 3–4 km from the Arabian Gulf. Cluster C3 consisted of GW samples with an seaf range of 3.9–6.2% and an average of 5.2%, revealing a low impact of SWI on GW quality. GW samples in cluster C3 were from shallow and middle aquifers within 3–4.7 km and 1–2 km of the Arabian Gulf, respectively. Cluster C4 consisted of GW samples with seaf ranging from 1.2–2.8% and an average value of 1.9%. The GW samples in cluster C4 were obtained from deep wells within 2–5 km of the Arabian Gulf (source of seawater). GW samples in C4 were from a deep aquifer, where SWI had very low or almost no impact on GW quality. Thus, SWI has not yet contaminated the deep layers of the multilayered aquifer system in the study area. Generally, the degree of SWI (seaf) was severe, intermediate, low, and very low in the GW samples in clusters C1, C2, C3, and C4, respectively.The degree of SWI into the GW sampled in the study area was measured using the seawater fraction (fsea) ,41,56. TThe salinity hazard of the GW was quantified using EC and PS. The salinity of irrigated water increases the osmotic pressure of soil solutions, hindering the absorption of water and nutrients by plants ,58. GW sseaf), with a greater influence on GW samples in C1, followed by C2, C3, and C4. Therefore, the SAR index may have more weight in assessing the water quality for irrigation in regions affected by SWI. sea)f. In general, the SAR index is the only Na hazard index that showed a trend of compatibility with the SWI index, indicating the higher reliability of this index for assessing water quality in coastal regions affected by SWI. Increased SAR concentrations with increased landward SWI have also been reported in the Nile Delta, Egypt [Scholars usually evaluate the sodium hazards of irrigated water using three indices: SAR, Na%, and KR ,59,60. Ia, Egypt . As a reseaf)  see . Accordi2+ and Mg2+ as calcite and magnesite, respectively [The PI is used to assess the detrimental effects of GW carbonates on the structure and permeability of the soil . Two exaectively . Althougectively ,  datasets to unravel the associated relationship. As shown in p-value < 0.05) correlations were observed between seaf and the EC, PS, and SAR indices  of 0.72, revealing a moderate impact of SWI on the magnesium concentration and the hazards of the tested GW. A low correlation was observed between the seaf and Na% and KR indices, with a correlation coefficient of 0.44 and 0.45, respectively. This indicates that these indices may not be suitable for representing sodium hazards in regions affected by SWI. No correlation was observed between seaf and PI, revealing that SWI did not contribute to increasing the carbonate hazards of the tested GW. The results of the correlation between irrigation hazards are presented in The impact of SWI on GW quality for irrigation was determined through multivariate statistical analyses, including correlation and PCA. Pearson correlation analysis was performed on the irrigation hazard index  and SWI index (seaf), to define index groups that can effectively assess GW quality for irrigation in coastal regions, and to determine the degree of SWI impact on irrigated GW and its hazardous levels. seaf, which correlated significantly with PC1 with correlation coefficients of 0.93, 0.93, 0.99, 0.70, and 0.94, respectively  in PC1 were grouped with the SWI index, seaf, reflecting the inverse impact of SWI on GW quality for irrigation. Thus, PC1 can be interpreted as a factor that primarily describes the salinity, sodium, and magnesium hazard variations in the GW of the study area. The second group of indices consisted of Na%, KR, and PI, which were mainly correlated with PC2 with correlation coefficients of 0.71, 0.71, and 0.90, respectively. These indices contributed to constructing the PC2, with a total percentage of 77.1%. Therefore, PC2 can be used to assess carbonate hazards in the tested GW. According to the results of the PCA analysis, the first group of irrigation hazard indices  coupled with the SWI index seaf can be used effectively to construct a water quality index for GW quality assessment in the region affected by SWI.PCA was performed in order to evaluate the relationship between the irrigation hazard indices and SWI (ectively . Table 3seaf) to assess the impact of SWI on groundwater (GW) resources in the eastern coastal aquifers of Saudi Arabia. The tested GW was classified into four main clusters, namely C1, C2, C3, and C4, representing different degrees of SWI from high to low. The results of the irrigation hazard indices, including EC, PS, SAR, Na%, KR, and MAR, revealed that GW is unsuitable for irrigation owing to salinity hazards. In contrast, GW displayed suitable categories for sodium, magnesium, and carbonate hazards. However, an increasing trend in salinity, sodium, and magnesium hazards was associated with an increasing degree of SWI, in ascending order, in clusters C4, C3, C2, and C1.In the current study, we utilized integrated clustering, irrigation index analysis, and the seawater intrusion (SWI) index (seaf) to understand the influence of SWI on GW quality. Salinity (EC and PS) and sodium hazards showed a significant increase due to SWI; however, a moderate increase in magnesium hazards (MAR) was observed. In contrast, no carbonate hazards (PI) are associated with SWI.The irrigation hazard indices were statistically correlated with the SWI index  to be applied in water quality assessments in coastal regions.The PCA resulted in two groups of irrigation indices. The main groups were the EC, PS, SAR, and MAR indices. They are associated with the SWI index