A receptor-positive modulator and its regulatory action on the HPA axis stress response has been reported in numerous preclinical and clinical studies. We previously demonstrated that 3α,5α-THP down-regulation of HPA axis activity during stress is sex-, brain region- and stressor-dependent. In this study, we observed a deleterious submersion behavior in response to 3α,5α-THP (15 mg/kg) during forced swim stress (FSS) that led us to investigate how 3α,5α-THP might affect behavioral coping strategies engaged in by the animal. Given the well-established involvement of the opioid system in HPA axis activation and its interaction with GABAergic neurosteroids, we explored the synergic effects of 3α,5α-THP/opiate system activation in this behavior. Serum β-endorphin (β-EP) was elevated by FSS and enhanced by 3α,5α-THP + FSS. Hypothalamic Mu-opiate receptors (MOP) were increased in female rats by 3α,5α-THP + FSS. Pretreatment with the MOP antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2  reversed submersion behavior in males. Moreover, in both males and females, CTAP pretreatment decreased immobility episodes while increasing immobility duration but did not alter swimming duration. This interaction between 3α,5α-THP and the opioid system in the context of FSS might be important in the development of treatment for neuropsychiatric disorders involving HPA axis activation.The neurosteroid 3α,5α-THP is a potent GABA Diagnostic and Statistical Manual of Mental Disorders (DSM–5), PPD is deemed present when a patient has a major depressive episode along with the onset of pregnancy or within four weeks of delivery and five depressive symptoms are present for at least two weeks [A receptors, and its ability to attenuate stress-induced effects has been well reported in the past [Eighty years after the neurosteroid was identified in the adrenal gland , the FDAwo weeks . The FDAwo weeks . 3α,5α-Tthe past ,7. Moreothe past ,9,10,11.We previously demonstrated that 3α,5α-THP regulation of the hypothalamic-pituitary-adrenal (HPA) axis is both sex- and region-dependent . 3α,5α-TPrevious studies have demonstrated that activation of the HPA axis leads to the synthesis and release of endogenous opioid peptides , includiIn the present study, we recorded the behavior of male and female rats over 10 min of FSS after administering 15 mg/kg of 3α,5α-THP, followed by molecular analysis of opioid system components, such as circulating β-endorphins and hypothalamic Mu-opioid receptors (MOP). To establish whether 3α,5α-THP had a sedative effect at this dose, we assessed locomotor activity in a separate group of animals. Finally, we scored the behavior during the FSS, pre-treating the animals with the MOPs antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) at two different doses , 30 min before administering 3α,5α-THP. Our results suggest that 3α,5α-THP + FSS activates the Mu-opioid receptor system to a different extent in male and female rats, but overall, 3α,5α-THP interaction with the opioid system affected the animals’ behavior during the FSS. This could be a clinically relevant discovery in relation to the therapeutic applicability of 3α,5α-THP in neuropsychiatric disorders that involve concurrent opioid system activation. All animal use was approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. Male and female (PN80-100) Sprague–Dawley rats used in all experiments were bred in-house from Envigo stock . A total of 90 male and 94 female rats were used to perform the experiments in this study. To avoid fluctuations in endogenous 3α,5α-THP due to the estrus cycle, and given that the main progesterone peak in the rat estrous cycle occurs in the early evening of proestrus and returns to basal levels by the morning of estrus ,29, all w/v in water) was prepared by dissolving 3α,5α-THP at a concentration of 7.5 mg/mL and administered to the rats via intraperitoneal (IP) injection. Solutions were prepared the day before the experiment and kept under stirring at 4 °C overnight. Animals were randomly divided into two groups, and according to their weight, all rats received an IP injection of 3α,5α-THP (15 mg/kg)  or an equivalent volume of vehicle (VEH). This dose was chosen based on our previous work [3α,5α-THP was prepared as previously described in Boero et al., 2022 . Brieflyous work  and is kous work ,15 but nous work ,17. FiftD-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) was used as a selective antagonist of Mu-opioid receptors (MOPs) in experiment 3. CTAP  was dissolved in 1 mL of sterile MilliQ water, following the instructions from the manufacturer. The animals were randomly divided into two groups, and according to their weight, all rats received an IP injection of CTAP (0.5 or 2 mg/kg) or an equivalent volume of vehicle . The lower dose was chosen based on previous data , while tSprague–Dawley rats were subjected to FSS . The animals swam in a standard clear plastic cylinder , filled with water to a depth ~30 cm, so that the animals could neither touch the bottom with their tails nor escape from the top. Between each test, the cylinders were thoroughly cleaned. The water temperature was kept at 25 ± 1 °C. Animals were randomly assigned to control or FSS groups. Fifteen min after 3α,5α-THP or vehicle administration, rats were gently picked up by the tail from the home cage and rapidly placed into the middle of the cylinder. The test lasted for 10 min, then the animals were dried with absorbent towels and returned to their home cage for about 20 min. All animals were first-time swimmers and none were used for multiple FSS exposures. To prevent any adverse incidents, animals were closely monitored, and any rat that showed distress for more than 2 sec was removed from the cylinder immediately. One animal was not able to complete the 10 min task and was excluded from the data analysis. Rats were euthanized via decapitation 45 min after 3α,5α-THP or vehicle administration (20 min after the end of the FSS exposure). The entire FSS session was videotaped for subsequent behavioral analysis.Rats  were handled for 3 to 5 days prior to the start of the experiment to minimize handling stress. All rats received 2 days of vehicle injections to habituate them to the holding cage, and injection. 3α,5α-THP doses  were delivered in a counterbalanced order with a 48-h wash out period between injections. During wash-out periods, animals were left undisturbed in their home cages. On the day of the experiment, rats were injected with vehicle or 3α,5α-THP IP and, 15 min later, gently placed in a chamber (floor space: 42 cm × 42 cm). Locomotor activity was performed for 10 min with lights on and the white noise of the room ventilation, at room temperature. The chambers were equipped with cameras to record locomotor activity for subsequent behavioral analysis. The 10 min FSS sessions were videotaped and subsequently analyzed. Immobility and swimming behavior were evaluated using ANY-maze video tracking system 7.20 . Submersion, climbing, and exploration behavior were analyzed blindly by a trained observer. Duration (sec) and total number of episodes for submersion, immobility, swimming, climbing, and exploration behavior were calculated. We also compared the rats’ position inside the cylinder, measuring the angle (α) during immobility 5 min after the beginning of the FSS for each rat.(1)Definition of submersion. Submersion was scored when the entire body of the animal, including the head, was upright but underwater, with no possibility of the rat breathing. If the rat showed distress underwater for longer than 2 s, it was removed from the cylinder immediately. Submersion was considered an involuntary passive behavior.(2)Definition of immobility. A rat was considered immobile when floating passively, keeping its head above the surface, making only the necessary movements to maintain balance. For the analysis, immobility behavior was measured starting from the first bout of immobility lasting longer than 1 s. Immobility was considered a passive behavior.(3)Definition of swimming. Swimming was identified as active swimming movements, more than were necessary to maintain the head above the water. Swimming was considered an active behavior.(4)Definition of climbing. Climbing was defined as the animal making upward-directed movements of the forepaws against the walls of the cylinders, trying to escape. Climbing was considered an active behavior.(5)Definition of exploration. Exploration was identified if the animal intentionally swam underwater, exploring the environment looking for a way to escape. Exploration was considered an active behavior.(6)Angle during immobility. The angle (α) during immobility was defined as the position of the animal with respect to the water surface, as previously described by Chen et al., 2015 . One frag for 15 min at 4 °C. Serum was transferred to Eppendorf Flex 2 mL tubes and frozen at −80 °C until the ELISA assay. Twenty min after the end of the FSS exposure, rats from experiments 1 and 3  were euthanized via decapitation, and blood and whole brains were collected. The whole brain was extracted and frozen at −80 °C until the immunoblotting or ELISA assays were performed. The blood samples were collected in glass tubes , allowed to clot on ice, then centrifuged at 1750× g for 30 min at 4 °C. Lysate supernatants were collected and transferred to new tubes, and the total protein concentrations were determined via bicinchoninic acid assay . The proteins (40 μg/lane) were denatured at 95°C for 5 min in LDS sample buffer (2.5 µL per 10 µL sample) (Thermo Fisher Scientific #NP0007) and with sample reducing agent (1 µL per 10 µL sample) (Thermo Fisher Scientific #NP0009) and were separated via NuPAGE™ 10% Bis-Tris Midi Protein Gel  electrophoresis at 125V for 10 min and 165V for the remainder of the process, then transferred to polyvinylidene fluoride membranes  via the iBlot 2 Dry Blotting System (Thermo Fisher Scientific). Membranes were blocked with 5% Blotting-Grade Blocker  in PBS-T (0.5% Tween-20) for 2 h (room temperature) and subsequently incubated overnight at 4 °C with an anti-MOP antibody . The blots were washed 3 times  in PBS-T (0.5%) after incubation with primary antibody and subsequently incubated with horseradish peroxidase-labeled secondary anti-mouse IgG antibody  for 1h at room temperature. After another wash in PBS-T (0.5%), the immunoreactive bands were visualized with Western Lightning Plus  and detected with enhanced chemiluminescence . Bands were analyzed using the software program ImageQuant TL v8.1.0.0. The ratio of each protein was calculated by dividing the densitometric measurement of the protein of interest by the corresponding β-Actin. All immunoblotting results are presented as % change vs vehicle no stress ± SEM. Whole brains were dissected and prepared as previously described for immunoblotting or ELISA . The hypSerum β-endorphin (β-EP)  was measured via enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions. Results are expressed in nanograms/milliliter (ng/mL). The minimum detectable quantity is 15.625 pg/mL. p < 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism 9 . Multiple groups of data were analyzed using a one-way or two-way analysis of variance (ANOVA) and significant interactions were followed up with Tukey’s Honest Significant Differences (HSD) test. A value of p < 0.0001; Sex F  = 1.849, p = 0.1824; Interaction between factor F  = 1.962, p = 0.1698)  = 50.81,  0.1698) a.p = 0.0598; Sex F  = 0.4651, p = 0.4996; Interaction between factors F  = 0.8794, p = 0.3546). However, the total time in immobility was higher in the 3α,5α-THP-treated groups  = 31.09, p < 0.0001; Sex F  = 0.02754, p = 0.8692; Interaction between factors F  = 2.287, p = 0.1395)  = 3.778,  0.1395) b. During the behavioral analysis, we observed remarkable differences in the animals’ floating body posture. We quantified these differences by calculating the angle between the surface of the water and the animals’ tail–nose axis during the first immobile episode 5 min after the beginning of the FSS . p < 0.0001; Sex F  = 1.160, p = 0.2887; Interaction between factors F  = 0.06033, p = 0.8074). These differences in body posture were positively correlated with submersion behavior, indicating the increase in α predicted the increase the number of episodes in male  = 13.34, p = 0.0065; α degrees/total time: R2 = 0.4566, F = 6.72, p = 0.0320) and female rats  = 24.02, p = 0.0012; α degrees/total time: R2 = 0.499, F = 7.969; p = 0.0224)  = 21.78,  0.0224) .This result suggests that the change in the position of the body in the water induced by 3α,5α-THP contributes to the increase in submersion behavior. p < 0.0001; Sex F  = 0.04369, p = 0.8356; Interaction between factors F  = 2.535, p = 0.1201)  = 32.38,  0.1201) a.p = 0.1046; Sex F  = 0.1432, p = 0.7074; Interaction between factors F  = 0.09163, p = 0.7639) or the total time for climbing  = 0.9446, p = 0.3376; Sex F  = 0.2973, p = 0.5889; Interaction between factors F  = 1.240, p = 0.2729)  = 2.772,  0.2729) b.p = 0.0004; Sex F  = 0.1701, p = 0.6825; Interaction between factors F  = 1.531, p = 0.2240) and duration  = 11.03, p = 0.0021; Sex F  = 1.708, p = 0.1995; Interaction between factors F  = 3.636, p = 0.0645)  = 15.03,  0.0645) c.p = 0.1324; Sex F  = 3.919, p = 0.0543; Interaction between factors F  = 0.08655, p = 0.9173). This result suggests that 3α,5α-THP alone did not affect locomotor activity in male and female rats  serum levels after 3α,5α-THP administration in male and female rats exposed to forced swim stress.p = 0.0300; Stress F  = 88.78, p < 0.0001; Interaction between factors F  = 10.99, p = 0.0026). Similar effects were observed in the female group, where FSS induced an increase in β-EP serum levels and the combination of stress with 3α,5α-THP administration resulted in a greater effect on β-EP serum concentrations in female rats  = 12.72, p = 0.0013; Stress F  = 105.3, p < 0.0001; Interaction between factors F  = 11.56, p = 0.0020). In both male and female rats, the exposure to FSS significantly raised β-EP serum levels. In the male group, the animals treated with 3α,5α-THP and exposed to swim stress 15 min later showed a greater increase in β-EP serum levels  = 5.246, p < 0.01). Moreover, the stress effect was greater in male than female rats, probably due to the higher baseline   a.p = 0.8103; Stress F  = 1.778, p = 0.1936; Interaction between factors F  = 0.7503, p = 0.3940. Females Treatment F  = 8.886, p = 0.0067; Stress F  = 0.2511, p = 0.6211; Interaction between factors F  = 6.899 × 10−5, p = 0.9934). These results suggest that the increase in β-EP levels is dependent on the type of stress (forced swim stress) and the interaction between this type of stress and 3α,5α-THP treatment  = 5.498, p = 0.0248; Interaction between factors F  = 1.920, p = 0.1746). In contrast, in female rats, 3α,5α-THP or FSS per se did not affect hypothalamic MOP levels, however the combination of the 3α,5α-THP treatment and the exposure to the forced swim stress induced an increase in the MOP receptor  = 6.774, p = 0.0133; Stress F  = 1.646, p = 0.2077; Interaction between factors F  = 8.992, p = 0.0049)  = 15.29,  0.0049) a.p = 0.2329; Stress F  = 0.1261, p = 0.7261; Interaction between factors F  = 40.62, p < 0.0001. Females Treatment F  = 0.6257, p = 0.4352; Stress F  = 1.024, p = 0.3197; Interaction between factors F  = 53.49, p < 0.0001)  = 1.509,  0.0001) b.To assess the hypothesis that 3α,5α-THP interaction with the opioid system induced the submersion behavior, we blocked the Mu-opioid receptors using the selective MOP antagonist CTAP at two different doses (0.5 and 2 mg/kg) prior to 3α,5α-THP treatment.p = 0.0001)  completely reversed the effect of 3α,5α-THP on submersion behavior, reducing the number of episodes  = 7.780,  0.0001) a. This d 0.0001) a.p < 0.0001). Nevertheless, the fact that the total number of episodes was clearly reduced and not different from the effects of vehicle alone suggests that CTAP regulates the 3α,5α-THP-induced effect on submersion behavior in female animals as well  in conjunction with prior 3α,5α-THP treatment. However, although the statistical analysis resulted in an overall significant effect, no significant changes were detected with the multiple comparison test of both doses of CTAP on submersion behavior  = 8.111,  as well b.p = 0.0011). The lower CTAP dose (0.5 mg/kg) did not change the 3α,5α-THP-induced increase in the total time for immobility in male rats. However, the higher CTAP dose (2 mg/kg) followed by 3α,5α-THP treatment induced a greater increase than 3α,5α-THP per se in this parameter  = 21.57, p < 0.00010 . However, the same dose of CTAP increased the duration of immobility in female rats  = 9.650, p < 0.0001)  reversed the 3α,5α-THP-induced increase in the number of immobility episodes  = 6.296,  0.0001) b.p = 0.0035)  = 4.727,  0.0035) c. p = 0.0065)  = 4.180,  0.0065) .p < 0.0001)  did not change the 3α,5α-THP-induced decrease in the duration spent swimming. Moreover, the higher CTAP (2 mg/kg) dose prior to 3α,5α-THP administration enhanced this effect, further reducing the swimming total time  = 14.70,  0.0001) a.p = 0.00130)  = 5.505, 0.00130) b.p = 0.0359) and total time  = 4.263, p = 0.0062)  = 2.886,  0.0062) a. p = 0.0003; duration: Treatment F  = 9.794, p < 0.0001)  = 6.775,  0.0001) b. p = 0.1881; total time: Treatment F  = 1.946, p = 0.1233)  = 1.627,  0.1233) a. p = 0.0058) and the time spent exploring at both CTAP doses  = 6.295, p = 0.0006)  = 4.317,  0.0006) b.We previously demonstrated that 3α,5α-THP regulation of the HPA axis stress response is sex- and stressor-dependent . In thisIt has previously been demonstrated that 3α,5α-THP administration at different doses  30 min prior to FSS exposure decreased the duration of immobility . This re3α,5α-THP decreased time spent swimming in both male and female rats while climbing behavior was not affected by 3α,5α-THP administration. Finally, exploring behavior was not significantly affected by 3α,5α-THP in male rats; however, in female rats, 3α,5α-THP decreased both the number of episodes and the time spent exploring. Together, the data show that 3α,5α-THP impacts passive coping behaviors during FSS (submersion and immobility) in both male and female animals. In contrast, 3α,5α-THP did not influence active behaviors in male rats. Although 3α,5α-THP reduced swimming behavior in males and females, 3α,5α-THP did not change the other active behavior parameters (climbing and exploring) in male rats. In female rats, 3α,5α-THP did not affect climbing but reduced active coping behavior to explore the environment to find a way to escape. Since it has been demonstrated that the 3α,5α-THP dose of 15 mg/kg had no hypnotic effect  in rats,It is important to note that FSS is routinely used to screen drugs with antidepressant activity and that only in recent years has it been proposed to test measures of stress coping strategies rather than depression-like phenotypes . It has Our data showed that swim stress increased circulating β-EP levels in both male and female rats. Interestingly, the combination of 3α,5α-THP and FSS enhanced this effect, suggesting that the participation of the opioid system is responsible for the behavioral outcome we observed. The increase in β-EP might account for the increase in submersion behavior observed in this study. Moreover, β-EP levels did not change after restraint stress (RS) exposure, suggesting that the activation of the HPA axis stress response is stressor-dependent, as we previously showed . One impAdministration of the selective MOP antagonist CTAP at two different doses prior to 3α,5α-THP administration changed the behavioral outcome during FSS. In male rats, the high dose of CTAP completely reversed the 3α,5α-THP effect on submersion behavior, suggesting, once again, that the contribution of the Mu-opioid receptor system resulted in this specific behavioral effect. In female rats, CTAP led to the same trend observed in male rats, but it was not statistically significant. Overall, pharmacological manipulation of MOPS with CTAP seemed to produce an effect on specific behaviors . Interactions between neurosteroids and the opioid system have previously been reported. In fact, previous studies in male mice showed that levels of progesterone directly increase sigma1 receptor effects ,38. OtheWe observed no sex differences in β-EP levels after 3α,5α-THP administration and swim stress exposure; however, MOP expression showed a sex dependent regulation. In fact, in male rats, both 3α,5α-THP treatment per se or swim stress alone induced a decrease in hypothalamic MOP expression, but the combination of 3α,5α-THP + FSS did not produce any effect. In contrast, in female animals, we did not observe any change due to 3α,5α-THP treatment or swim stress; however, the combination of 3α,5α-THP + FSS resulted in an increase in hypothalamic MOP levels. It is possible that the timing and/or the brain region studied here were not related to the behavioral effects we observed. For example, it might be interesting to analyze MOP expression in brain regions involved in decision making processes, such as the prefrontal cortex and projecting targets. In fact, it has been demonstrated that a possible mechanism responsible for the cognitive function impairment due to opioid drug use is the suppression of cortical interneuron spiking by mu-opioid receptors; this effect results in an increase in glutamate that led to a disorganization in control and decision processes . FurtherThis is the first study that has shown a possible deleterious behavior due to the interaction between 3α,5α-THP and the opioid system in the context of FSS. Further studies are needed to understand this phenomenon; this study is an indirect observation, and further direct investigation of 3α,5α-THP and opiate interactions are required. It would be interesting, for example, to explore the effect of the combination of 3α,5α-THP and opioid agonists (such as fentanyl) at sub-threshold doses for multiple behavioral effects. These findings might be important to better understand the limitations of 3α,5α-THP in the treatment of neuropsychiatric disease. Several neuropsychiatric disorders, such as major depression, post-traumatic stress disorder, and substance use disorders share a dysregulation of the HPA axis as well as an imbalance between the glutamatergic excitatory inputs and GABAergic inhibitory transmission across brain . The ratThis study suggests that 3α,5α-THP has a deleterious effect combined with FSS, which together induced an exacerbation of the activation of the opioid system, resulting in submersion behavior. This effect was prevented with the Mu-opioid receptor antagonist CTAP in rats. These results might be an important contribution to the development of new treatments for patients with neuropsychiatric disorders that involve HPA axis activation. The data, in fact, suggest that 3α,5α-THP/opiate system interactions may lead to negative behavioral outcomes. Future studies are needed to better support this hypothesis. Forthcoming perspectives to confirm our theory should be activating the opioid system using an opioid agonist concurrently with 3α,5α-THP administration, to evaluate the level of anesthesia and the interaction between the two drugs.


TM, Cupertino, CA, USA), the participants were randomly evaluated on a treadmill at three different walking speed intensities for 10 min at each one, SS − 20%/SS + 20%/ SS, with 5 min of passive recovery in-between. Heart rate (HR) was monitored and normalized as %HRmax, while the rate of perceived exertion (RPE)  was asked after each trial. Kinematic analysis was performed, assessing the Contact Time (CT), Swing Time (ST), Stride Length (SL), Stride Cycle (SC), and Gait Variability as Phase Coordination Index (PCI). RPE and HR increased as the walking speed increased . CT and SC decreased as the speed increased , while ST remained unchanged (p = 0.277). SL increased with higher walking speed (p = 0.0001). Conversely, PCI was 3.81 ± 0.88% (high variability) at 3.96 ± 0.47 km·h−1, 2.64 ± 0.75% (low variability) at SS (4.94 ± 0.58 km·h−1), and 3.36 ± 1.09% (high variability) at 5.94 ± 0.70 km·h−1 (p = 0.001). These results indicate that while the metabolic demand and kinematics variables change linearly with increasing speed, the most effective GV was observed at SS. Therefore, SS could be a new methodological approach to choose the individual walking speed, normalize the speed intensity, and avoid a gait pattern alteration.Gait variability (GV) is a crucial measure of inconsistency of muscular activities or body segmental movements during repeated tasks. Hence, GV might serve as a relevant and sensitive measure to quantify adjustments of walking control. However, it has not been clarified whether GV is associated with walking speed, a clarification needed to exploit effective better bilateral coordination level. For this aim, fourteen male students  took part in this study. After three days of walking 1 km each day at a self-selected speed (SS) on asphalt with an Apple Watch S. 7 (Apple The analysis of human locomotion during activities such as pedaling ,2, walki−1 or km·h−1) and physiological response (heart rate) were not reported and studied. Furthermore, in that study, the preferred walking speed was assessed on the treadmill only. The critical aspect concerning walking speed lies in the fact that each participant exhibits a distinct preferred walking speed. Therefore, the preferred walking speed should be assessed on overground to be more realistic . Lastly, to compensate for the association between ϕ_ABS and ϕ_CV, the phase coordination index (PCI) was obtained as PCI = ϕ_CV + Pϕ_ABS, where Pϕ_ABS = 100 × (ϕ_ABS/180). Further details about the association between ϕ_ABS and ϕ_CV can be found in Plotnik and coll. [To evaluate the level of consistency in phase generation across all strides for each participant, the coefficient of variation of the mean of nd coll. . NotablyMAX, RPE, CT, ST, SC, SL, PCI, ϕ_CV, ϕ_ABS, and ϕ over the 3 different walking conditions (SS − 20/SS/SS + 20%), a one-way, repeated-measures analysis of variance (RM-ANOVA) was used. When a significant F-value was found, post-hoc analysis (LSD) between conditions was performed. The ANOVA effect size was also calculated .Results are expressed as mean ± standard deviation (SD). The Shapiro–Wilk test was used to verify the normality of the distribution. The reliability of the SS and PCI was assessed by an Intra-Class Correlation Coefficient (ICC) and classified as poor, if <0.05; moderate, if between 0.50 and 0.75; good, if between 0.75 and 0.9; and excellent, if >0.9, according to Koo and Li . To asse (>0.14) . The sig−1. The speed during SS − 20 was 3.96 ± 0.47 (min/max: 3.20–5.00) km·h−1 and the speed during SS + 20 was 5.80 ± 0.73 (min/max: 4.80–7.44) km·h−1. RM-ANOVA showed differences among the three walking speeds for RPE  as well as HR .The SS was 4.94 ± 0.58 (min/max: 4.00–6.20) km·h1,12 = 8.232 and p = 0.013/F1,12 = 4.974, and p = 0.044, respectively), while ST was unchanged . SL increased as the speed increased . Conversely, the bilateral coordination . The ICC for SS was 0.998 (CI 95%: 0.994–0.999).CT and SC decreased as the speed increased . These metrics quantify the ability of young males to coordinate left–right stepping on flat terrain at different speeds and evaluate the precision and coherence of the gait pattern [A similar U-shaped trend in gait variability, analyzed using long-range correlation through detrended fluctuation analysis, has been observed in both running and walking gaits for female participants , althoug pattern .These metrics was able to quantify the ability of young males to coordinate left–right stepping on flat terrain at different speeds. Simultaneously, the PCI assesses both the precision  of anti-Our study demonstrated that walking at a specific self-selected pace requires the participant to continuously adjust the force produced and its timing relative to the foot position . Reasona


The samples were analyzed using X-ray diffraction (XRD), atomic force microscope (AFM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and energy dispersive X-ray (EDX) techniques. The impact of TiO2 on the electrical impedance, conductivity, permittivity, and energy efficiency of the PVA/Cs was determined in a frequency range of 100 Hz to 5 GHz. The XRD, FTIR, and EDX results showed the successful fabrications of the PVA/Cs/TiO2. The SEM and AFM images illustrated that the TiO2 was loaded and distributed homogenously in PVA/Cs chains. In addition, the electrical conductivity was enhanced from 0.04 × 10−7 S.cm−1 of PVA/Cs to 0.25 × 10−7 S.cm−1 and 5.75 × 10−7 S.cm−1, respectively, for the composite PVA/Cs/0.01TiO2 and PVA/Cs/0.1TiO2, and the dielectric constant grew from 2.46 for PVA/Cs to 7.38 and 11.93, respectively. These results revealed that modifications were made to the produced films, paving the way for using the composite PVA/Cs/TiO2 films in different energy applications, such as electronic circuits and supercapacitors.This research paper aims to fabricate flexible PVA/Cs/TiO Nanocomposites are getting a lot of focus in storage energy due to their exceptional mechanical and electrical characteristics . Polymer2 composite is a promising material with a wide variety of applications due to its properties of biodegradable, mechanical, optical characteristics, and thermal stability. PVA/chitosan are frequently discussed in the literature. For instance, Wu et al. [PVA/Cs/TiOu et al.  created u et al. . Fan et u et al.  showed tu et al.  determinBecause of its improved process ability and reversible conductivity control, polyvinyl alcohol (PVA) is one of the polymers with promising applications . PVA is Chitosan (Cs) is a natural polymer that is widely used in the biotechnology industry. Its benefits in life science, including its readily available, biocompatible, and biodegradable nature, drive its increased use . AdvantaIncorporating nanofillers in the polymer has paved the way for tailoring nanocomposites’ features. Furthermore, the nanofillers conduct linkages inside the polymer chains, improving the polymer matrix’s dielectric properties. The host properties can be modified thanks to the nanofillers embedded in the polymeric chains . Moreove2) as a conductive filler has shown considerable promise in the devices of electronic chips, supercapacitors, and solar cells. There has been growing concern in recent years about using TiO2 in several applications, particularly in detectors and lighter emitters [2 is the most promising material for usage in photovoltaic cells, sensors, and electronic devices [2 dispersion colloids in different solvents is to include TiO2 in PVA/Cs [2 is widely used in photo-electrochemical and cell air-water purification because it is a powerful redox material [Titanium dioxide  was made by dissolving PVA in deionized water at 88 °C for 1.5 h. The Cs solution (6% w/v) was dissolved in deionized water at 88 °C with agitation for 1.5 h, as discussed before [v/v acetic acid solution was stirred into a 1.1% w/v Cs solution. The polymeric combination was obtained by combining the PVA and Cs solutions and stirring the mixture for 5 h. The produced polymer solution was then combined with TiO2NPs of varying concentrations to produce a PVA/Cs/TiO2 solution, which was then cross-linked using glutaraldehyde, as described before [The chemicals, PVA with a molecular weight of 86,500–90,500 g/mol and hydrolysis of 99.4%, TiOd before . Overnigd before . At 50 °The structural properties were analyzed using XRD , while the chemical changes were investigated using FTIR . SEM  and AFM  investigated the morphology and geometric shape of the samples, as well as the surface roughness. In addition, the dielectric characteristics of all films were recorded using an LCR meter  operating in frequencies of 100 Hz to 5 GHz.2 composite are shown in XRD patterns as in 2 showed a new diffraction peak of TiO2 at 25.6° correlated to (111). In addition, a shift in the peak of PVA/Cs with intensity demonstrated crystallinity due to the interaction of TiO2 in the PVA/Cs [2 nanoparticles caused a shift in the peaks because it altered the electron–hole recombination rate caused by the presence of the defects introduced into the crystal structure. The average crystallite size (D) of TiO2 was calculated using the Debye-Scherrer formula by [Films of PVA/Cs and PVA/Cs/0.1TiOe PVA/Cs . The insrmula by (1)D=0.92 are given in −1 of PVA/Cs were to −OH groups and at 2922 cm−1 for C–H stretching vibrations. The peak at 1635 cm−1 was for vibrational C=C. Another peak at ~1415 cm−1 and 1075 cm−1 referred to vibrations of −OH and C=O, respectively. The band 830 cm−1 referred to CH2 stretches. Due to the interaction between TiO2 and PVA/Cs levels, these peaks were seen in all spectra with a slight shift [2 composite films had a different intensity than pure PVA/Cs [The chemical structure and functional groups of the produced composites are studied with FTIR. The FTIR of pristine PVA/Cs and the composite PVA/Cs//TiOht shift . Moreovee PVA/Cs . When na2 in PVA/Cs as analyzed using EDX mapping. The three constituent elements, i.e., O, C, and Ti, were all detectable and homogenous distributions. As shown in 2 nanoparticles on the PVA/Cs. Additionally, the EDX mapping showed the dispersion of Ti elements on the structure as a sign of attachment of TiO2 to the structure [tructure ,35. 2 in 3D dimensions. The AFM images of PVA/Cs were smoother, as shown in 2, as shown in 2 increased the surface roughness from 13 nm for PVA/Cs to 32 nm for PVA/Cs/0.1TiO2. The addition of TiO2 in PVA/Cs caused an increase in surface roughness, demonstrating the mixing of TiO2 in the PVA/Cs blend. Hydrophilic in PVA/Cs blend are efficiently covered by TiO2, making PVA/Cs/TiO2 composite rougher to be more convenient in different applications. 2 and PVA/Cs/0.05TiO2 compared to PVA/Cs/0.1TiO2 ε*=ε′2 are shown in The vided by :(3)ε′=c.d fields .2. The dielectric constant of pure PVA/Cs at 100 Hz was 2.46 and enhanced to 7.38, 9.07, 9.76, and 11.93, respectively, for PVA/Cs/0.01TiO2, PVA/Cs/0.025TiO2, PVA/Cs/0.05TiO2, and PVA/Cs/0.1TiO2, as shown in 2 in PVA/Cs films contributed significantly to the enhancement of the dielectric constant of the composite PVA/Cs/TiO2 films. The interfacial polarizability was also enhanced due to the rise in dipole density caused by adding TiO2. As a result, the polarization and dielectric of the PVA/Cs polymer matrix were improved by the presence of these flaws. Atta [The dielectric ws. Atta  found thThe dielectric loss (given by .(4)ε″=ε2 is presented in 2, and 10.58 for PVA/Cs/0.1TiO2. The enhancement of 2 was due to an increase in coupling strength across the grain boundary [2.The requency . Howeverrequency . In addiboundary , which rThe electrical modulus (M*) is determined by t .(5)M*=1The real M′ and imaginary M″ modules are given by :(6)M′=ε′2. In the low-frequency range, as depicted in the image, M′ was very close to zero. At very high frequencies, M′ increased at an exponential rate with frequency. Polymers’ electrical dipoles were strongly attracted to an external electric field in the low-frequency area but much less in the high frequencies zone. Furthermore, the relaxation conductivity of electrode polarization affected the electric modulus [2 was present, the actual electric modulus M′ decreased. The modulus M′ of pure PVA/Cs was 0.174 and reduced to 0.089 and 0.039, respectively, for PVA/Cs/0.01%TiO2 and PVA/Cs/0.1%TiO2. The electric modulus permittivity decreased as TiO2 concentration rose because of the dipolar contribution of charge carriers. modulus . As show2 is shown in 2. The contribution of dipolar groups to the permittivity decreased as the frequency rose because more dipolar groups became un-orientable at higher frequencies. By increasing TiO2, the M″ decreased from 0.101 for the PVA/Cs to 0.073 for PVA/Cs/0.01TiO2 and to 0.007 for PVA/Cs/0.1TiO2. The addition of TiO2 shifted the PVA/Cs relaxation peak, which reduced the relaxation τr=1ωd et al.  investigm easier .2, PVA/Cs/0.025TiO2, PVA/Cs/0.05TiO2, and PVA/Cs/0.1TiO2 are shown in 2 reduced the Z′ value because TiO2 increased conductivity and decreased the film impedance directly. The Z′ was decreased from 2.65 × 107 for the pristine PVA/Cs to 2.07 × 107 and 1.06 × 107, respectively, to PVA/Cs/0.01TiO2 and PVA/Cs/0.1TiO2. This finding was because TiO2 processed the dipole mobility contribution of charge carriers.The complex impedance relation :(9)Z*=Z′2. The Z″ dropped with frequency, as is evident from identical actions of 7, which reduced, respectively, to 6.5 × 107 and 2.3 × 107 for the composite PVA/Cs/0.01TiO2 and PVA/Cs/0.1TiO2. This reduction was because TiO2 incorporation induced changes in polarizability and due to the rise in dipole density caused by adding TiO2.achieved . The valU) is computed using this relation [The energy density (relation :(10)U=12E is the electric field, and U at various frequencies for pristine PVA/Cs and the composite PVA/Cs/TiO2 are shown in 2 increased the energy density from 1.05 × 10−5 J/m3 for PVA/Cs to 3.16 × 10−5 J/m3, and 3.89 × 10−5 J/m3 and 5.12 × 10−5 J/m3 for PVA/Cs/0.01TiO2, PVA/Cs/0.025TiO2 and PVA/Cs/0.1TiO2. The results demonstrated that TiO2 was successfully incorporated into the PVA/Cs chains, which enhanced its dielectric responses and capabilities for energy storage.The electrical conductivity given by .(11)σac2 with frequency. Because the applied electric field increased the mobility of charge carriers, the 2 incorporation [−7 S/cm, increased to 0.25 × 10−7 S/cm, 0.96 × 10−7 S/cm, and reached 5.75 × 10−7 S/cm, respectively, by increasing the content of TiO2 to 0.01, 0.025, and 0.1 in PVA/Cs, as shown in 2 [−4 S/cm for PVA, which improved for PVA/MWCNT to 26 × 10−5 S/cm. This result was due to MWCNT-generated charge carriers, which improved conductivity.ll films . In addiporation . At 100 own in 2 . The σaca et al.  at 1 MHzThe maximum height  formula :(12)Wm=−The m value is given by the slopes of the line ln 2, PVA/Cs/0.025TiO2, PVA/Cs/0.05TiO2, and PVA/Cs/0.1TiO2 films. With TiO2 concentration, the predicted potential barrier 2, PVA/Cs/0.025TiO2, PVA/Cs/0.05TiO2, and PVA/Cs/0.1TiO2 films. Moreover, the effects of incorporating on crystal structure and defect density of TiO2 were responsible for this decrease in potential barrier 2 in PVA/Cs improved its electrical conductivity by lowering the energy needed for charge carrier hopping.The value of m changed from 0.098 for PVA/Cs to 0.132, 0.156, 0.112, and 0.133, respectively, for PVA/Cs/0.01TiO2 composite films successfully. The films were then analyzed using various methods, such as XRD, FTIR, EDX, SEM, and AFM. The XRD, FTIR, and EDX methods verified that the PVA/Cs/TiO2 polymeric composite films were fabricated successfully. Furthermore, the AFM results demonstrated that TiO2 increased the surface roughness from 13 nm for PVA/Cs to 32 nm for PVA/Cs/0.1TiO2. The SEM analysis illustrated that TiO2 was successfully integrated into PVA/Cs. The incorporation of TiO2 was shown to improve electrical characteristics. Moreover, the electric modulus and complex impedance were found to be sensitive to changes in TiO2 content. When the percentage of TiO2 was raised from 0.025 to 0.1, the dielectric dispersion properties were enhanced as a result of the migration of charge carriers. The addition of TiO2 increased the energy density from 1.05 × 10−5 J/m3 for PVA/Cs to 3.16 × 10−5 J/m3, 3.89 × 10−5 J/m3, and 5.12 × 10−5 J/m3 for PVA/Cs/0.01TiO2, PVA/Cs/0.025TiO2, and PVA/Cs/0.1TiO2. The results demonstrated that TiO2 was successfully incorporated into the PVA/Cs chains, which enhanced its dielectric responses and capabilities for energy storage. The results of this research open up opportunities for the flexible PVA/Cs/TiO2 nanocomposite films to be used in a wide range of potential devices, such as batteries and supercapacitors.The solution casting method is used to create flexible PVA/Cs/TiO


The in vivo antidiabetic efficacy of the synthesized compounds was assessed on streptozotocin-induced diabetic mice using standard protocols, and their effect on weight gain was also studied. Molecular docking and molecular dynamics (MD) simulation studies were performed to investigate the binding interactions of the title compounds with the PPAR-γ receptor and to establish their binding mechanism. Antidiabetic activity results revealed that compounds 5d and 5e possess promising antidiabetic activity comparable with the standard drug rosiglitazone. No compound showed considerable effect on the body weight of animals after 21 days of administration, and the findings showed statistical difference (p < 0.05 to p < 0.0001) among the diabetic control and standard drug rosiglitazone groups. In molecular docking study, compounds 5c and 5d exhibited higher binding energies  than the native ligand, non-thiazolidinedione PPAR-γ partial agonist (nTZDpa) . MD simulation further authenticated the stability of compound 5c-PPAR-γ complex over the 150 ns duration. The RMSD, RMSF, rGyr, SASA, and binding interactions of compound 5c-PPAR-γ complex were comparable to those of native ligand nTZDpa-PPAR-γ complex, suggesting that the title compounds have the potential to be developed as partial PPAR-γ agonists.Peroxisome proliferator-activated receptor-γ (PPAR-γ) partial agonists or antagonists, also termed as selective PPAR-γ modulators, are more beneficial than full agonists because they can avoid the adverse effects associated with PPAR-γ full agonists, such as weight gain and congestive heart disorders, while retaining the antidiabetic efficiency. In this study, we designed and synthesized new benzylidene-thiazolidine-2,4-diones while keeping the acidic thiazolidinedione (TZD) ring at the center, which is in contrast with the typical pharmacophore of PPAR-γ agonists. Five compounds ( Although several antidiabetic drugs of different classes are available in the market, most of them are associated with minor to serious side effects3. One of the drug classes used for type 2 DM is thiazolidinediones (TZDs) or glitazones, which act as insulin sensitizers. They regulate the transcription of genes responsible for glucose and lipid metabolism via binding to the peroxisome proliferator-activated receptor-γ (PPAR-γ)4. Through PPAR-γ stimulation, glitazones also regulate cell development, angiogenesis, and inflammation, which broadens their restorative potential4.One of the major growing threats for the human population worldwide is diabetes mellitus (DM), and a considerable percentage of human population is currently suffering from type 1 or 2 DM6. TZDs have also been reported to have other serious adverse effects, such as cardiotoxicity, congestive heart failure, and even conceivable carcinogenesis in a few cases8. Hence, the pleiotropic effects of TZDs must be clarified to ensure medication safety and facilitate novel drug design and development. Selective PPAR-γ modulators (SPPARMs), also known as the partial agonists/antagonists of PPAR-γ subtype, disengage insulin sensitization from triglyceride (TG) accumulation in people with type 2 DM9. SPPARMs also help avoid the unfavorable effects of using full agonists, such as body weight gain and cardiotoxicity.The positive correlation between adipogenesis and PPAR-γ action signifies the undesirable weight gain linked to the use of TZDs in people with DM and obesity11. In this study, we designed new benzylidene-2,4-thiazolidinediones by keeping the acidic polar head in between the lipophilic ring and the linker instead of placing it at the end, which is typical for full agonists. This new strategy was expected to have effect on the binding to the receptor, which was studied using molecular docking by investigating the plausible interactions between the molecule and protein. The antidiabetic potential of the newly synthesized derivatives was assessed using an in vivo antidiabetic rodent model.Several new derivatives with or without the TZD ring system have been designed and tested for their partial agonistic activities on PPAR-γ, resulting in the identification of various pharmacophores that could bind partially at the binding site of the protein1H NMR and 100 MHz frequency for 13C NMR analysis using dimethyl sulfoxide (DMSO) as the solvent and tetramethylsilane as the internal standard. The mass spectra (MS) were recorded using a triple quadrupole mass spectrometer . The completion of reactions was monitored using thin-layer chromatography (TLC) on precoated (F-254) aluminum-supported TLC plates . Benzene: methanol (9:1) was used as the solvent system, and the retardation factor (Rf) was calculated.All the chemicals, reagents, and solvents used in this study were purchased from Sigma Aldrich  and used without further purification. The melting points of all synthesized compounds were measured by the open capillary method using a melting point apparatus . The infrared (IR) spectra of the compounds were recorded using a Fourier transform-infrared spectrometer (FT-IR) coupled with attenuated total reflectance (FTIR–ATR) . The nuclear magnetic resonance (NMR) spectra of the synthesized compounds were obtained using NMR spectrometer  operating at 500 MHz frequency for In vivo studies were performed on male Swiss albino mice obtained from the central animal house facility, Jazan University, Saudi Arabia and were kept in the animal house at the College of Pharmacy until experiments were performed. All the animals were acclimatized for 1 week under standard conditions with a temperature of 22 ± 1 °C and humidity of 50% ± 5% with a 12 h light/dark cycle and had free access to commercial food pellets and fresh tap water. The experimental protocol was approved by the Standing Committee for Scientific Research Ethics-Jazan University (HAPO-10-Z-001) prior to conducting the experiments with an approval number REC42/1/027. All the procedures were in accordance with the guidelines set by the Organization for Economic Cooperation and Development and International Conference on Harmonization. The results of the experiments were reported in accordance with the ARRIVE 2.0 guidelines.All the titled compounds were synthesized using the scheme shown in Fig. Thiazolidine-2,4-dione (0.25 mol) and an appropriate proportion of aryl aldehydes (0.25 mol) were mixed in 50 mL of hot glacial acid, followed by the addition of fused sodium acetate (1.8 g) to the resulting solution. The reaction mixture was then refluxed for 5 h. The progress of the reaction was continuously monitored using TLC. Upon the completion of the reaction, the reaction mixture was allowed to cool at room temperature and then added to ice-cold water. Thus, a solid product was obtained, filtered, dried, and purified by recrystallization from glacial acetic acid.2a–e, 0.02 mol), ethyl chloroacetate (0.04 mol), and potassium carbonate (0.04 mol) were mixed in acetone (250 mL) and refluxed for 16 h. The inorganic base was filtered off, and the filtrate was evaporated to dryness under reduced pressure using a rotavapor. The obtained solid residue was recrystallized using methanol to afford the corresponding acetates (3a–e).Substituted-benzylidene thiazolidine-2,4-diones  were added to hydrazine hydrate (10 mL), then mixed with an appropriate quantity of absolute ethanol (30 mL), and stirred to achieve a clear solution. The resulting solution was refluxed for 7 h. After the reaction was completed, ethanol was concentrated under vacuum to obtain the crude compound, which was then stirred into n-hexane, filtered at reduced pressure, and dried. Finally, the obtained desired compound was purified by recrystallization under ethanol.Substituted-benzylidene thiazolidine-2,4-dione ethyl esters (4a–e) and acetophenone were dissolved in absolute ethanol (50 mL), followed by the addition of glacial acetic acid (3 mL). The resulting mixture was then refluxed for 10 h, and the progress of the reaction was monitored by TLC using ethyl acetate and petroleum ether (2:1) as solvents. After the reaction was completed, the reaction mixture was cooled to room temperature, concentrated under reduced pressure, and recrystallized using ethanol to afford the corresponding hydrazides as titled compounds (5a–e).Equimolar quantities (6 mmol) of substituted-benzylidene thiazolidine -2,4-dione hydrazides (−1): 3244(–NH), 3058(Ar–CH), 3005 (CH), 2925(CH2), 1705, 1659, 1602 (C=O), 1556 (C=C), 1492 (C=N); 1H NMR : 1.72 , 4.13 , 7.10 , 7.46 , 7.96–7.05 ; 13C NMR : 15.9 (CH3), 40.36 (CH2), 126.94 , 127.1, 127.2, 128.5, 128.7, 128.9, 129.0, 130.2, 130.9, 131.0, 131.3, 135.9, 137.2 , 137.3 (methylidene C=C), 153.7, 156.2, 159.1 (C=O), 160.1 (C=N); Mass m/z (M + H)+: 380.15; Analysis calculated for C20H17N3O3S: C (63.31); H (4.52); N (11.07); O (12.65); S (8.45); Rf value = 0.75; Log P = 4.31.Color: light brown; Yield: 38%; Melting point (MP): 120–122 °C. FT-IR : 3334(–NH), 3193(Ar–CH), 3063(CH), 2924(CH2), 1774, 1682, 1604 (C=O), 1487 (C=C), 1443 (C=N), 759 (C–Cl); 1HNMR : 2.08 , 3.86 , 7.91–7.14 , 7.20 , 7.86 ; 13CNMR : 15.6 (CH3), 40.19 (CH2), 126.93 , 127.1, 127.2, 128.7, 128.8, 120.0, 129.1, 130.2, 130.9, 131.0, 131.1, 131.3, 135.6 , 135.7 (methylidene C=C), 153.7, 157.7, 159.1 (C=O), 160.06 (C=N); Mass m/z (M + H)+: 414.17; Analysis calculated for C20H16ClN3O3S: C (58.04); H (3.90); Cl (8.57); N (10.15); O (11.60); S (7.75); Rf value = 0.70; Log P = 5.02.Color: light brown; Yield: 35%; MP: 115–117 °C; FT-IR : 3343(–NH), 3063(Ar–CH), 2957(CH), 2921(CH2), 1723, 1683, 1604 (C=O), 1568 (C=C), 1490 (C=N), 1025(C–O–C); 1HNMR : 2.2 , 3.67 , 3.88 , 7.92–7.04 , 7.11 , 7.84 ; 13CNMR : 15.1 (CH3), 40.60 (CH2), 111.46 , 112.0, 112.2, 126.3, 126.92, 126.99, 128.6, 128.7, 128.8, 129.1, 129.9, 130.2, 133.6, 137.2 , 138.3 (methylidene C=C), 152.5 (C=O), 157.7 (C=N); Mass m/z (M + H)+: 410.26; Analysis calculated for C21H19N3O4S: C (61.60); H (4.68); N (10.26); O (15.63); S (7.83); Rf value = 0.64; Log P = 4.23.Color: deep brown; Yield: 40%; MP: 112–114 °C; FT-IR : 3190(–OH), 3100(–NH), 2950(Ar–CH), 2924(CH), 2849(CH2), 1720, 1650, 1606 (C=O), 1507 (C=C), 1434 (C=N); 1HNMR : 1.6 , 2.57 , 4.12 , 7.90–7.07 , 7.4 , 7.73 ; 13CNMR : 21.9 (CH3), 40.1 (CH2), 116.17 , 115.9, 116.17, 116.5, 122.3, 123.5, 127.12, 131.6, 131.7, 132.0, 132.6, 134.2, 139.0 , 147.9 (methylidene C=C), 160.3, 165.2, 167.3 (C=O), 174.29 (C=N); Mass m/z (M + H)+: 396.19; Analysis calculated for C20H17N3O4S: C (60.75); H (4.33); N (10.63); O (16.18); S (8.11); Rf value = 0.60; Log P = 3.64.Color: cream; Yield: 45%; MP: 110–112 °C; FT-IR : 3190(–NH), 2975(Ar–CH), 2914(CH), 2801(CH2), 1760, 1679, 1601 (C=O), 1520 (C=C), 1442 (C=N), 1227 (C–N); 1HNMR : 2.26 , 2.91 , 4.12 , 7.96–7.01 , 7.4 , 7.70 ; 13CNMR : 21.9 (CH3), 40.1 (CH2), 39.8, 39.9 (N-CH3), 111.6 , 112.9, 113.7, 115.3, 115.9, 116.1, 123.5, 126.8, 127.1, 127.3, 128.5, 128.7, 128.8 , 131.7 (methylidene C=C), 144.5, 153.9, 155.0 (C=O), 167.0 (C=N); Mass m/z (M + H)+: 423.23; Analysis calculated for C22H22N4O3S: C (62.54); H (5.25); N (13.26); O (11.36); S (7.59); Rf value = 0.60; Log P = 4.47.Color: deep brown; Yield: 50%; MP: 118–120 °C; FT-IR  using a reported procedure14. After a 16 h fast, each mouse had their blood glucose level (BGL) checked, and those with BGLs between 60 and 90 mg/dL were divided into 12 groups of six mice each. The mice in the experimental groups were given oral doses of 50 mg/kg and 100 mg/kg body weight of synthesized chemicals dissolved in 5% DMSO. The same amount of DMSO was given to the test, positive control, and negative control group. Rosiglitazone (12.5 mg/kg body weight) was administered orally to the positive control group. A glucose load (2.5 g/kg body weight) was given orally to each animal 30 min after the test sample or vehicle was administered. The blood glucose profile of each mouse was measured using a glucometer at 0, 30, 60, 90, and 120 min after glucose administration. The BGL of each mouse was assessed three to four times, and the average was calculated. During the trial, the cages were devoid of food but not of water. OGT was used to screen the test compounds for hypoglycemic action in normal, healthy mice at two dosage levels (50 and 100 mg/kg body weight) to establish the therapeutic dose. For future studies, the dosage that improves glucose tolerance in a similar way to the standard drug was chosen.Oral glucose tolerance test (OGT) was performed on male Swiss albino mice weighing 24 ± 2.5 g16. First, nicotinamide was dissolved in normal saline and administered at a dose of 120 mg/kg body weight into the mice fasted overnight. At 15 min post nicotinamide administration, STZ was injected intraperitonially at 60 mg/kg body weight dose. The STZ solution  was prepared fresh by dissolving an appropriate amount of STZ in citrate buffer. The fasting blood glucose (FBG) level was measured after 72 h in each mouse by taking the blood samples from the tail vein using a glucometer. Each mouse’s BGL was monitored three to four times, and only those with average BGL over 200 mg/dL were chosen for the study and designated as STZ-induced diabetic animals.DM was induced in the mice through a single intraperitoneal (i.p.) injection of nicotinamide and streptozotocin (STZ)5a–e) dissolved in DMSO. All the animals were fasted overnight, and their FBG levels on day 0 were determined. The test compounds were administered individually to the assigned groups at a dose of 100 mg/kg orally every day at a fixed time for 21 days. The vehicle was given to the negative control group, and rosiglitazone was administered at a dosage of 12.5 mg/kg body weight to the positive control group. Blood samples were withdrawn on days 0, 7, 14, and 21 from all the animals, and the percentage decrease in FBG was determined. On each sampling day, blood samples were collected from the tail vein of each mouse 3–4 times, and the average FBG was calculated.The selected STZ-induced diabetic mice were weighed again before the study and split randomly into seven groups with six animals each. Group 1, the diabetic control group, was orally given 5% DMSO only as vehicle. Group 2 served as the positive control group and was given the standard drug rosiglitazone. Groups 3–7 orally received the synthesized compounds . Chain A was deleted, and chain B was used for processing the receptor structure for docking. The binding coordinates of the native ligand nTZDpa were marked and retained for binding site identification. After the removal of native ligand and solvent, the receptor was energy-minimized by applying Amber ff14SB force field for standard residues and Gasteiger charges for other residues in Chimera 1.13.117. The prepared receptor was saved in *.pdbqt format for further use. The structures of ligands under investigation were drawn in ChemDraw and saved in protein data bank (PDB) format. The native ligand nTZDpa structure for redocking was retrieved from PubChem in SMILES format. All ligand structures were processed in Chimera to obtain a minimum energy conformer through 100 steepest descent steps with the step size of 0.02 Å, followed by 10 conjugate gradient steps of size 0.02 Å. The stabilized ligand conformers were also saved in *.pdbqt format.The 3D structure of PPAR-γ bound with its partial agonist nTZDpa (PDB ID: 2Q5S) was downloaded from the Research Collaboratory for Structural Bioinformatics database , the higher the reliability. The docked complexes of 2Q5S were probed for the intermolecular binding interactions in Discovery Studio.5c and 5d. The 3D pharmacophore model was generated for compound 5c first by loading the corresponding docked complex onto the macromolecular view. The ligand was then checked for missing bonds, if any, by focusing on the yellow box on the ligand. Afterward, the 3D pharmacophore model was created by selecting the create model option. The ligand and the pharmacophore model were saved to the alignment mode. The 3D structure of 2Q5S complexed with compound 5d was then loaded, and the procedure was repeated. Both pharmacophore models from compounds 5c and 5d were aligned, and the fingerprint features were shared and merged to create a 3D pharmacophore model with merged structural features18.The 3D pharmacophore model was analyzed with LigandScout 4.3 software using the 3D structures of 2Q5S docked to compounds https://www.schrodinger.com/products/desmond) for MD. To better understand the conformational changes caused by the test ligand binding, the apo form of the PPAR-γ and native partial agonist nTZDpa-PPAR-γ complex were also simulated. Inside an orthorhombic box with a 10 Å buffer region between protein atoms and box sides, the apo form of PPAR-γ and compound 5c-PPAR-γ or nTZDpa-PPAR-γ complexes were inserted. The box was then filled with the required number of water molecules in accordance with the system setup procedure.MD simulation was used to examine the thermodynamic behavior and stability of the best-ranked conformation of compound 5c in contact with PPAR-γ provided by the docking study. We used the Desmond 7.4 software integrated with Maestro of Schrödinger, Inc.  ensemble. The simulation time was customized to 150 ns, and trajectories were recorded every 150 ps. For short-range van der Waals and Coulomb interactions, a cut-off radius of 9.0 was chosen. The system temperature and pressure were maintained using the Nose–Hoover thermostat and Martyna–Tobias–Klein techniques, respectively. For bonded and non-bonded interactions inside the short-range cut-off, the reversible reference system propagator algorithm (RESPA) integrator was employed with an inner time step of 2.0 fs to integrate the equations of motion. The system was minimized and equilibrated to Desmond's default protocols. The Desmond package's simulation interaction diagram module was used to examine the trajectory files.A simple point charge (SPC) model and OPLS3e force field were adopted for the MD computationsp) < 0.05 were significant.The data obtained for the antidiabetic activity using the in vivo STZ-induced diabetic model and the effect of synthesized compounds on the body weight were analyzed by two-way mixed analysis of variance (ANOVA) using RStudio program version 1.4.1564 . The mean ± standard error of the mean (SEM) values were compared and cross-classified by two independent categorical variables, including one between the groups and one within the group. Values (5a–e) were synthesized first by reacting thiazolidine-2,4-dione and appropriate aromatic aldehydes (1a–e) to afford compounds 5-(4-substituted-benzylidene) -thiazolidine-2,4-diones (2a–e). The corresponding ethyl esters (3a–e) were then synthesized by reacting benzylidene-thiazolidine-2,4-diones (2a–e) with ethyl chloroacetate. The substituted  acetic acid hydrazides (4a–e) were synthesized by refluxing the ethyl esters (3a–e) with hydrazine hydrate. Finally, the target benzylidene-2,4-thiazolidinediones (5a–e) were synthesized by reacting the acetic acid hydrazides (4a–e) with acetophenone.New acetic acid (1-phenylethylidene)hydrazides , 3343–3100 (− NH), 3193–2950 (Ar–CH), 3063–2914 (CH), 2925–2801 (CH2), 1774–1601 (C = O), 1568–1487 (C = C), 1492–1434 (C = N), 1227 (C-N), 1025 (C–O–C), and 759 (C–Cl). The 1H-NMR spectra confirmed the presence of − CH3, − CH2, CONH, and − C-H groups by showing singlets at δ 1.6–2.26, δ 2.57–4.13, δ 7.1–7.4, and δ 7.46–7.86 ppm, respectively. Meanwhile, the Ar–H groups were confirmed by detecting multiplets at δ 7.96–7.01 ppm. Singlets for − OCH3 and OH were observed at 3.67 and 4.12 ppm, respectively. The 13C-NMR spectra confirmed the presence of − CH3, − CH2, C = C (thiazolidinedione), C (benzene), methylidene (C = C), and (C = N). Electrospray ionization (ESI) in MS revealed the protonated molecular ions in the positive ion mode. Mass m/z (M + H)+ peaks at 380.15, 414.17, 410.26, 396.19, and 423.23 were observed for compounds 5a, 5b, 5c, 5d, and 5e, respectively.All the synthesized compounds were characterized using various spectral techniques such as FT-IR, 5a–e) derivatives were administered in a series of doses ranging 10–500 mg/kg, followed by the measurement of weight before and after the treatment. The mice were monitored for 14 days and exhibited no symptoms of harm. Furthermore, no deaths occurred throughout the follow-up study. No significant variation in mouse weight was observed pre- and post-treatment, indicating the median lethal dose (LD50) of each compound to be more than 500 mg/kg.The acute oral toxicity test was performed to evaluate the acute physical, fatal, or behavioral changes caused by the newly synthesized compounds when administered orally. The synthesized benzylidene-2,4-thiazolidinedione (5a–e) derivative was evaluated at 50 and 100 mg/kg doses in normal mice, and the results are summarized in Table 5e at 100 mg/kg dose showed the greatest percent decrease (59.79%) in mice at 120 min. This percent decrease was even higher than that for the standard drug rosiglitazone (41.90%) tested at 12.5 mg/kg dose after 120 min. Table The blood glucose lowering action of each benzylidene-2,4-thiazolidinedione (5a–e) were performed on the STZ-induced diabetic murine model. The test compounds (100 mg/kg) and rosiglitazone (12.5 mg/kg) were administered to the diabetic mice for 21 days at a set time each day. FBG levels were measured at the same time on days 0, 7, 14, and 21, and the findings are presented in Table 5d (% decrease = 59.28%), followed closely by 5e (% decrease = 55.67%).The in vivo antidiabetic screening of synthesized benzylidene-2,4-thiazolidinediones  reduction in BGL comparable with the standard drug rosiglitazone. The maximum percent change in FBG level was observed with compounds 5d (59.28%) and 5e (55.67%). Furthermore, a pairwise comparison was performed between different times within groups to investigate the effects of treatment and time on the FBG, and the findings are depicted in Figs. Statistically significant two-way interactive effects were observed between treatment group and time on FBG with F18, 42) = 31.4 and  = 31.4 a5a–e) and the standard drug rosiglitazone on the body weight of STZ-induced diabetic animals were studied for 21 days, and the results are presented in Table The effects of the test compounds  change in body weight relative to that of the control at this time. This effect continued after 14 days, with rosiglitazone inducing a highly significant (****p < 0.0001) change in body weight due to its opposite effects on body weight over time. For the test compounds, compound 5a caused a significant (**p < 0.01) change in body weight relative to the control after 14 days. Compounds 5c, 5d, and 5e also induced lesser but statistically significant changes in body weight (*p < 0.05) compared with that of the control. At the end of the study period (21 days), the level of significance for compounds 5d and 5e increased (**p < 0.01), and a similar trend was observed for compound 5a (***p < 0.001) after 21 days.The change in body weight induced by the test compounds was examined relative to the body weight of the control at different time points to determine the level of significance. None of the test compounds induced any significant difference in body weight compared with that of the control after 7 days; however, rosiglitazone caused a statistically significant (*5a–e) under investigation engaged the same binding site as the native ligand nTZDpa, a partial agonist. Compounds 5c and 5d were predicted to exhibit better binding affinities to PPAR-γ than the native ligand as shown in Table The applied molecular docking protocol was successful in predicting the bioactive conformations of all investigated ligands. As shown in Fig. 5c and 5d) were probed further to understand the nature of these binding interactions and the role of the ligand’s structural motifs in enhancing the binding to PPAR-γ. Figure 5c and 5d, respectively.The binding interactions of the native and the top two ligands  to its methyl ether derivative (5c) slightly increased the binding affinity. However, similar electrostatic hydrogen bond interactions with the receptor were predicted because the oxygen atom of these groups was essential for the formation of hydrogen bond as the hydrogen bond acceptor, not the donor hydrogen. Chlorine (5b) and dimethylamino (5e) substituents on the phenyl ring did not improve the binding affinity. The thiazolidine ring and a keto group on the thiazolidine ring were important for binding to the receptor. The two terminal aromatic phenyl rings also played a crucial role in the PPAR-γ partial agonistic activity of 5c and 5d. The prototype of antihyperglycemic drugs is TZDs such as rosiglitazone and pioglitazone, which act as full agonists of the PPAR-γ receptor. However, they suffer from the disadvantages of causing heart failure, weight gain, edema, and obesity as side effects. PPAR-γ partial agonists are beneficial over full agonists because they exhibit the dose-related weak transactivation of the receptor by binding to different binding sites and utilizing different coactivators, leading to their distinguished effects24. Molecular docking results suggested that the partial PPAR-γ agonistic activity of compounds 5c and 5d were better than that of the native ligand. These findings were in good correlation with the in vivo results supporting the successful design of novel benzylidene 2,4-thiazolidinedione derivatives as PPAR-γ partial agonists for type 2 DM.The structure–activity relationship deduced from docking suggested that the conversion of the phenol  and root-mean-square fluctuations (RMSF) of the protein backbone atoms, as shown in Fig. 9. Compound 5c-PPAR-γ complex had an average RMSD value of 2.31 Å, whereas the nTZDpa-PPAR-γ complex and the unbound PPAR-γ protein exhibited an average RMSD value of 2.87 Å and 2.27 Å, respectively. The RMSD results demonstrated greater fluctuation of the nTZDpa-PPAR-γ complex than the compound 5c-PPAR-γ complex  illustrates the dynamics of each atom moving because of numerous hydrogen and hydrophobic connections with nearby residues. The unique conformational modes of interactions are a result of the position variation of the ligand molecules. As seen in Fig. 5c and nTZDpa were calculated to be 1.23 Å, 1.07 Å and 1.17 Å, respectively, for all amino acids of PPAR-γ protein over 150 ns simulated trajectories.The fluctuations of apo and compound . The greater spreading area of the protein backbone's atoms accounts for the higher gyration values. The lower rGyr value, on the other hand, denotes the compactness of the protein backbone structure and the stability of the complex. The values of rGyr range between 4.70 and 6.46 Å in compound 5c-PPAR-γ complex whereas the rGyr ranged between 4.24 and 4.56 Å for nTZDpa-PPAR-γ complex, as shown in Fig. The root mean square distance between the center of mass of each atom in the protein backbone is used to calculate the radius of gyration (rGyr). The rGyr of the compound 5c-PPAR-γ complex concerning the protein backbone is shown in Fig. 5c and the native ligand nTZDpa with PPAR-γ. More number of hydrogen bonds and hydrophobic interactions were predicted in compound 5c-PPAR-γ complex than in nTZDpa- PPAR-γ complex. Furthermore, the nature of binding interactions and their stability were analyzed from the extracted trajectories of MD. The 2D depiction of binding interactions in compound 5c-PPAR-γ complex and nTZDpa- PPAR-γ complex is shown in Figs. Figure 5c and PPAR-γ, which docking failed to predict. Moreover, MD unveiled a hydrogen bond interaction between keto oxygen of thiazolidinedione moiety and Ser342 alike nTZDpa. There are slight differences in the binding role of polar functional groups of compound 5c and the binding residues of PPAR-γ predicted in docking and MD, but the number of hydrogen bonds predicted by docking and MD correlated well. The hydrogen bonds formed by compound 5c with Cys285, Leu340, and Ser342 were stable and existed for 54%, 44%, and 29% of the time of MD. Figure Figure 5c and PPAR-γ residues during the simulation period of 150 ns. A deeper shade of orange indicates specific residues making more than one contact with the ligand. Fig. 5c inside the binding site of PPAR-γ during MD simulation. In summary, MD simulations indicated that compound 5c existed in a stable conformation inside the PPAR-γ binding site establishing stable hydrogen bonds with critical residues, confirming its potential to serve as partial agonist of PPAR-γ which requires further experimental validations.The supplementary Fig. The developed benzylidene 2,4-thiazolidinediones proved to be promising antidiabetic compounds that partially agonize the PPAR-γ receptors. The results of in vivo and in silico investigations were in agreement with those from the structure–activity relationship studies, leading to the identification of derivatives with comparable antidiabetic activity to the standard drug rosiglitazone. The molecular docking and molecular dynamics studies suggested that the title compounds bind to the same location as the native ligand, acting as a partial agonist at the receptor. The title compounds mainly interacted with the receptor via hydrophobic interactions; the introduction of a hydrogen bond acceptor, such as oxygen, to the compound resulted in hydrogen bonding and increased binding energy. All the required characteristic features of a partial agonist were present in the most active tested compounds, suggesting their partial PPAR-γ agonistic mechanism. Further specific in vitro and ex vivo molecular level studies are required to establish the exact mechanism. The most active compounds of this series can act as potential leads whose structure can be modified to further improve their efficacy.Supplementary Figures.Supplementary Video 1.


Objective: To observe the clinical efficacy and safety of Yiqi Yangxue formula (YQYXF) on knee osteoarthritis (KOA), and to explore the underlying therapeutic mechanism of YQYXF through endogenous differential metabolites and their related metabolic pathways.Methods: A total of 61 KOA patients were recruited and divided into the treatment group  and the control group . Effects of these two drugs on joint pain, swelling, erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) were observed, and their safety and adverse reactions were investigated. In animal experiments, 63 SD rats were randomly divided into normal control (NC) group, sham operation (sham) group, model (KOA) group, Cxb group, as well as low-dose (YL), medium-dose (YM), and high-dose groups of YQYXF (YH). The KOA rat model was established using a modified Hulth method. Ultra-high-performance liquid chromatography/Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass (UHPLC-QE-MS)-based metabolomics technology was used to analyze the changes of metabolites in plasma samples of rats. Comprehensive (VIP) >1 and t-test p < 0.05 conditions were used to screen the disease biomarkers of KOA, and the underlying mechanisms of YQYXF were explored through metabolic pathway enrichment analysis. The related markers of YQYXF were further verified by ELISA (enzyme-linked immunosorbent assay).Results: YQYXF can improve joint pain, swelling, range of motion, joint function, Michel Lequesen index of severity for osteoarthritis (ISOA) score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, ESR, and CRP. No apparent adverse reactions were reported. In addition, YQYXF can improve cartilage damage in KOA rats, reverse the abnormal changes of 16 different metabolites, and exert an anti-KOA effect mainly through five metabolic pathways. The levels of reactive oxygen species (ROS) and glutathione (GSH) were significantly decreased after the treatment of YQYXF.Conclusion: YQYXF can significantly improve the clinical symptoms of KOA patients without obvious adverse reactions. It mainly improved KOA through modulating lipid metabolism-related biomarkers, reducing lipid peroxidation and oxidative stress. Knee osteoarthritis (KOA) is the most prevalent form of arthritis characterized by a degeneration of articular cartilage resulting in the development of osteophytes, or bone spurs . The gloAlthough the mechanisms of degenerative changes are better-understood thanks to numerous biochemical and genetic studies, drugs that can stop the degenerative cascade remain unknown. So far, arthritis have been managed pharmacologically and non-pharmacologically, including common pharmacotherapies, surgery, and lifestyle changes. All available forms of KOA therapy are based on symptomatic treatment, such as pain relief and joint function improvement . Pain meIn the present study, we aimed to evaluate the clinical efficacy and safety of YQYXF in patients with KOA. We observed the effects of YQYXF and celecoxib (Cxb) on visual analogue scale (VAS) score, swelling, range of motion (ROM) and joint function, Michel Lequesen index of severity for osteoarthritis (ISOA) score, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Kellgren-Lawrence score, erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) index of KOA patients . The safSixty-one KOA patients (Kellgren-Lawrence score I-III) were recruited from Yunnan Provincial Hospital of Traditional Chinese Medicine. The patients were divided into a treatment group with 30 patients (YQYXF) and a control group with 31 patients . All patients fulfilled the American College of Rheumatology criteria for primary KOA, and the subjects agreed to sign the informed clinical consent. The age was between 38 and 70 years old—patients who discontinued NSAIDs for 7 days or more. The exclusion criteria included patients with other rheumatic diseases, such as rheumatoid arthritis, Sjögren’s syndrome, and gouty arthritis. In addition, patients with allergies or severe other systemic diseases will not participate in this study. The design scheme of this project has been approved by the Medical Ethics Committee of Yunnan University of Traditional Chinese Medicine (ethics number: 2019YXLL005).YQYXF granules, provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd. YQYXF granules consist of astragalus 30 g, codonopsis 15 g, tangerine peel 10 g, cohosh 10 g, bupleurum 10 g, angelica 20 g, cassia twig 15 g, white peony 15 g, atractylodes 15 g, licorice 5 g, divaricate saposhniovia root 15 g, ligusticum wallichii 15 g, rhizoma drynariae 15 g, epimedium 15 g. The Cxb capsules, provided by Ruihui Pharmaceutical Co., Ltd., are approved by Chinese medicine J20080059. In this study, the treatment group was given YQYXF, one bag/time, three times a day. The control group was given 200 mg/day of Cxb capsules once times a day.All patients were evaluated using the VAS score and ISOA score, WOMAC, Kellgren-Lawrence score, range of motion (ROM), and joint function grades before the treatment and at 4 weeks after treatment . ESR andThe 14 herbs in YQYXF are provided by the Chinese Pharmacy of the First Affiliated Hospital of Yunnan University of Traditional Chinese Medicine, and their composition and dosage are the same as those of YQYXF granules. The high dose group of YQYXF was administered by gavage with an aqueous solution containing 18.4 g of crude drug/kg, the medium dose group was administered with 9.2 g of crude drug/kg, and the low dose group was administered with 4.6 g of crude drug/kg of rats . In the positive drug group, 18 mg Cxb/kg aqueous solution was administered by gavage. Cxb capsules, provided by Ruihui Pharmaceutical Co., Ltd., approved by Chinese medicine J20080059.Compounds in YQYXF were analyzed using a UHPLC system  equipped with a waters UPLC BEH C18 column (1.7 μm 2.1 100 mm), and the flow rate was set to 0.5 mL/min, and an injection volume was set to 5 μL. Mobile phase A consisted of 0.1% formic acid solution, and mobile phase B was 0.1% formic acid in acetonitrile. The multi-step linear elution gradient program was as follows: 0–11 min, 85%–25% A; 11–12 min, 25%–2% A; 12–14 min, 2%–2% A; 14–14.1 min, 2%–85% A. During each acquisition cycle, the mass range was from 100 to 1,500, the top four of every process were screened, and the corresponding MS/MS data were further acquired. Sheath gas flow rate: 35 Arb, Aux gas flow rate: 15 Arb, Ion Transfer Tube Temp: 350°C, Vaporizer Temp: 350°C, Full ms resolution: 60,000, MS/MS resolution: 15,000, Collision energy: 16/32/48 in NCE mode, Spray Voltage: 5.5 kV (positive) or −4 kV (negative). An Orbitrap Exploris 120 mass spectrometer coupled with Xcalibur software was employed to obtain the MS and MS/MS data information of YQYXF based on the IDA acquisition mode. The raw data of mass spectra were imported into XCMS software for processing, such as retention time correction, peak identification, peak extraction, peak integration, and peak alignment. The peak information of compounds was searched through the in-house secondary mass spectrometry database provided by Shanghai BIOTREE Biotech Co., Ltd.Sixty-three SPF-grade female SD rats (180 ± 20 g) were purchased from Hunan Slike Jingda Laboratory Animal Co., Ltd., license number: SCXK (Xiang) 2019–0004. The experiments were conducted under full authorization from the Ethics Committee of Yunnan University of Chinese Medicine . The rats were randomly divided into seven groups, including normal control (NC), sham-operated (sham), model (KOA), Cxb (18 mg/kg), YQYXF low-, middle-, and high-dose groups . Each group had nine rats. The KOA models were established by the modified Hulth method . The antRoutine dewaxing, rinsed with tap water, stained with hematoxylin solution for 5 min, dehydrated in acid water and ammonia, rinsed with tap water, dehydrated and stained with eosin for 3 min. Dehydrated from low to high concentrations of alcohol, clear, sealed with neutral glue.Add 400 μL of extract (methanol: acetonitrile = 1:1 (V/V), containing isotope-labeled internal standard mixture) to 100 μL of plasma and vortex to mix for 30 s. Sonicate for 10 min and let stand at −40°C for 1 h, centrifuge at 12,000 rpm for 15 min at 4°C, and collect the supernatant for assay. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all plasma samples. UHPLC-QE-MS analyses were performed using a UHPLC system  with a UPLC BEH Amide column  coupled to Q Exactive HFX mass spectrometer . The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (A) and acetonitrile (B). The auto-sampler temperature was 4°C, and the injection volume was 2 μL. The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra in information-dependent acquisition mode in the control of the acquisition software . In this mode, the acquisition software continuously evaluated the full scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate as 30 Arb, Aux gas flow rate as 25 Arb, capillary temperature as 350°C, full MS resolution as 60,000, MS/MS resolution as 7,500, collision energy as 10/30/60 in NCE mode, spray Voltage as 3.6 kV (positive) or −3.2 kV (negative), respectively. The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. Then an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. The cutoff for annotation was set at 0.3. The Thermo Q Exactive HFX mass spectrometer is capable of primary and secondary mass spectral data acquisition under the control of the acquisition software .p < 0.05, the variable was defined as a significantly different metabolite between the two groups. The significantly different metabolites were used for plotting hierarchical clustering based on the Euclidean distance formula and drawn heat maps using the Pheatmap package in R studio. The volcano plots were used to filter the metabolites of interest based on Log 2 (fold change) and–Log 10 . The Kyoto Encyclopedia of Genes and Genomes  and MetaboAnalyst  databases were used for pathway enrichment analysis.Principal components analysis (PCA) and orthogonal correction partial least squares discriminant analysis (OPLS-DA) were conducted using the SIMCA16.0.2 software package . PCA, an unsupervised analysis that reduces the dimension of the data, was carried out to visualize the distribution and the grouping of the samples. A 95% confidence interval in the PCA score plot was used as the threshold to identify potential outliers in the dataset. In order to visualize group separation and find significantly changed metabolites, supervised orthogonal projections to latent structures-discriminate analysis (OPLS-DA) were applied. Then, 7-fold cross-validation was performed to examine the quality of the model. Permutation tests were used to test the validity of the model. The first principal component of variable importance in the projection (VIP) and Student’s t-test were obtained to refine the analysis. Suppose VIP>1 and The plasma of the rats was collected, and the levels of lipid peroxidation-related indexes, such as reactive oxygen species (ROS) and glutathione (GSH), were tested by enzyme-linked immunosorbent assay (ELISA). The ELISA kit  was used according to the manufacturer’s instructions.p < 0.05 was considered statistically significant.The data were processed and analyzed by using SPSS 26.0 software. If the data followed the normal distribution, they were presented as mean ± SD (standard deviation). Data were compared for differences using two independent samples t-tests or one-way ANOVA. If the data were not normally distributed, they were presented as median , and a non-parametric test was adopted, and p = 0.97). Among the treatment group, 4 males (13.33%) and 26 females (86.67%). While in the control group, 5 were males (16.13%) and 26 were females (83.87%). There was no significant difference in gender between the two groups (p = 0.76). The disease duration of the treatment and control groups were 46.33 ± 21.34 months and 45.29 ± 20.92 months, respectively. There was no significant difference in disease duration between the two groups (p = 0.84). In grading of severity of KOA assessed by Kellgren-Lawrence score grading, 6 cases were grade I, 20 were grade II, and 4 were grade III among the treatment group. While, in the control group, 7 cases were grade I, 19 were grade II, and 5 were grade III. There was no significant difference in severity of KOA between the two groups (p = 0.99). The joint function grades in the treatment group included 10 cases of grade I, 18 cases of grade II, and 2 cases of grade III. In comparison, the control group had 11 cases of grade I, 18 cases of grade II, and 2 cases of grade III. There was also no significant difference in joint function between the two groups (p = 0.87). Therefore, the age, gender, disease duration, severity of arthritis, and joint function of the two groups of patients are comparable, and follow-up research can be carried out.As illustrated in p > 0.05) (p = 2.81 × 10−14). The VAS score of patients in the control group was 4.35 ± 1.20 before administration and 1.86 ± 0.83 after 4 weeks of administration (p = 1.14 × 10−13). In the treatment group, the VAS score of physician was 4.20 ± 0.92 before administration and 1.73 ± 0.79 after 4 weeks of administration (p = 5.44 × 10−16). In the control group, the VAS score of physician was 4.10 ± 1.25 before administration, and the VAS score of physician was 1.97 ± 0.66 after 4 weeks of administration (p = 1.07 × 10−11). In the treatment group, the medium of swelling score was 2.00 before administration and 0.00 after 4 weeks of administration (p = 1.80 × 10−5). The swelling score of the control group was 2.00 before administration and 0 after 4 weeks of administration (p = 1.10 × 10−5). In the treatment group, the joint range of motion (ROM) was 2.00 before administration and 0.00 after 4 weeks of administration (p = 9.40 × 10−7). The ROM in the control group was 2.00 before administration and 0.00 after 4 weeks of administration (p = 0.002). In the treatment group, ISOA score was 9.00 ± 1.80 before administration and 3.07 ± 1.36 after 4 weeks of administration (p = 8.49 × 10−21). The ISOA in the control group was 8.74 ± 1.63 before administration and 4.10 ± 1.33 after 4 weeks of administration (p = 4.78 × 10−18). In the treatment group, the WOMAC score was 52.27 ± 6.74 before administration and 16.13 ± 3.85 after 4 weeks of administration (p = 3.24 × 10−33). The WOMAC score of the control group was 53.13 ± 6.38 before administration and 19.52 ± 3.71 after 4 weeks of administration (p = 9.10 × 10−34). In the treatment group, the ESR was 20.67 ± 8.21 (mm/h) before administration and 10.03 ± 4.80 (mm/h) after 4 weeks of administration (p = 7.21 × 10−18). The ESR of the control group was 20.90 ± 8.94 (mm/h) before administration and 10.61 ± 3.07 (mm/h) after 4 weeks of administration (p = 1.90 × 10−7). In the treatment group, the CRP was 8.56 ± 3.05 (mg/L) before administration and 2.06 ± 1.15 (mg/L) after 4 weeks of administration (p = 3.12 × 10−15). The CRP of the control group was 8.80 ± 2.81 (mg/L) before administration and 2.09 ± 1.06 (mg/L) after 4 weeks of administration (p = 2.71 × 10−17). After drug intervention, the joint function grades in the treatment group included 22 cases of grade I, 8 cases of grade II, and 0 cases of grade III, while the control group had 19 cases of grade I, 12 cases of grade II, and 0 cases of grade III and no significant difference was observed (p = 0.97).The clinical observation results showed that 3 (10%) cases were highly effective, 17 (56.67%) cases were moderately effective, 9 (30%) cases were lowly effective, and 1 (3.33%) case was ineffective, with a total effective rate of 96.67% in the treatment group. In the control group, 2 (6.45%) cases were highly effective, 15 (48.39%) cases were moderately effective, 12 (38.71%) cases were lowly effective, and 2 (6.45%) cases were ineffective, and the total effective rate was 93.55%  . The VASp > 0.05) , liver function , renal function , and electrocardiogram after 4 weeks of treatment  . Our resThe compounds in YQYXF were identified by the UHPLC-QE-MS method. A total of 447 compounds were characterized in YQYXF (p > 0.05). The point indicated by the arrow was the cartilage surface , which played an essential role in avoiding the over-fitting of the test model and evaluating the robustness of the model.To further explore the effect of YQYXF on the endogenous differential metabolites and their related metabolic pathways of KOA, we used the plasma of rats in the NC group, KOA group, and YH group for metabolomic analysis. We included QC samples throughout the experimental process to ensure the stability and reliability of the data and the system. The QC samples (yellow dots) were closely clustered together, indicating that the UHPLC-QE-MS system had good stability and was reliable for metabolomic analysis of the samples. LC-MS data obtained from the plasma samples were analyzed using PCA for metabolic changes between the NC, KOA, YH, and QC samples . The metabolic pathway enrichment analysis of differential metabolites showed that YQYXF mainly exerted its anti-inflammatory effect by regulating five pathways: linoleic acid metabolism, α-linolenic acid metabolism, sphingolipid metabolism, arachidonic acid metabolism, and glycerophospholipid metabolism  and ROS  were significantly increased  and the ROS of drug-treated groups was lower than that of the KOA group , suggesting that the YQYXF could regulate the plasma lipid metabolism disorder in the KOA rats. Hence, YQYXF may exert anti-KOA cartilage degeneration by regulating lipid peroxidation.The results of metabonomics experiments showed that YQYXF mainly regulated the signal pathway related to lipid metabolism. Therefore, it may play the role of anti-KOA cartilage destruction by repairing lipid metabolism disorder. We further explored the changes of oxidative stress and lipid peroxidation-related factors in KOA rats. The GSH of NC, sham, OA, YL, YM, YH, and Cxb group was 235.69 ± 33.23, 250.43 ± 24.85, 467.97 ± 29.25, 397.37 ± 22.89, 374.49 ± 29.29, 319.31 ± 18.88, 284.02 ± 28.56 mmol/L, respectively. The ROS of NC, sham, OA, YL, YM, YH, and Cxb group was 290.41 ± 50.77, 318.59 ± 31.99, 681.62 ± 25.38, 540.76 ± 48.31, 495.07 ± 47.84, 433.13 ± 51.34, 359.10 ± 30.95 pg/mL, respectively. Compared with the NC and sham group, the expression of GSH . While all PUFAs reduced markers of oxidative stress, omega-3 PUFAs additionally decreased prostaglandin production . The omeA study used liquid chromatography/mass spectrometry (LC/MS)-based metabolomics to explore the serum metabolomics in rats with KOA. The six biomarkers were identified, which were metabolized through tryptophan metabolism, glutamate metabolism, nitrogen metabolism, spermidine metabolism, and fatty acid metabolism pathways . Anothervia the Toll-like receptor 4/NF-κB pathway in KOA rats (in vivo (For note, the flavocoxid is a medical food consisting of plant-derived flavonoids which have anti-inflammatory activity and are used to treat chronic KOA. Studies have shown that flavocoxid was as effective as naproxen in managing the signs and symptoms of KOA . In our KOA rats . The useKOA rats . In addiKOA rats . Besides(in vivo . There avia autotaxin generates lysophosphatidic acid, an inflammatory and pain-producing signal. Endocannabinoids, endogenously produced lipid-derived compounds, could be beneficial for individuals with KOA to reduce pain symptoms. In turn, the endocannabinoids may result in the production of lysoPCs, which could promote joint pain due to metabolism of lysophosphatidic acid (For note, the metabolic pathways likely contribute to symptoms and pathology in KOA. Some studies demonstrated that metabolites in the synovial fluid and blood could be used as biomarkers for KOA incidence, prognosis, and response to therapy . Variousdic acid . In addidic acid . The lindic acid . In addidic acid  (Table 5The limitation of this study is that the components of traditional Chinese medicine are complex, with the characteristics of multiple targets and pathways. Although the components of YQYXF were identified, the analysis of members entering the blood and the study of pharmacokinetics still need to be clarified.In conclusion, YQYXF could be an effective and promising agent for treating KOA, which might exert its action by regulating multiple lipid metabolism-related pathways. Our study provides new insights into studying the underlying molecular mechanism of YQYXF against oxidative stress in the KOA model. However, further research exploration is needed.