Combining Ozonated Autohemotherapy with Pharmacological Therapy for Comorbid Insomnia and Myofascial Pain Syndrome: A Prospective Randomized Controlled Study

Objective To examine the efficacy and safety of ozonated autohemotherapy (O3-AHT) combined with pharmacological therapy for comorbid insomnia and myofascial pain syndrome (MPS). Materials and Methods One hundred and eighteen patients were randomly divided into two groups: the control group (N = 50) and the O3-AHT group (N = 53). Patients in both groups were given the same pharmacological management for three weeks. Patients in the O3-AHT group were treated with ozonated autohemotherapy (the concentration of ozone was 20 µg/ml in the first week, 30 µg/ml in the second week, and 40 µg/ml in the third week) combined with pharmacological therapy. Primary (the insomnia severity index (ISI) and visual analogue scale (VAS)) and secondary outcomes (the Epworth sleepiness scale (ESS), polysomnography data, the anxiety and preoccupation about sleep questionnaire (APSQ), the beck depression index (BDI), and the multidimensional fatigue inventory (MFI)) were examined at pretreatment, posttreatment, 1 month, and 6 months. Results Fifty patients in the control group and fifty-three patients in the O3-AHT group completed the study. In both groups, insomnia and pain symptoms were relieved significantly compared with pretreatment. Compared with the control group, the O3-AHT group had significantly improved sleep quality, pain, and negative mood at different time points. No adverse complications were observed in either group. Conclusion Compared with pharmacological therapy alone, ozonated autohemotherapy combined with pharmacological therapy can ameliorate insomnia, reduce pain intensity, improve negative mood, and alleviate fatigue more effectively without serious adverse complications.


Introduction
Myofascial pain syndrome (MPS), defned as a kind of painful and aseptic skeletal muscle infammation, is one of the main causes of chronic pain in clinics. Tis complicated syndrome is characterized by myofascial trigger points (MTrPs) within fascia and muscles, and pressing on MTrPs can induce localized and referred pain [1]. MPS is a common disease faced by pain physicians in clinical practice, with a prevalence of about 30%-85% in patients with musculoskeletal pain [2]. Insomnia, defned as at least 3 months of difculty in initiating and/or maintaining restorative sleep, is a common and debilitating comorbidity of musculoskeletal painful diseases [3]. Te comorbidity of insomnia and pain is a fairly destructive situation for patients, and there is a complex bidirectional connection between insomnia and pain [4]. Tey aggravate each other reciprocally or even generate a vicious circle wherein pain disrupts sleep rhythms and poor sleep lowers the pain perception threshold in turn [5]. Although the bidirectional relationship between insomnia and pain has been extensively acknowledged, little scientifc knowledge about the fundamental mechanisms underlying this complicated association has been entrenched. Recent researches consider that the neurochemical mechanisms involve immune-related infammatory reaction and multiple neurohumoral regulations including the opioid system, monoaminergic system, pineal melatonin system, nitric oxide signaling, etc [6]. Comorbid insomnia and pain afect the life quality of patients substantially, causing symptoms such as daytime fatigue, emotional distress, and various dysfunctions. Tese complexities make the management of comorbid insomnia and chronic pain difcult.
Te close relationship between insomnia and pain suggests the importance of efectively managing patients' sleep when treating patients with MPS. However, in clinical practice, pain physicians often focus on improving pain symptoms and ignore the potential harm caused by insomnia. Te overall goals of treating patients with comorbid insomnia and MPS are improving sleep quality, alleviating pain, relieving negative emotions, and reducing the risk of insomnia-related disorders. Current strategies for the management of insomnia include cognitive behavioral therapy, medication, acupuncture, and other complementary therapies [7]. Cognitive behavioral therapy for insomnia (CBT-I), which is usually recommended as the frst choice for the treatment of insomnia, has been reported to be efective in improving the symptoms of patients with comorbid insomnia and chronic pain as well [8]. CBT-I was generally considered to have few adverse complications compared with medication, but it has been reported that sleep restriction therapy, a standard behavioral strategy used within CBT-I, is associated with reduced objective total sleep time, increased daytime sleepiness, and objective performance impairment [9]. Pharmacological agents for insomnia include benzodiazepines such as alprazolam, nonbenzodiazepines such as zolpidem, and several antipsychotics [7]. Pharmacological agents for MPS include nonsteroidal anti-infammatory drugs (NSAIDs), tricyclic antidepressants (TCAs), and muscle relaxants [1]. Medication may cause adverse efects such as gastrointestinal bleeding, renal injury, vomiting, and dizziness. Terefore, it is necessary to fnd an efective, safe, and feasible treatment for comorbid insomnia and MPS.
Recently, a multitude of studies has focused on the therapeutic value of ozone. In general, ozone's mechanism of action can be summarized as antioxidant capacity, vascular and hematological modulation, pathogen inactivation, and immune system activation [10]. Ozonated autohemotherapy (O 3 -AHT) is a common pattern of ozone therapies during which autologous blood is ozonated and then injected back into the body [11]. Recently, several studies have investigated the efect of O 3 -AHT on coronavirus 19 [12]. Besides, O 3 -AHT has shown signifcant advantages in diverse diseases such as psoriasis, age-related macular degeneration, and multiple sclerosis [13][14][15]. In terms of painful diseases, O 3 -AHT has been reported to relieve pain in fbromyalgia and postherpetic neuralgia [16,17]. In addition, a study using low-dose ozone to treat insomnia in patients with coronary heart disease found that low-dose ozone therapy improved sleep quality and ameliorated depression by elevating the levels of brain-derived neurotrophic factor (BDNF) in blood serum [18].
As mentioned above, there is a complicated correlation between insomnia and pain, which poses a knotty challenge for management. We hypothesized that combining O 3 -AHT with pharmacological therapy can provide safe and efective relief of insomnia and pain in patients with comorbid insomnia and MPS. Terefore, we conducted a prospective, randomized, controlled study to evaluate the efcacy and safety of this combination therapy.

Ethics and Patients.
Tis study was approved by the Institutional Review Board and Ethics Committee of Shanghai East Hospital (ChiCTR1900021602). Te present clinical research was conducted in accordance with the Declaration of Helsinki. All authors had access to the study data and approved the manuscript. Written informed consent was obtained from all patients before the inclusion. From March 2019 to October 2021, we invited 176 patients to participate in our study on the basis of inclusion and exclusion criteria. Te inclusion criteria are as follows: (1) age >18 years; (2) sufering from back pain for at least 3 months; (3) underwent a pain physician interview and a thorough physical examination to confrm the MPS diagnosis [19]; (4) diagnosed with insomnia according to the International Classifcation of Sleep Disorders, Tird Edition (ICSD-3); and (5) willing to undergo randomization and follow instructions. Te exclusion criteria are as follows: (1) the presence of other sleep diseases such as sleep apnea and restless legs syndrome; (2) unstable psychiatric disorders such as bipolar disorder, mania, and schizophrenia; (3) severe system disorders such as heart failure, liver dysfunction, renal injury, hematologic disorders, and coagulation dysfunction; (4) abuse of psychotropic substances or analgesics; (5) sufering from other painful diseases, especially cancer pain; (6) participating in other psychological treatments and/or drug trials; (7) being allergic to anticoagulants such as sodium citrate; and (8) being pregnant or lactating women. All patients were randomly divided into the O 3 -AHT group or the control group using a computerized number generator.

Sample Size.
Because there was no reference for the efectiveness of ozonated autohemotherapy in patients with comorbid chronic insomnia and MPS, we conducted a preliminary trial before conducting the formal research. Te preliminary trial indicated that the efective rate of treatment was 45% (9/20) in the control group and 75% (15/ 20) in the O 3 -AHT group after treatment. Terefore, the sample size calculation was based on a 45% efective rate in the control group and a 75% efective rate in the O 3 -AHT group. Assuming a two-sideda � 0.05 and a statistical power of 0.9, the sample size was calculated to be 38 for each group. Considering a 15% loss to follow-up, the sample size was at least 44 in each group.

Terapeutic Method.
All patients included in the study underwent routine examinations after admission to the hospital, including a blood routine examination, coagulation function, liver function, and kidney function.

Pharmacological Management.
Patients in both groups were given the same pharmacological management for 3 weeks. For insomnia management, patients were allowed to take 0.5-2 mg of estazolam if they found it extremely difcult to fall asleep. For pain management, patients with a VAS score <5 were given 60 mg of loxoprofen, with a maximum of 3 tablets per day. Patients with a VAS score of ≥5 were given tramadol 100 mg tablets, up to a maximum of 3 tablets per day.

Ozonated Autohemotherapy.
A special operator collected 100 ml of blood via the median cubital vein and injected it into a blood bag with an anticoagulant (10 ml of 3.8% sodium citrate). Blood was then mixed with a prepared 100 ml O 2 -O 3 mixture by the ozone medical apparatus (Forefront Medical Equipment Co., Zibo, Shandong Province, China) for 3 minutes. Afterward, the ozonated autologous blood was transfused back into the patient within 10 minutes. Patients received ozonated autohemotherapy three times a week for 3 weeks. Te concentration of ozone for each week was as follows: 20 µg/ml in the frst week, 30 µg/ml in the second week, and 40 µg/ml in the third week. 100 ml of the blood of patients in the control group was also collected without mixing with ozone, and then the sham infusion was performed. Only the special operator was aware of each patient's actual infusion condition.

Primary Outcomes.
Te insomnia severity index (ISI) was the primary outcome for evaluating the severity of insomnia in our study. Tis self-report questionnaire includes 7 items with a total score range of 0-28. Higher scores indicate more serious insomnia in the past two weeks.
Te visual analogue scale (VAS) was the primary outcome for evaluating the severity of pain in our study. One end of a 10 cm straight line is written "painless," and the other end is "the most severe pain." According to the degree of pain they felt, patients made a mark at a certain point on the straight line to indicate the intensity of pain and psychological impact. Te distance from the starting point to the mark is the amount of pain, 0-10 points. Te higher the scores, the heavier the degree of pain.

Secondary
Outcomes. Te Epworth sleepiness scale (ESS) is a questionnaire used to evaluate the degree of subjective somnolence. Patients recalled their recent experiences of drowsiness and scored the severity of drowsiness in diferent situations, such as sitting still, lying fat, watching TV, and driving. Ten, we evaluated the severity of daytime drowsiness according to the total score. Te sum score was calculated (range 0-24), with higher scores indicating more dangerous somnolence. We used ESS as the secondary outcome to evaluate sleep quality.
We utilized a polysomnography system (PolySmith, Neurotronics Co., FL, USA) to record the sleep data of patients at four time points (pretreatment, posttreatment, 1monthfollow-up, and 6-month follow-up). At each time point, this instrument monitored the patient's sleep for 3 nights. Ten, we collected the following data and took the following average: (1)  Te anxiety and preoccupation about sleep questionnaire (APSQ) consists of 10 self-reports of insomnia patients about their distress and anxiety about sleep, each with a score of 0-10. Individuals compared the consistency between these self-reports and their own conditions in the past three days. Te total score is the sum of the 10 answers, with a range of 0-100. Higher scores indicate worse anxiety and preoccupation in the past three days.
Te Beck depression inventory (BDI) contains 21 questions about the severity of depression in the past week, each with a score of 0-3. Te total score ranges from 0-63, and higher scores indicate more severe depression.
Te multidimensional fatigue inventory (MFI) assesses the following 5 aspects of fatigue: general fatigue, physical fatigue, reduced activity, reduced motivation, and mental fatigue. Tere are 20 questions in this questionnaire, each with a score range of 1-5. Higher scores indicate worse fatigue.
Te treatment efect was assessed 6 months after treatment according to the following criteria: "remarkable"-the symptoms, especially insomnia and pain, almost disappeared, with good quality of life restored; "valid"-the symptoms, especially insomnia and pain, were relieved, with quality of life improved; "invalid"-no improvement in the symptoms, signs, or quality of life. Te efective rate (%) � ((remarkable + valid)/n) * 100%.

Adverse Complications.
Possible complications, such as an allergic reaction, puncture point infection, hypotension, myocardial infarction, blood contamination, and abnormal blood potassium, were evaluated throughout the observation period.

Statistical
Analysis. All data were analyzed using SPSS 26.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Te data were presented as the mean ± SD or n (%). Te independent-samplet-test was used for statistical analysis of the continuous data, and the chi-squared test was used for statistical analysis of the categorical data. Te repeatedmeasures analysis of variance (ANOVA) test was used to compare data between the two groups before and after treatment. A P-value of <0.05 was considered to represent a statistically signifcant diference.

Preoperative Patient Characteristics.
We invited 176 patients to participate in this study, and 118 of them were randomized. Finally, 103 patients (50 in the control group and 53 in the O 3 -AHT group) completed each follow-up ( Figure 1). No signifcant diference was found in the demographic and clinical characteristics between groups at baseline (P > 0.05) ( Table 1).

Insomnia Severity Index.
Tere was no signifcant diference between the two groups before treatment. In the control group, the ISI scores showed the lowest posttreatment and then gradually increased back. At each time point, the ISI scores of the O 3 -AHT group were signifcantly lower than pretreatment and showed the lowest at the 6monthfollow-up. Meanwhile, the ISI score of the O 3 -AHT group was signifcantly lower than the control group at each time point after treatment (Figure 2(a)).

Visual Analogue Scale.
In the O 3 -AHT group, the VAS scores decreased gradually with time, and the VAS score was signifcantly lower than pretreatment at each time point. Similarly, compared with the baseline, an improvement in the VAS score was observed in the control group. However, at a 6-monthfollow-up, the VAS scores showed an increase compared with the 3-monthfollow-up. Between-group comparisons revealed that the VAS scores of the O 3 -AHT group were signifcantly lower than the control group at each time point after treatment (Figure 2(b)).

Secondary Outcomes
3.3.1. Epworth Sleepiness Scale. Before treatment, there was no signifcant diference between the two groups. Compared with pretreatment, the ESS scores of the two groups decreased signifcantly at each time point. Between-groups comparisons revealed that the ESS scores of the control group were higher than those of the O 3 -AHT group at each time point (Figure 2(c)).

Polysomnography.
Tere was no signifcant diference between the two groups for TST, SOL, WASO, SE, and NOA before treatment. Between groups, comparisons revealed that, compared with the control group, data from the O3-AHT group were signifcantly improved at posttreatment, 1monthfollow-up, and 6-monthfollow-up. Within-group comparisons revealed a signifcant improvement for TST, SOL, WASO, SE, and NOA of the O 3 -AHT group at each time point versus pretreatment. For TST and SOL, patients in the control group showed a signifcant improvement only at posttreatment. For WASO, except for the 1-monthfollowup, patients in the control group had no improvement compared with pretreatment. For SE, patients in the control group gained signifcant improvement at posttreatment and the 6-monthfollow-up. However, for NOA, there was no diference between pretreatment and any time point after treatment in the control group (Table 2).

Anxiety and Preoccupation about Sleep Questionnaire.
Te APSQ scores of the control and O 3 -AHT groups did not difer from each other before treatment. At the time of posttreatment, the APSQ scores of the O 3 -AHT group were lower than those of the control group. At the 1-and 6monthfollow-ups, APSQ scores of the O 3 -AHT group showed a signifcant decrease compared with the control group. Within-group comparisons showed that the APSQ scores of the control group showed signifcant improvement only at posttreatment. APSQ scores of the O 3 -AHT group decreased signifcantly at each time point compared with pretreatment ( Figure 2(d)).

Beck Depression Inventory.
Te BDI scores of the control and O 3 -AHT groups did not difer from each other before treatment. At each time point after treatment, the BDI scores of patients in the control group were signifcantly higher than those in the O 3 -AHT group. Within-group comparisons revealed that in the control group, the BDI scores did not difer from pretreatment at the time point of posttreatment and even increased at the 1-monthfollow-up compared with pretreatment. At the 6-monthfollow-up, it showed a signifcant improvement compared with pretreatment. In the O 3 -AHT group, the BDI scores decreased signifcantly compared with pretreatment at each time point (Figure 2(e)).

Multidimensional Fatigue Inventory.
Tere was no diference between the MFI scores of the two groups before treatment. Te MFI scores of patients in the control group were signifcantly higher than those in the O 3 -AHT group at each time point after treatment. Within-group comparisons showed that MFI scores of patients in the control group improved at posttreatment and 1-monthfollow-up but did not difer from pretreatment at 6-monthfollow-up. Te MFI scores of the O 3 -AHT group decreased signifcantly compared with pretreatment at each time point (Figure 2(f )).

Te Efective Rate.
Te efective rate was 44% in the control group and 84.90% in the O 3 -AHT group 6 months after treatment (Table 3).

Adverse Complications.
No adverse complications, such as an allergic reaction, puncture point infection, hypotension, myocardial infarction, blood contamination, or abnormal blood potassium, occurred in either group during the treatment and follow-up period. No patients withdrew from the treatment because of adverse complications.

Discussion
In our study, we comprehensively assessed the efects of O 3 -AHT on improving insomnia, ameliorating pain, and reducing negative emotions in patients with comorbid insomnia and MPS. Previous studies have generally regarded the improvement of insomnia symptoms as an additional outcome measure of O 3 -AHT in the treatment of painful diseases. In this trial, we put the assessment of sleep in a quite important position, and polysomnography was frst utilized to record TST, SOL, WASO, SE, and NOA. Pharmacological therapy alone improved the ISI score to some extent after treatment, but from a long-term perspective, such improvement is inadequate and susceptible. Meanwhile, polysomnography results showed that pharmacological therapy alone only partially and temporarily improved symptoms of insomnia. By comparison, the advantages of O 3 -AHT combined with pharmacological therapy are refected in more striking short-term improvements, a more comprehensive range of infuence, and more sustained and incremental efects. Te change in ESS and MFI scores also show that O 3 -AHT has a signifcant and lasting efect on improving insomnia-related somnolence and fatigue.
Te improvement of pain in the two groups was consistent with expectations. Te two groups both had therapeutic efcacies of diferent degrees. Te VAS score was used to assess an individual's pain severity. Te results of the VAS score show that the combined application of O 3 -AHT on the basis of pharmacological therapy can materially improve the pain relief efect. Patients with chronic myofascial pain are more likely to develop depression or anxiety [1,2,20]. Meanwhile, there is a close association between insomnia and negative emotions as well [21]. In our study, the APSQ and BDI scores of patients in the O 3 -AHT group showed a signifcant improvement, whereas there was no such improvement in patients treated by pharmacological therapy alone. Tese results are in line with our expectations.   Current studies believe that psychological, social, cultural, and biological factors will afect the pain perception of patients with MPS [22]. When chronic pain diseases such as MPS and insomnia occur together, the quality of life of patients will be greatly afected. A large number of studies have been carried out to explore the relationship between insomnia and pain, and there is no doubt that a bidirectional and mutually promoting relationship between insomnia and pain exists [4]. Pain can directly afect patients' sleep quality, and insomnia will reduce patients' pain perception threshold and increase their pain sensitivity. In addition, negative emotions, including depression, anxiety, and boredom, are important mediators connecting the relationship between pain and insomnia [23]. Pain, insomnia, and negative emotions form a triangular relationship wherein these three aggravate each other.
Contrary to popular knowledge, recent research suggests that sleep disorders may have a stronger infuence on pain than vice versa [24]. Insomnia not only directly aggravates pain intensity but also afects the development of painful conditions. According to a large population-based study of Norwegian women who did not have fbromyalgia, there is a strong dose-dependent association between sleep disorders and the risk of fbromyalgia [25]. Another longitudinal cohort study in Norway analyzed the infuence of insomnia at baseline on the risk for headache 11 years later and demonstrated a particularly strong association between insomnia and headache [26]. In conclusion, current evidence suggests that sleep disorders increase the risk of newonset chronic pain in asymptomatic individuals and deteriorate the long-term prognosis of existing pain. When pain physicians treat headaches, musculoskeletal pain, or neuropathic pain, it is necessary to pay close attention to the accompanying insomnia. Te essential role of insomnia in the onset and development of chronic pain is supposed to be of concern, which is why we regarded the assessment of sleep condition and insomnia severity as such an important component in our study.
Ozone therapy has been gradually applied in the feld of medicine since its identifcation, although the process was circuitous and controversial [10]. Ozonated autohemotherapy (O 3 -AHT), the most mature and widespread form of ozone therapy in clinical practice, combines precollected blood with O 2 -O 3 in vitro and then administers ozonated blood back to the patient. O 3 -AHT has been well received because it allows for a predetermined amount of blood to be taken and a precise concentration of O 2 -O 3 to be infused [27]. It has been reported that O 3 -AHT can efciently alleviate pain and improve functional scores in patients with low back pain, causing only occasional and slight adverse reactions [28].
Our study indicates the efcacy and safety of O 3 -AHT in the treatment of comorbid insomnia and MPS. Tis clinical efect may involve the antioxidant, anti-infammatory, oxygen supply, and microcirculation mechanisms of ozone. Ozone has a strong antioxidant capacity. To be specifc, after being dissolved in the aqueous component of plasma, ozone can initiate transient oxidative stress and an increased release of microparticles from blood cells [29]. In response, an endogenous cascade is induced, and then diverse bioactive substances are released. Ozone can react with polyunsaturated fatty acids (PUFA) and produce reactive oxygen species (ROS) and lipid ozonation products (LOPs) [30]. ROS and LOPs, which are considered signifcant in the ozone biotransformation, increase activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and, thus, relieve pain [31]. Te antioxidant capacity of ozone may play an important role in the alleviation of insomnia as well. By scavenging free radicals and limiting oxidative stress, ozone is benefcial for improving total antioxidant levels in the blood, which had been weakened by abnormal melatonin regulation [32]. Owing to these potential antioxidant functions, in our study, signifcant improvements in insomnia and pain were found. Sleep restriction can elevate infammatory markers, including interleukin-6 (IL-6), tumor necrosis factor (TNF), and C-reactive protein (CRP), which are considered to be related to the onset and mediation of pain [33,34]. Ozone has the capacity to be anti-infammatory and can decrease proinfammatory cytokines [35]. By anti-infammatory effects, ozone may ameliorate insomnia severity and local infammatory reaction. Meanwhile, the association between insomnia and increased pain experience may be weakening. Another famous infammatory mediator-Prostaglandins (PG), especially PGE2, are not only important endogenous substances that regulate pain perception but have also been found to be elevated in patients with sleep disorders [36]. PG and its precursor, arachidonic acid, are classic PUFA, and as previously mentioned, ozone can react with PUFA after being dissolved in plasma. Terefore, it is reasonable to speculate whether ozone can alleviate insomnia and pain by afecting the metabolism of PG.
Ozone can improve the oxygen supply for tissues. O 3 -AHT can promote the activity of glycolysis and increase 2, 3-diphosphoglycerate (2, 3-DPG) in red blood cells [37]. 2, 3-DPG plays a key role in the rightward shift of the oxyhemoglobin dissociation curve and allows oxygen to be transferred to oxygen-starved tissues. On the one hand, increased oxygen supply to brain tissues can directly relieve insomnia. On the other hand, increased oxygen supply to localized tissues of pain regions, especially MTrPs, may alleviate hypoxia and hypermetabolism caused by continuous contraction of muscle fbers [38], so as to alleviate pain. In addition, ozone can improve the microcirculation in brain tissues and damage local tissues by increasing levels of vasodilators such as prostacyclin and nitric oxide [39]. Te improvement in brain oxygen supply may be one of the reasons for the relief of anxiety and depression. To sum up, the efect of O 3 -AHT on insomnia and pain is complicated and comprehensive, and further studies are needed to clarify its specifc mechanisms.
Consistent with previous studies [16,17], we did not observe adverse complications caused by O 3 -AHT throughout the study. However, it is still necessary for researchers to carry out O 3 -AHT in a standardized procedure and actively notice any potential risk.
Tere are also the following limitations to our study: (1) the patients were recruited from a single centre; (2) double blinding was not used in this research; and (3) the patients were only followed for 6 months after treatment. we did not comprehensively analyse the interaction between insomnia and pain during treatment. Terefore, it is necessary to be cautious about the results of this study and the explanation for our fndings.

Conclusions
In conclusion, our research demonstrated that ozonated autohemotherapy combined with pharmacological therapy was an efective and safe treatment for comorbid insomnia and myofascial pain syndrome. Compared with pharmacological therapy alone, this combination therapy can ameliorate insomnia, reduce pain intensity, improve related negative mood, and alleviate daily fatigue more signifcantly.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.