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. 2021 Jun 10;6(3):177–195. doi: 10.1089/can.2020.0105

Table 2.

In Vivo Study Characteristics Including Species, Model, Specimen Examined, Cannabis Intervention Details, Study Methodology, Measures, Inflammatory Cytokines, and Results

Study Animal species/sex/age Model Specimen Cannabis administration/dosage Methods Measures Inflammatory cytokine(s) measured/effect directionality Results
Al-Ghezi et al.23 Female C57BL/6 mice (6–8 weeks) EAE Spleen; serum; cecal content CBD+THC (10 mg/kg each; 1:1 ratio); vehicle; daily i.p. injection, beginning 10 days after disease induction EAE was induced using MOG/CFA emulsion (subcutaneous injection) and Bordetella Pertussis toxin (i.p. injection). The role of the microbiota was studied in relation to the mechanism of cannabinoids on clinical symptoms of MS Histopathology; inflammatory cytokines; microbiome profiling; LPS and SCFA quantification ↓ IL-17A
↑ IL-10
No change: IFN-γ
CBD+THC treatment reduced EAE severity by enhancing anti-inflammatory responses reflected in IL-10 and TGF-β levels. Treatment also limited gut microbiota such as Akkermansia muciniphila linked to EAE reflected by reduced microbial metabolites and LPS while increasing SCFA production
Arruza et al.47 Male long white Landrace piglets (1–2 days) HI brain damage Brain; lung; BALF CBD (1 mg/kg); CBD+WAY100635 (5-HT1A receptor antagonist, 0.1 mg/kg); vehicle; single IV administration Carotid arteries were exposed and wrapped with elastic bands. HI was induced by pulling out the elastic bands until complete interruption of carotid blood flow occurred for 30 min. Total lung compliance, oxygenation index, and extravascular lung water content were monitored for 6 h after HI brain damage Respiratory and cardiac functional outcome measures; histopathology; IL-1β; oxidative stress markers CBD vs. vehicle: ↓ IL-1β (lung tissue)
CBD+WAY100635 vs. vehicle (lung tissue): No change
CBD mitigated negative HI-induced effects on total lung compliance, oxygen index and reduced histological lung damage. For CBD alone, decreases in IL-1β were observed, combined with reduced protein content in BALF and extravascular lung water. Positive effects are hypothesized to be owing to anti-inflammatory changes in the brain induced by CBD only treatment
Barichello et al.38 Male Wistar rats (estimated 8–10 weeks based on weight) Neuroinflammation-pneumococcal meningitis Brain, CSF CBD (2.5, 5, or 10 mg/kg) once, or daily for 9 days after meningitis induction; placebo; i.p. injection Meningitis was induced by inoculating rats with Streptococcus pneumoniae. Rats (single dose group) were killed 6 h after meningitis induction. On day 10, rats receiving chronic CBD treatment completed the inhibitory avoidance task before killing and collection of hippocampal and frontal cortical tissue Inflammatory cytokines; BDNF; behavioral assessment (inhibitory avoidance task) Acute treatment: no change: TNF-α (hippocampus); IL-6; IL-1β
Chronic treatment: ↓ TNF-α (frontal cortex); unchanged in hippocampus
Acute CBD treatment did not revert increases in TNF-α, IL-6, or IL-1β resulting from pneumococcal meningitis induction.
Chronic treatment with CBD (10 mg/kg) prevented cognitive impairment in rats induced with pneumococcal meningitis. CBD administration at 2.5, 5 and 10 mg/kg reduced TNF-α concentrations in the frontal cortex
Borrelli et al.24 Male ICR mice (estimated 7–9 weeks based on weight) Colitis (IBD) Colon CBD (1–10 mg/kg daily for gross macroscopic evaluations and 5 mg/kg for all other measures) for six consecutive days, commencing 3 days before DNBS administration; control NR Colitis was induced with intracolonic administration of DNBS. Mice were killed 3 days after DNBS administration. Macroscopic evaluation and histology performed Histopathology; inflammatory cytokines; nitrites; oxidative stress markers; endocannabinoid levels ↓ IL-1β; ↑ IL-10 5 mg/kg CBD reduced pro-inflammatory IL-1β and increased anti-inflammatory IL-10 concentrations in full-thickness colonic tissues. Attenuation of DNBS-induced damage in colon tissue was greatest at 5 mg/kg CBD treatment. CBD reduced nitrite production, oxidative stress marker and endocannabinoid levels, but no effect on COX-2 and FAAH mRNA expression
Borrelli et al.25 Male ICR mice (estimated 7–9 weeks based on weight) Colitis (IBD) Colon Preventative protocol: CBG (1–30 mg/kg) daily for six consecutive days commencing 3 days before DNBS administration; curative protocol: CBG (1–30 mg/kg) injected for two consecutive days starting 24 h after DNBS administration As per Borrelli et al.24 Inflammatory cytokines; oxidative stress markers; nitrites; intestinal permeability; SOD activity; histopathology; IHC ↓ IL-1β; IFN-γ; ↑ IL-10 Preventative and curative protocols (30 mg/kg): CBG reduced colon injury, colon weight:length ratio (from 1 mg/kg preventative protocol and from 5 mg/kg curative protocol).
Curative protocol only (30 mg/kg): CBG reduced pro-inflammatory IL-1β and IFN-γ and increased anti-inflammatory IL-10, improved IHC, intestinal permeability, decreased MPO, SOD, iNOS, but had no effect on COX-2.
Preventative protocol only (30 mg/kg): CBG partially improved IHC
Britch et al.39
Experiment 1
Male and female Sprague–Dawley rats (aged 60–90 days) Localized inflammatory pain Hind-paw THC (0.0–4.0 mg/kg); CBD (0.0–10.0 mg/kg) i.p. administration twice daily for 3 days (repeated dose group). On day 4, naive rats received single dose of THC, CBD, or vehicle (acute dose group) and assessments performed Inflammation was induced by intraplantar injection of CFA. Assessments were carried out 0.5–4.0 h postinjection on day 4 Allodynia; nociceptive behavior; weight-bearing; locomotor activity; body weight Not measured THC and CBD increased mechanical threshold, and weight bearing on inflamed paw. THC had anti-hyperalgesic effects and reduced body weight; CBD did not. THC reduced locomotor activity while CBD showed the opposite effect
Britch et al.39
Experiment 2
Male and female Sprague–Dawley rats (60–90 days) Systemic inflammation Serum THC (2.0 mg/kg); CBD (10 mg/kg); vehicle; i.p. administration twice daily for 3 days. On day 4 (4 h after treatment) serum samples obtained for cytokine analysis Inflammation was induced by intraplantar injection of CFA or mineral oil (no pain control was present) Inflammatory cytokines THC: No change: IFN-γ; TNF-α; IL-1β; IL-6; IL-10
CBD: ↓ IFN-γ; IL-10; IL-1β; TNF-α (only male in CFA model); ↑ IL-6
Sex, dosing frequency, and treatment varied cytokine responses independently. Serum cytokines were largely unaffected by THC. In contrast, CBD generally exerted reduction in serums in IFN-γ, IL-10, and IL-1β production, and to some extent with TNF-α, with increases in IL-6
Costa et al.40 Male Wistar rats (200–220 g) (estimated ∼6 weeks based on weight) Chronic neuropathic pain; inflammatory pain Hind-paw; spinal cord; plasma Chronic neuropathic pain: CBD (2.5, 5, 10, and 20 mg/kg); Inflammatory pain: 20 mg/kg); vehicle; oral administration daily for 7 days, beginning on day 7 after disease induction Unilateral neuropathic pain was induced by constriction injury of the sciatic nerve in the right hind paw. Inflammatory pain was induced using CFA (intraplantar injection). 14 days after disease induction (24 h after last CBD dose), behavioral evaluations carried out and rats killed. Biochemical assays and hind paw tissue assessed TNF-α; oxidative stress markers; nociceptive behavior No change: TNF-α (measured in neuropathic model only) Hyperalgesia to mechanical and thermal stimuli was reduced in both pain models after daily oral CBD treatment. CBD activity was associated with reduced pro-oxidant markers and normalized overall antioxidant capacity. No reductions in TNF-α or NF-κB were observed
Elliott et al.26 Female C57BL/6 mice (6–8 weeks) EAE Serum CBD (20 mg/kg); vehicle; i.p. administration daily, beginning 9 days after disease induction, until day 25 Disease induction as per Al-Ghezi et al.23 Clinical scores of EAE assessed. Serum cytokines measured Inflammatory cytokines Serum: ↓ IFN-γ; IL-17 CBD treatment delayed onset of disease, and led to reduction in EAE severity, T cell infiltration and pro-inflammatory markers
Gallily et al.27 Female Sabra mice (6–8 weeks old) Inflammatory pain Hind-paw; plasma Parenteral dose (single i.p. injection): Purified CBD (1, 5, 25, and 50 mg/kg); Cannabis clone 202 (5, 25 and 50 mg/kg); vehicle.
Oral dose (single administration): Purified CBD (10, 25, 50 and 100 mg/kg); Cannabis clone 202 (10, 25, 50, 100, and 150 mg/kg); vehicle
Inflammation was induced using 60 μg zymosan injection into the subplanter surface of the right paw. Therapeutic agents were given immediately after disease induction and compared with aspirin and tramadol. Paw swelling and pain perception were assessed 2-, 6-, and 24-h postinjection TNF-α; paw edema; nociceptive behavior ↓ TNF-α CBD at 5 mg/kg achieved maximal effect on inhibition of inflammation (evident by reduced TNF-α) and antinociceptive effect. These beneficial effects were greater compared with tramadol and aspirin
Li et al.28 C57BL mice; sex and age NR (weight 22–26 g) Acute pancreatitis Pancreas; lungs; plasma CBD (0.5 mg/kg) or O-1602 (synthetic cannabinoid; 10 mg/kg); 2 doses 30 min before the first cerulein injection and immediately before the fifth (of 6) cerulein injection; saline; all i.p. administration Pancreatitis was induced by hourly cerulein (i.p. injection) for a total of six doses. At 3 h after final cerulein injection, mice were killed. Plasma, lungs and pancreas were collected for assessment Histopathology; inflammatory cytokines; pancreatic enzyme activity; MPO activity; GPR55 expression CBD: ↓ IL-6; TNF-α
O-1602: ↓ TNF-α
Either CBD or O-1602 attenuated acute pancreatitis pathophysiology, pancreatic enzyme activity, reduced MPO activity, protein expression, and rescued IHC outcomes.
Plasma: CBD reduced IL-6 and TNF-α and O-1602 reduced TNF-α only.
Pancreatic tissue: CBD reduced IL-6, O-1602 did not
Napimoga et al.41 Male Wistar rats (age NR; estimated ∼7–10 weeks based on weight) Experimental periodontitis Gingival tissue/bone CBD (5 mg/kg); vehicle, i.p. administration daily for 30 days after disease induction Periodontal disease was induced using a ligature around the first molars of each treated rat. Rats were killed after 30 days where mandibles and gingival tissue were taken for further assessment Histopathology; inflammatory cytokines; MPO activity ↓ IL-1β; TNF-α CBD-treated animals had reduced bone loss in periodontal disease along with decreased expression of RANKL/RANK; an NF-κB activator. The MPO demonstrated a lessening of neutrophil migration, associated with reductions in levels of IL-1β and TNF-α
Ribeiro et al.30 Male C57BL/6 mice (∼60 days) Acute lung injury Lung; BALF CBD (0.3, 1.0, 10, 20, 30, 40, and 80 mg/kg); vehicle, single i.p. administration 60 min after disease induction Acute lung injury was induced by intranasal instillation of Escherichia coli LPS. Total inflammatory cells combined with BALF was used to assess leukocyte migration to the lungs Inflammatory cytokines; MPO activity; protein concentration (for normalizing read-out in BALF samples) ↓ TNF-α; IL-6 CBD administration reduced lung leukocyte migration, the production of pro-inflammatory cytokines, and vascular permeability, mediated by adenosine A2A receptor signaling to promote anti-inflammatory effects
Ribeiro et al.29 Male C57BL/6 mice (8–10 weeks) Acute lung injury Lung; BALF CBD (20 and 80 mg/kg); vehicle, single i.p. administration 6 h after disease induction As per Ribeiro et al.30 Mice were killed 24 h after disease induction. Pulmonary mechanics were assessed and various biochemical assays performed Inflammatory cytokines; pulmonary mechanics; MPO activity; histopathology; protein concentration (for normalizing read-out in BALF samples) ↓ TNF-α (CBD 20 and 80 mg/kg); IL-6 (80 mg/kg CBD) CBD (both doses or 80 mg/kg CBD only) successfully reduced inflammatory cytokines and chemokines, leukocyte migration, and MPO activity in BALF. Both doses of CBD improved pulmonary mechanics and lung function
Soares et al.42 Male adult Wistar rats (estimated 10–12 weeks based on weight) Ischemia/reperfusion kidney injury Kidney CBD (5 mg/kg); saline, single infusion via the aorta immediately before the release of renal pedicle (clamped bilaterally for 45 min) Kidney injury was induced by incising the abdominal wall and dissecting the renal pedicle bilaterally. A clamp was applied to pedicle bilaterally for 45 min followed by reperfusion. Rats were killed 24 h after disease induction. Kidneys were retrieved for further analysis Inflammatory cytokines; MPO activity; oxidative stress markers; cannabinoid receptor signaling ↓ TNF-α; IL-1β CBD exhibited a protective effect in kidney injury and reduced pro-inflammatory cytokines induced by ischemia/reperfusion. CBD reduced damage caused by oxidative stress, but had no effect on nitrate levels. No changes were observed in the expression of cannabinoid receptors after treatment
Sonego et al.31 Male Swiss mice (10 weeks) Experimental tardive dyskinesia Brain Experiment 1: CBD (60 mg/kg) and haloperidol (2–3 mg/kg) i.p. administration daily for 21 days; vehicle.
Experiment 2: CBD (60 mg/kg) or GW9662 (PPAR-γ antagonist; 2 mg/kg) and haloperidol (3 mg/kg) i.p. administration daily for 21 days; vehicle
Orofacial dyskinesia was induced by haloperidol. Behavioral assessments carried out 24 h after final injection, then striatum extracted for analyses Inflammatory cytokines; oxidative stress markers; locomotor activity; vacuos chewing movements ↓ TNF-α; IL-1β
No change: IL-6
↑ IL-10 (only in conjunction with haloperidol 3 mg/kg)
Chronic CBD administration alleviated orofacial dyskinesia but did not improve locomotor activity. In striatum, CBD attenuated microglial activation, oxidative stress, and pro-inflammatory cytokines TNF-α; IL-1β, and increased anti-inflammatory IL-10. CBD reduced TNF-α and IL-6 mRNA expression; no treatment affected IL-1β mRNA expression. When mice were pretreated with PPAR-γ antagonist, this blocked positive behavioral effects of CBD, suggesting PPAR-γ receptor involvement
Verrico et al.48 Female, C57BL/6J mice (6–10 weeks). Note: canine data not reported as inflammatory cytokines not assessed Experimental inflammation (local and systemic) Ear; serum Croton oil-induced inflammation: CBD oil (100 μL of 10 mg/mL); vehicle; single topical administration 2 h after disease induction.
LPS-induced inflammation: CBD (1, 10, and 100 μg) i.p. administration or CBD (100 μg) topical administration at LPS injection site 2 h after disease induction; control
Croton oil (localized) and LPS (systemic) models of experimental inflammation (localized applied topically to ear and systemic via i.p. injection, respectively). Collection of ear tissue 1–4 h after croton oil and serum samples 2 h after LPS administration Inflammatory cytokines; oxidative stress markers; MPO activity; in vivo assessment of cannabidiol bioavailability ↓ TNF-α; IL-6
↑ IL-10
Local CBD administration reduced MPO activity, circulating TNF-α, and the development of edema. Systemic TNF-α and IL-6 were reduced in a dose-dependent manner; 100 μg topical CBD generated similar anti-inflammatory effects to i.p. administration. CBD alone increased IL-10. Liposomal CBD was more bioavailable than naked CBD and was able to reduce inflammatory cytokines at a faster rate
Vuolo et al.43 Male adult Wistar rats (8 weeks) Experimental asthma Serum CBD (5 mg/kg); vehicle, i.p. administration once daily on final 2 days of OVA challenge Asthma was induced by inoculation with OVA (i.p. injection). 14 days after initial injection, rats were boosted with OVA or alum. After another 7 days, rats were challenged with aerosol with OVA or saline (30 min/day for 3 days). 24 h after the final OVA challenge, animals were killed and blood samples taken for inflammatory marker assessment Inflammatory cytokines ↓ Th1 cytokines: TNF-α; IL-6; and Th2 cytokines: IL-4; IL-5; IL-10; IL-13
Note: IL-10 results taken from Figure 2C; abstract reports opposing effect
CBD treatment reduced all measured cytokines (all of which are specifically implicated in asthma pathogenesis). BALF and lung function were not quantified
Vuolo et al.32 BALB/c mice; sex and age NR (weight 20–25 g) Experimental asthma Lung; BALF CBD (5–10 mg/kg); OVA; vehicle, single i.p. administration Mice were inoculated with sterile OVA (i.p. injection). Mice were anesthetized and airway responsivity after methacholine challenge was assessed 24 h after the final treatment Inflammatory cytokines; lung function; cannabinoid receptors; metalloproteinase protein content ↓ IL-4; IL-5; IL-13 CBD generally reduced inflammatory cytokines; this effect was not reversed when CB1 and CB2 receptors were individually blocked. Double blockade of CB1 and CB2 receptors did reverse effects of CBD and increased levels of IL-4, IL-5, and IL-13 in BALF. CBD reversed increases in collagen fiber content in alveolar septa. CBD at the highest dose (10 mg/kg) reversed changes in static lung elastance and inflammatory cell infiltration in lung tissue
Wang et al.33 Female C57BL/6J mice (10–12 weeks) Hepatic steatosis Liver; serum CBD (5 or 10 mg/kg); vehicle, daily i.p. administration for 11 days during ethanol exposure Chronic plus binge ethanol feeding was used to induce liver injury and steatosis TNF-α; biochemical assays; MPO activity; oxidative stress markers; histopathology ↓ TNF-α
Note: only fold change; absolute concentration not presented
CBD treatment attenuated both oxidative and nitrative stress together with ethanol-induced liver steatosis. Leukocyte infiltration and neutrophil-mediated inflammation was also reduced
Watt et al.34 Male C57BL/6J×C3H/Hej mice (12 months) AβPPswe/PS1ΔE9 AD model Brain (hippocampus and cortex) CBD (50 mg/kg) daily; vehicle, daily i.p. injection for a total of 7 weeks Double transgenic mice were used for AD model. Treatment commenced 3 weeks before behavioral testing and continued throughout, for a total of 7 weeks Inflammatory cytokines; behavioral assessments; disease markers (Aβ40,42); protein expression of IBA1; BDNF; PPAR-γ No change: TNF-α; IL-1β CBD treatment restored deficits in spatial learning and social recognition memory, and marginally reduced hippocampal insoluble Aβ40, but no effect on other measures
Wei et al.44 Sprague-Dawley rats (sex and age NR; estimated males ∼9–10 weeks based on weight). Note: C57/BL mouse data NR as inflammatory cytokines not assessed Colitis Colon; serum CBD (1 mg/kg); O-1602 (synthetic atypical cannabinoid, 10 mg/kg control (ethanol solution), i.p. administration daily for 5 days Colitis was induced by TNBS via intrarectal instillation daily. CBD/O-1602 treatment was administered 30 min before drug injection Inflammatory cytokines; histopathology; MPO activity; GPR55 gene and protein expression; ATP-mediated colon muscular activity ↓ IL-6
No change: TNF-α
CBD and O-1602 treatment reduced disease severity mediated by decreased MPO activity and IL-6 but not TNF-α levels. CBD also increased GPR55 expression and reversed abnormal colonic mobility induced by TNBS
Weiss et al.35 Female NOD/LtJ (known as NOD/ShiLtJ since 2007) mice (6–12 weeks) Autoimmune type 1 diabetes Pancreatic tissue; plasma CBD (5 mg/kg); vehicle, daily i.p. administration (5 times/week for 2–4 weeks) Urinary glucose was tested 1–2 times/week. Plasma was obtained from treated and untreated mice and assayed for inflammatory cytokines Inflammatory cytokines; urinary glucose, pancreatic histopathology ↓ TNF-α; IFN-γ CBD treated mice had lower incidence of diabetes, delayed disease onset and reduced severity of insulitis and reduction of pro-inflammatory cytokines TNF-α, and IFN-γ in plasma
Weiss et al.36 Female NOD/LtJ and BALB/c mice (all 11–14 weeks) Autoimmune type 1 diabetes Pancreas; plasma CBD (5 mg/kg); vehicle, daily i.p. administration (5 times/week for 4 weeks) Mice were observed until 24 weeks of age. Urinary glucose was tested 1–2 times/week. Plasma was obtained from treated and untreated mice and assayed for cytokines IL-6; urinary glucose; histopathology ↓ IL-6 CBD-treated mice were less prone to diabetes development, had normal intact islet with limited immune cell infiltration along with reductions in IL-6 levels in plasma compared with untreated mice
Wong et al.45 Australian Albino Wistar rats (sex and age NR; estimated males ∼56 days based on weight) Obesity Adipose, plasma THC (10 mg/kg); vehicle, daily i.p. administration for 10 days Method of inducing obesity not reported. After a 48-h washout period (after final injection), rats were killed. Epididymal fat pads were removed for analyses Histopathology; TNF-α; obesity markers; apoptosis marker No change: TNF-α THC improved obesity markers reflected by reduced overall body weight and food intake, free fatty acid levels, and increased gluconeogenesis. THC increased macrophage infiltration, TNF-α expression (gene but not plasma conc.) and hypertrophy in adipose tissue
Zhang et al.37 BALB/c mice (6 weeks); sex NR Retinal damage Retina THC (1 or 2 mg/kg); vehicle; i.p. administration daily for 2 months Pathological and morphological changes in the retina induced by THC administration were evaluated Inflammatory cytokines; oxidative and ER stress markers; apoptosis markers; histopathology; retinal function ↑ TNF-α; IL-1β; IL-6
Note: only fold change; absolute concentration not presented
THC caused retinal damage evident by presence of apoptotic photoreceptor cells. THC also enhanced oxidative and ER stress along with increased pro-inflammatory cytokines in retinas
Zhou et al.46 Female Lewis rats (9–14 weeks) EAE Spinal cord THC:CBD (10:10 and 1:20; 215 mg/kg); oral gavage daily on days 6–18 after disease induction Disease induction by myelin basic protein gp 69–88 (subcutaneous injection) in CFA. Measures of neuropathic pain induced behavioral changes and neurological disability scoring was used to assess therapeutic effect of CBD TNF-α; neurological disability scoring; thermal sensory testing; behavior for neuropathic pain; BDNF activity ↓ TNF-α; THC:CBD 10:10 more effective at the initial time point than THC:CBD 1:20 Both combination cannabinoid formulas decreased TNF-α and increased BDNF and improved behavioral assessments and neurological disability scoring. Both formulations were able to delay peak onset of disease, however the THC:CBD 10:10 formulation delayed time to peak neurological disability by an additional day compared with THC:CBD 1:20

Aβ, beta-amyloid; AD, Alzheimer's disease; BALB, Bagg Albino; BALF, bronchoalveolar lavage fluid; BDNF, brain-derived neurotrophic factor; CB1, cannabinoid type 1; CB2, cannabinoid type 2; CBD, cannabidiol; CBG, cannabigerol; CFA, complete Freund's adjuvant; COX-2, cyclooxygenase-2; CSF, cerebral spinal fluid; DNBS, 2,4,6-dinitrobenzene sulfonic acid; EAE, experimental autoimmune encephalitis; ER, endoplasmic reticulum; FAAH, fatty acid amide hydrolase; HI, hypoxic-ischemic; IBA1, ionized calcium binding adaptor molecule 1; IBD, inflammatory bowel disease; ICR, Institute of Cancer Research; IFN-γ, interferon gamma; IHC, immunohistochemistry; IL, interleukin; iNOS, inducible nitric oxide synthase; i.p., intraperitoneal; IV, intravenous; LPS, lipopolysaccharide; MOG, myelin oligodendrocyte glycoprotein; MPO, myeloperoxidase; mRNA, messenger RNA; MS, multiple sclerosis; NF-κB, nuclear factor-κB; NO, nitric oxide; NOD, nonobese diabetic; NR, not reported; OVA, ovalbumin; PPAR-γ, peroxisome proliferator-activated receptor gamma; RANKL/RANK, receptor activator of nuclear factor kappa-B ligand; SCFA, short chain fatty acid; SOD, superoxide dismutase; TGF-β, transforming growth factor beta; THC, delta 9-tetrahydrocannabinol; TNBS, 2,4,6-trinitrobenzenesulfonic acid; TNF-α, tumor necrosis factor alpha.